InteractiveFly: GeneBrief

A kinase anchor protein 200 : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

Gene name - Akt1

Synonyms - RacPK, PKB

Cytological map position - 89B9-89B9

Function - signaling

Keywords - growth response, insulin signaling pathway, anti apoptotic, oncogene

Symbol - Akt1

FlyBase ID: FBgn0010379

Genetic map position -

Classification - S/T kinase, Src homology 2 domain

Cellular location - cytoplasmic

NCBI links:   Precomputed BLAST |  Entrez Gene
Recent literature
Slade, J. D. and Staveley, B. E. (2015). Compensatory growth in novel Drosophila Akt1 mutants. BMC Res Notes 8: 77. PubMed ID: 25889856
The insulin receptor signalling pathway with its central component, the Akt1 kinase, and endpoint regulator, the transcription factor Foxo, plays a significant role in the control of growth. Imprecise excision of a PZ P-element inserted in the upstream region of Akt1 generated several mutations that lead to small, viable flies that presented with delays in development. Suppression of this phenotype by the directed expression of Akt1- indicates that the phenotypes observed are Akt1 dependent. Somatic clones of the eyes, consisting of homozygous tissue in otherwise heterozygous organisms that develop within a standard timeframe, signify that more severe phenotypes are masked by an extension in the time of development of homozygous mutants. Generation of flies having the hypomorphic Akt1 alleles and a null allele of the downstream target foxo result in a phenotype very similar to that of the foxo mutant and do not resemble the Akt1 mutants. It is concluded that developmental delay of these novel Akt1 hypomorphs results in a latent phenotype uncovered by generation of somatic clones. The compensatory growth occurring during the extended time of development appears to be implemented through alteration of foxo activity. Production of clones is an effective and informative way to observe the effects of mutations that result in small, viable, developmentally delayed flies.

Jevtov, I., et al. (2015). TORC2 mediates the heat stress response in Drosophila by promoting the formation of stress granules. J Cell Sci [Epub ahead of print]. PubMed ID: 26054799
The kinase TOR is found in two complexes, TORC1, involved in growth control, and TORC2 with less well defined roles. This study asked whether TORC2, disrupted by use of Rictor mutant flies, has a role in sustaining cellular stress. TORC2 inhibition in Drosophila was shown to lead to a reduced tolerance to heat stress. Accordingly, upon heat stress, both in the animal and Drosophila cultured S2 cells, TORC2 is activated and is required for the stability of its known target Akt/PKB. The phosphorylation of the stress activated protein kinases is not modulated by TORC2, nor is the heat-induced upregulation of heat shock proteins. Instead, it was shown, both in vivo and in cultured cells, that TORC2 is required for the assembly of heat-induced cytoprotective ribonucleoprotein particles, the pro-survival stress granules. These granules are formed in response to protein translation inhibition imposed by heat stress that appears less efficient in the absence of TORC2 function. It is proposed that TORC2 mediates heat resistance in Drosophila by promoting the cell autonomous formation of stress granules.

Johnson, J. L., Huang, W., Roman, G. and Costa-Mattioli, M. (2015). TORC2: a novel target for treating age-associated memory impairment. Sci Rep 5: 15193. PubMed ID: 26489398
Memory decline is one of the greatest health threats of the twenty-first century. Because of the widespread increase in life expectancy, 20 percent of the global population will be over 60 in 2050 and the problems caused by age-related memory loss will be dramatically aggravated. However, the molecular mechanisms underlying this inevitable process are not well understood. This study shows that the activity of the recently discovered mechanistic target of rapamycin (mTOR) complex 2 (mTORC2, see Drosophila TOR and Rictor) declines with age in the brain of both fruit flies and rodents and that the loss of mTORC2-mediated actin polymerization contributes to age-associated memory loss. Intriguingly, treatment with a small molecule that activates mTORC2 (A-443654; a specific Akt inhibitor that activates mTORC2-mediated phosphorylation of Akt) reverses long-term memory (LTM) deficits in both aged mice and flies. In addition, pharmacologically boosting either mTORC2 or actin polymerization enhances LTM. In contrast to the current approaches to enhance memory that have primarily targeted the regulation of gene expression (epigenetic, transcriptional, and translational), the data points to a novel, evolutionarily conserved mechanism for restoring memory that is dependent on structural plasticity. These insights into the molecular basis of age-related memory loss may hold promise for new treatments for cognitive disorders.
Mensah, L. B., Goberdhan, D. C. and Wilson, C. (2017). mTORC1 signalling mediates PI3K-dependent large lipid droplet accumulation in Drosophila ovarian nurse cells. Biol Open [Epub ahead of print]. PubMed ID: 28302666
Insulin and insulin-like growth factor signalling (IIS), which is primarily mediated by the PI3-kinase (PI3K)/PTEN/Akt kinase signalling cassette, is a highly evolutionary conserved pathway involved in co-ordinating growth, development, ageing and nutrient homeostasis with dietary intake. It controls transcriptional regulators, in addition to promoting signalling by mechanistic Target of Rapamycin (mTOR) Complex 1 (mTORC1; see Tor), which stimulates biosynthesis of proteins and other macromolecules, and drives organismal growth. Previous studies in nutrient-storing germline nurse cells of the Drosophila ovary showed that a cytoplasmic pool of activated phosphorylated Akt (pAkt) controlled by Pten, an antagonist of IIS, cell-autonomously regulates accumulation of large lipid droplets in these cells at late stages of oogenesis. This study shows that the large lipid droplet phenotype induced by Pten mutation is strongly suppressed when mTor function is removed. Furthermore, nurse cells lacking either Tsc1 or Tsc2, which negatively regulate mTORC1 activity, also accumulate large lipid droplets via a mechanism involving Rheb, the downstream G-protein target of TSC2, which positively regulates mTORC1. It is concluded that elevated IIS/mTORC1 signalling is both necessary and sufficient to induce large lipid droplet formation in late-stage nurse cells, suggesting roles for this pathway in aspects of lipid droplet biogenesis, in addition to control of lipid metabolism.

Organism size is determined by a tightly regulated mechanism that coordinates cell growth, cell proliferation and cell death. The Drosophila insulin receptor/Chico/Dp110 pathway regulates cell and organism size. Chico, an adaptor protein that binds to the Insulin-like receptor, and Phosphotidylinositol 3 kinase 92E (Dp110), an enzyme that phosphorylates lipids, are both involved in transmitting insulin receptor signals downstream to cellular effectors. The subject of this overview, the phosphoinositide-3-OH-kinase-dependent serine/threonine protein kinase Akt1 (also known as protein kinase B or PKB) affects cell and organ size in Drosophila in a cell autonomous manner (Verdu, 1999). PKB has a PH domain that binds 3-phosphorylated inositol lipids (phosphatidylinositol 3,4,5-trisphosphate also known as PIP3), and the translocation of the mammalian homolog of Drosophila Akt1 to the plasma membrane is an important part of its activation. PKB is also phosphorylated by a PIP3-activated phosphoinositide-dependent protein kinase (PDK-1), which has a PH domain that binds PIP3. Thus there are two independent contributions of 3-phosphorylated inositol lipids to the activation of PKB, one via PDK-1 and the other involving PKB itself (Irvine, 1998 and references therein). Akt appears to stimulate intracellular pathways that specifically regulate cell and compartment size independent of cell proliferation in vivo (Verdu, 1999).

To determine whether Drosophila Akt1 participates in insulin-receptor signal transduction, Akt1 activity was measured in Schneider (S2) cells. Insulin stimulates Akt1 activity sevenfold in S2 cells overexpressing a wild-type Akt1 transgene. Furthermore, membrane localization of Akt1 by addition of an src myristoylation sequence to its amino terminus is sufficient to confer constitutive kinase activity. In contrast, kinase-deficient Akt1 shows activity neither in the basal state nor after insulin stimulation, thus indicating that the measured phosphotransferase activity is not due to a contaminating kinase. These observations confirm that Akt1 is regulated in a way similar to that of its mammalian homolog. Consistent with this proposal, pretreatment with the PI(3)K inhibitor wortmannin blocks Akt1 activation by insulin. These data indicate that, as in mammalian cells, Drosophila PI(3)K is a component required for mediating the activation of Akt1 (Verdu, 1999).

To determine whether ectopic expression of Akt1 increases the size of tissues, Akt1 was targeted to the wing using a 71BGAL4 line. This resulted in a marked enlargement of the wing imaginal disc and an expansion of the surface of the adult wing blade as well as an increase in vein thickness. This increase in size is often accompanied by a mild disruption of the proximo-distal alignment characteristic of the hairs present on the wing-blade surface. Morphometric analysis of 71BGAL4/UAS-Akt1 wings reveals a 29% increase in wing surface area. Furthermore, ectopic expression of Akt1 along the anteroposterior boundary of the wing imaginal disc results in enlargement of only the corresponding region of the adult wing. In spite of the increased size of the wing in 71BGAL4/UAS-Akt1 flies, there is no change in the number of cells, resulting in a cell density in 71BGAL4/UAS-Akt1 flies that is 15% lower than that in 71BGAL4/+ controls. Together, these observations show that ectopic expression of Akt1 increases the size of the wing imaginal disc, leading to enlargement of the adult wing. The question of whether the effect of Akt1 on compartment growth in the wing is cell autonomous was addressed further. Targeting of Akt1 to the posterior compartment of the wing imaginal disc with an engrailed-GAL4 line results in a marked expansion of this region, whereas the anterior compartment remains unaffected (Verdu, 1999).

To evaluate the Akt1-selective increase in cell size more quantitatively, Akt1 was expressed in the posterior compartment of wing imaginal discs; measured were compartment areas, cell size and cell number, the latter two by flow cytometry. Expression or inactivation of cell-cycle regulators, such as E2F, RBF and Cdc2, in the posterior compartment affects cell size and number without altering compartment size. Akt1 expression increases the area occupied by the posterior compartment concomitant with a marked enlargement of its cells as measured by forward light scatter. Strikingly, no changes in the number of cells in the posterior compartment are detected. Thus, overexpression of Akt1 affects compartment size by altering cell growth without a concomitant increase in the final number of cells within the compartment. Studies of mammalian cells have indicated that Akt may positively regulate cell-cycle progression. However, in the wing imaginal disc, no differences were found in cell proliferation between control cells in the anterior compartment and cells expressing Akt1 in the posterior compartment, as judged by the pattern or frequency of bromodeoxyuridine incorporation (Verdu, 1999).

Akt overrides G1 arrest induced by PTEN (see Drosophila Pten) and by interleukin-2 deprivation in cell-culture models. To determine whether ectopic Akt1 could bypass cell-cycle arrest in imaginal tissues, a population of physiologically arrested cells in the wing imaginal disc, the zone of non-proliferating cells (ZNC), was studied. Expression of positive regulators of the cell cycle, such as the phosphatase Cdc25string and cyclin E, bypasses both G1 and G2 arrests in the ZNC. Interestingly, Akt1 expression in the posterior compartment does not rescue the cells of the ZNC from their G1 arrest. As a more quantitative assay of Akt1 effects on cell-cycle progression, wing imaginal discs ubiquitously expressing Akt1 were dissected and cellular DNA content was measured by flow cytometry. The proportions of cells in G1, S and G2 phase remain indistinguishable in cells expressing Akt1 and wild-type cells, despite the differences in compartment size (Verdu, 1999).

Compartments function as an independent units of growth and size control. Ectopic expression of Akt1 overrides the intrinsic control mechanisms regulating the final size of posterior compartment. To circumvent potential compartment controls on cell number, clones of cells overexpressing Akt1 were generated in the wing imaginal disc. Clone size was assessed 48 h after induction by heat-shock. Akt1 markedly increases clonal size through an enlargement of the cells rather than an increase in the cell number. As a more sensitive assay of cell number, clones of cells expressing Akt1 were induced in the wing disc 72 h after egg deposition; cell number was assessed 48 h later. Analysis reveals that the increase in clonal size induced by ectopic Akt1 expression is due to a selective increase in cell size but not cell number. Thus, it is concluded that Akt1 affects compartment size by increasing cell growth (that is, cell size) without altering cell proliferation (Verdu, 1999).

Several lines of evidence indicate a requirement for components of the protein-synthetic regulatory apparatus for cell growth. The large-cell and small-cell phenotypes resulting from increasing or removing Akt activity, respectively, are consistent with concomitant alterations in the translational machinery. In mammals, Akt appears to influence the rate of protein synthesis through mTOR (for mammalian target of rapamycin)-mediated activation of p70S6kinase (see Drosophila RPS6-p70-protein kinase) and inhibition of the 4E-binding protein-1 (4E-BP1 or PHAS-1), a repressor of translation initiation. These results implicate Akt as an activator of messenger RNA translation and indicate that regulation of this pathway could be relevant to the ability of Akt to promote cell growth in vivo. A critical question is whether increases in protein synthesis are merely permissive for expansion of cell size, implying the existence of a distinct growth-regulatory mechanism, or whether Akt-dependent enhancement of protein translation is in itself sufficient to cause an increase in organ size. Alternatively, the augmentation in cell growth produced by Akt could be the result of activation of a concerted anabolic program, for which protein synthesis would be a vital component (Verdu, 1999 and references therein).

An important question arising from this and other papers is how signaling from the insulin receptor regulates compartment size. From the data presented here it can be concluded that manipulation of Akt levels affects compartment size by increasing cell growth without significant changes in cell number. Similar findings have been obtained from study of wing discs with reduced levels of S6 kinase (Montagne, 1999). The insulin receptor, Chico and Dp110 appear to influence both cell size and number in the Drosophila wing. Thus, a plausible scenario is that the pathway bifurcates directly upstream of Akt, which is required for cell growth (through a Drosophila TOR, S6 kinase and 4E-BP1), while a second branch mediates cell proliferation through a parallel pathway. However, it is not yet clear that activation of the insulin-receptor signaling pathway promotes cell proliferation in Drosophila. Reduction in levels of 1) the insulin receptor, 2) Chico or 3) Dp110 negatively affects cell growth and cell number. Nonetheless, it remains unclear whether this is a direct result of modulation of the cell-cycle machinery, or secondary to an impairment in cell growth. Inadequate cell growth may well function as a mitotic checkpoint, or render the cell more susceptible to apoptosis as cell division proceeds unabated. Either mechanism would result in a decrease in cell number. Interestingly, ectopic expression of Dp110 in clones of cells in the wing imaginal disc results in a dramatic increase in cell and clone size, with no effects in cell number. In any case, clearly the phenotypes resulting from ectopic expression of cell-cycle regulators in the wing disc do not resemble those reported for ectopic expression of Dp110, Akt and S6 kinase. Thus, the effects of the insulin-receptor pathway on cell growth are unlikely to be secondary to alterations in cell cycle, but probably represent the major biological output for Chico, Dp110 and Akt in Drosophila. Other regulatory pathways probably function as primary determinants of proliferation (Verdu, 1999 and references therein).

PDK1 regulates growth through Akt and S6K in Drosophila

The insulin/insulin-like growth factor-1 signaling pathway promotes growth in invertebrates and vertebrates by increasing the levels of phosphatidylinositol 3,4,5-triphosphate through the activation of p110 phosphatidylinositol 3-kinase. Two key effectors of this pathway are the phosphoinositide-dependent protein kinase 1 (PDK1) and Akt/PKB. Although genetic analysis in C. elegans has implicated Akt as the only relevant PDK1 substrate, cell culture studies have suggested that PDK1 has additional targets. In Drosophila, PDK1 controls cellular and organism growth by activating Akt1 and S6 kinase, dS6K (FlyBase name: RPS6-p70-protein kinase). Furthermore, dPDK1 genetically interacts with dRSK but not with dPKN (FlyBase name: Protein kinase related to protein kinase N), encoding two substrates of PDK1 in vitro. Thus, the results suggest that dPDK1 is required for dRSK but not dPKN activation and that it regulates insulin-mediated growth through two main effector branches, dAkt and dS6K (Rintelen, 2001).

To analyze the function of dPDK1 in Drosophila, gain- and loss-of-function alleles of the kinase were generated. Drosophila contains a single gene that encodes a kinase that is highly homologous to PDK1 in its primary sequence and its domain structure. Initially, two EP transposable elements in the 5' region of the endogenous Drosophila PDK1 gene dPDK1 were identified. These EP elements drive expression of dPDK1 under the control of the Gal4 system, allowing a test whether dPDK1 and dAkt cooperate in promoting growth in Drosophila. Overexpression of either kinase in the eye imaginal disc during the last cell division cycle and subsequent differentiation shows little effect on the size or the structure of the eye. Co-overexpression of dAkt and dPDK1, however, leads to a significant increase in eye size. Furthermore, analysis of clones of cells in the eye overexpressing dPDK1 and/or dAkt reveals that the observed effect on cell size is strictly autonomous. These results indicate that overexpression of dPDK1 does not interfere with the normal differentiation of eye disc cells and that it promotes local growth through dAkt activation (Rintelen, 2001).

To generate loss-of-function alleles of dPDK1, the dominant eye size phenotype caused by co-overexpression of dPDK1 and dAkt was reverted by using EMS mutagenesis, leading to three partial or complete loss-of-function mutations. dPDK13 causes a G(352) to S substitution in the conserved DFG motif in the kinase subdomain VII. The D residue in this motif is essential for kinase activity by orienting the ATP-Mg2+ complex for phosphotransfer. dPDK14 causes a P(441) to L substitution in a conserved residue in kinase subdomain VIII. In the dPDK15 allele, a Q codon at position 437 in kinase subdomain VIII is mutated to a STOP codon. Because this latter mutation results in the formation of a truncated dPDK1 protein lacking part of the kinase domain and the Pleckstrin-homology domain, dPDK15 is likely to be a null mutation. A fourth allele EP(3)3091 (dPDK11), from the Berkeley Drosophila Genome Project, has an EP element located in the third intron of dPDK1 and is homozygous lethal. It failed to complement dPDK15, and the lethality was reversed by EP element excision (Rintelen, 2001).

Combinations of loss-of-function alleles provide mutants of varying strengths. Larvae homozygous for the dPDK15 null allele or larvae of the dPDK11/5 heteroallelic combination die during the second instar stage. A less severe reduction in dPDK1 function (dPDK14/5) permits development of viable dPDK1 mutant flies that are delayed 1 day in development and smaller than their heterozygous siblings, having an 18% reduction in body weight. By measuring the cell density in the wing, the reduction in size and weight apparently is primarily caused by a decrease in cell size, because cell number is only slightly affected. The lethality associated with the dPDK1 null allele and the size defect of dPDK1 hypomorphs was rescued by ubiquitous expression of a wild-type dPDK1 transgene with armadillo (arm)-Gal4 as a driver. dPDK14/5 male flies are almost completely sterile, although they show no obvious defect in sperm morphology and motility and in mating behavior. That loss of zygotic dPDK1 function results in larval lethality is in contrast to a recent analysis of two dPDK1 mutations caused by the EP insertion EP(3)3091 (dPDK11) or a 10-kb deletion (dPDK12), which are homozygous embryonic lethal. It is possible that the embryonic lethality observed by Cho (2001) is not caused by loss of dPDK1 function but by a linked lethal mutation on the same chromosome, because no rescue was attempted, and the phenotype was only analyzed in homozygotes. Consistent with this observation, larvae homozygous for a dPDK11 mutant chromosome, which has been cleaned from second hits by recombination, die during the second instar stage. Although it is very likely that dPDK1 functions during embryogenesis, like dAkt, maternal transcripts may be sufficient to support embryonic development (Rintelen, 2001).

To determine whether the effects of loss of dPDK1 function on cell growth and organ development are autonomous events, loss of dPDK1 was analyzed in clones of cells by using the FRT mitotic recombination system. In contrast to organism lethality, clones of cells homozygous for the dPDK1 null allele dPDK15 survive to adulthood. These cells show no defect in their ability to differentiate into photoreceptor cells or accessory cells, but mutant photoreceptor cells are ~30% smaller than the heterozygous cells outside the clone, a strictly cell autonomous effect. To test whether an entire body part could develop in the absence of dPDK1 function, dPDK1 was selectively removed in much of the head primordium by using the ey-Flp system. Heads homozygous mutant for anyone of the three alleles, dPDK13, dPDK14, and dPDK15, are reduced in size, which indicates that entire organs differentiate and develop in the absence of dPDK1 function, but that the final size of these organs autonomously depends on the amount of dPDK1 activity. The reduction in head size was most severe with dPDK15 followed by dPDK14 and dPDK13, with the complete removal of dPDK1 function similar to that observed for loss-of-function mutations in the Drosophila insulin receptor (dInr), Dp110/PI(3)K, and dAkt (Rintelen, 2001).

The pronounced effect of loss of dPDK1 function on head size suggests that it is a dominant constituent in the dInr pathway. To test this possibility, the ability of complete and partial loss-of-function alleles of dPDK1 to reverse phenotypes caused by either overexpression of dInr or by mutations in dPTEN, the 3-phosphatidylinositide phosphatase, was evaluated. Overexpression of a wild-type dInr cDNA under the control of GMR-Gal4 leads to a marked increase in eye size and a slightly rough eye surface, an effect dominantly suppressed by removing one copy of dPDK1. Further reduction of dPDK1 function by the dPDK11/4 heteroallelic combination reduces the eye to almost wild-type size, suggesting that the amount of dPDK1 protein is rate-limiting for the dInr overgrowth phenotype. Null mutations in dPTEN cause lethality, and removal of dPTEN function in clones stimulates cell autonomous growth, suggesting that increased levels of PIP3 promote growth and are the likely cause of lethality. Thus, if dPDK1 is an essential target of PIP3, mutations in dPDK1 may suppress the dPTEN phenotype. Surprisingly, some dPTEN/dPDK1 double mutant flies survive to adulthood, indicating that the presumed PIP3-induced lethality is primarily caused by the hyperactivation of dPDK1 or of one of its targets (Rintelen, 2001).

The fact that the growth phenotypes of dPDK1 mutations are similar to those caused by mutations in genes coding for dS6K, and dAkt, and that S6K1 is a mammalian PDK1 substrate, raises the possibility that dPDK1 may independently control growth through dS6K. This possibility was tested in the wing, which is composed of a dorsal and a ventral epithelial sheet that are tightly attached to each other through extracellular matrix. Selective overexpression of a wild-type dS6K cDNA in the dorsal wing epithelium with the apterous (ap)-Gal4 driver leads to a bending down of the wing blade, probably because of a cell-size increase in the dorsal surface. This phenotype is suppressed by a reduction of dPDK1 function. Although ap-Gal4 induced overexpression of wild-type dPDK1 alone had little effect on wing morphology, overexpression of a dPDK1A467V variant is sufficient to cause a bent-wing phenotype. The corresponding amino acid substitution in the C. elegans PDK1 is thought to cause a hyperactivation of the kinase. The dPDK1A467V-induced bent wing phenotype depends on normal levels of dS6K and dAkt, because null mutations in either of the corresponding genes dominantly suppress the phenotype. Together with the biochemical evidence in both cultured cells and in vivo, that dPDK1 controls the activity of dAkt and dS6K, these results provide functional evidence that dPDK1 is a key regulator in the control of growth and cell size by regulating the activity of two AGC kinases, dAkt and dS6K (Rintelen, 2001).

The effects of dPDK1 on dS6K raised the possibility that dPDK1 controls the activity of other AGC kinases in vivo, such as dRSK and dPKN, which have been implicated as mammalian PDK1 substrates. Because the developing eye depends on endogenous levels of dPDK1, whether lowering the dose of dPDK1 is sufficient to suppress dominantly the rough eye phenotype caused by overexpression of dRSK and dPKN under GMR-Gal4 control was tested. Reduction of dPDK1 activity in a viable dPDK1 mutant combination is sufficient to suppress the rough eye phenotype of dRSK but not of dPKN overexpression. These results suggest that at least in this in vivo assay, dRSK activity critically depends on dPDK1 function, whereas dPKN activity is not changed by a reduction in dPDK1 levels. This idea is in line with the recent finding that in PDK1-/- embryonic stem cells the protein kinase C-related protein kinase PRK2 (CG2049), which shares extensive homology with PKN, is still partially phosphorylated at its T loop residue, indicating that PDK1-independent mechanisms may exist for the phosphorylation of the T loop of certain AGC kinases including dPKN (Rintelen, 2001).

These results show that dPDK1 is an essential component in the insulin signaling pathway in the control of cell growth and body size through its two substrates, dAkt and dS6K. These results are distinct from the genetic evidence in C. elegans where Akt is the primary target of PDK1 in dauer formation. Because mutations in the insulin signaling pathway do not show an autonomous alteration of cell size in C. elegans, the regulation of the rate of protein synthesis through S6K does not seem to be a primary target of this pathway. However, the fact that dPDK1 may yet have additional substrates is suggested by the genetic interaction with dRSK gain-of-function mutations and because viable dPDK1 males are almost completely sterile. Although mutations in components of the insulin signaling pathway such as dInr, chico, Dp110/PI(3)K, and dAkt cause female sterility, male sterility is not observed. Further genetic dissection of dPDK1 function is required to determine the role of dPDK1 in male fertility. These findings in Drosophila are consistent with the absence of insulin growth factor-1-induced activation of S6K, Akt, and RSK in mammalian PDK1-/- embryonic stem cells, and therefore provide evidence for the functional conservation of branch points in kinase networks during evolution (Rintelen, 2001).


Drosophila Phosphoinositide-dependent kinase-1 acts upstream of Akt1

Phosphoinositide-dependent kinase-1 (PDK-1) is a central mediator of the cell signaling between phosphoinositide 3-kinase (PI3K) and various intracellular serine/threonine kinases including Akt/protein kinase B (PKB), p70 S6 kinases, and protein kinase C. Recent studies with cell transfection experiments have implied that PDK-1 may be involved in various cell functions including cell growth and apoptosis. However, despite its pivotal role in cellular signalings, the in vivo functions of PDK-1 in a multicellular system have rarely been investigated. Drosophila PDK1) and Ser473 within the carboxyl-terminal hydrophobic motif by an unknown kinase. This study shows in Drosophila and human cells the target of rapamycin (TOR) kinase and its associated protein rictor are necessary for Ser473 phosphorylation and that a reduction in rictor or mammalian TOR (mTOR) expression inhibits an Akt/PKB effector. The rictor-mTOR complex directly phosphorylated Akt/PKB on Ser473 in vitro and facilitated Thr308 phosphorylation by PDK1. Rictor-mTOR may serve as a drug target in tumors that have lost the expression of PTEN, a tumor suppressor that opposes Akt/PKB activation (Sarbassov, 2005).

The Akt/PKB kinase is a well-characterized effector of phosphoinositide 3-kinase (PI3K), and its deregulation plays important roles in the pathogenesis of human cancers. PI3K is necessary for the activation of Akt/PKB, and current models suggest that phosphatidylinositol-3,4,5-triphosphates produced upon growth factor stimulation recruit Akt/PKB to the plasma membrane by binding to its N-terminal pleckstrin homology (PH) domain. At the membrane, Akt/PKB is phosphorylated on two key residues: Thr308 (T308) of the activation loop by PDK1 and Ser473 (S473) in the hydrophobic motif of the C-terminal tail by a kinase whose identity has been elusive. The role of S473 phosphorylation is controversial, but there is an emerging view that it precedes the phosphorylation of T308 and is important for the recognition and activation of Akt/PKB by PDK1 (Sarbassov, 2005 and references therein).

The molecular identity of the S473 kinase (S473K), at times referred to as 'PDK2' or the 'hydrophobic motif (HM) kinase,' has been hotly debated for many years. Several candidate S473Ks have been proposed, including PDK1, integrin-linked kinase (ILK), Akt/PKB itself, and, most recently, DNA-PKcs. Many lines of evidence argue that neither PDK1, ILK, nor Akt/PKB is the physiological S473K, and for several reasons, DNA-PKcs is also unlikely to have this function. There is no Drosophila ortholog of DNA-PKcs, and, thus, if DNA-PKcs is a physiological S473K in mammals, a distinct kinase must play that role in flies even though all other core components of the pathway (e.g., PI3K, Akt/PKB, PDK1, and PTEN) are well conserved. Moreover, it has not been shown that DNA-PKcs phosphorylates full-length Akt/PKB, and DNA-PKcs null mice do not suffer the growth retardation or insulin signaling defects associated with Akt1/PKB1 or Akt2/PKB2 (Sarbassov, 2005).

Mammalian TOR (mTOR) is a large protein kinase that exists in two distinct complexes within cells: one that contains mTOR, GβL, and raptor and another containing mTOR, GβL, and rictor. The raptor-containing complex is sensitive to the drug rapamycin and regulates cell growth, in part by phosphorylating the hydrophobic motif of S6K1, a member of the same family of kinases to which Akt/PKB belongs. The rictor-containing complex does not appear to be rapamycin-sensitive, and its cellular function is just beginning to be understood. Despite its structural similarity to S6K1, Akt/PKB phosphorylation is not sensitive to acute rapamycin treatment, and thus mTOR has not previously been considered as the S473K (Sarbassov, 2005).

This study used RNA interference (RNAi) in cultured Drosophila cells to determine the role of TOR pathway components in the phosphorylation of the hydrophobic motif sites of Drosophila Akt/PKB (dAKT/dPKB) and S6K (dS6K). In mammals and Drosophila, S6K suppresses signaling through the PI3K/Akt pathway so that inhibition of S6K boosts Akt/PKB phosphorylation. Knockdown of dS6K or Drosophila Raptor expression with double-stranded RNAs (dsRNAs) inhibited the phosphorylation and activity of dS6K and increased the phosphorylation of dAkt/dPKB. Despite reducing dS6K phosphorylation to the same extent as did dRaptor dsRNA, the dTOR dsRNA failed to increase dAkt/dPKB phosphorylation and, surprisingly, decreased it by a small amount. The contrasting effects on dAkt/dPKB phosphorylation by the dTOR and dRaptor dsRNAs suggest that dTOR has an unexpected positive role in dAkt/dPKB signaling that is not shared with dRaptor and that dTOR is required for the increase in dAkt/dPKB phosphorylation caused by dS6K inhibition. Consistent with the dRaptor-independent role for dTOR in dAkt/dPKB phosphorylation, a knockdown of dRictor reduced dAkt/dPKB phosphorylation (Sarbassov, 2005).

Because basal dAkt/dPKB phosphorylation is low in Drosophila Kc167 cells, the roles of dRictor and dTOR were verified in cells in which dAkt/dPKB phosphorylation was enhanced by decreasing the expression of dPTEN, the negative regulator of the PI3K/Akt pathway. Knockdown of dS6K or dRaptor expression in dPTEN-depleted cells further boosted dAkt/dPKB phosphorylation. In contrast, knockdown of dRictor expression almost completely prevented the dramatic increase in dAkt/dPKB phosphorylation caused by a dPTEN knockdown, whereas the knockdown of dTOR expression caused a slightly smaller suppression. Also, dRictor and dTOR were required for the increase in phosphorylation of dAkt/dPKB caused by a knockdown in the expression of dRaptor (Sarbassov, 2005).

The results in Drosophila cells suggest that dTOR and dRictor have a shared positive role in the phosphorylation of the hydrophobic motif site of dAkt/dPKB. This finding was unexpected, because previously no decrease was observed in the phosphorylation of the hydrophobic motif site of Akt/PKB after reducing mTOR expression in human cells with small interfering RNAs (siRNAs). In retrospect, however, these experiments were undertaken when RNAi-mediated knockdowns of expression in mammalian cells were relatively inefficient. In this study, with the use of a lentiviral short hairpin RNA (shRNA) expression system that robustly suppresses gene expression, results in human cell lines were obtained analogous to those in Drosophila cells. In human HT-29 colon and A549 lung cancer cells, knockdown of rictor or mTOR expression using two different sets of shRNAs decreased phosphorylation of both S473 and T308 of Akt/PKB. Mammalian cells may try to compensate for the effects of the rictor and mTOR knockdowns by boosting Akt/PKB expression. The decrease in T308 phosphorylation is consistent with the importance of S473 phosphorylation for T308 phosphorylation and with the fact that the Ser473 --> Asp473 mutant of Akt/PKB is a better substrate than the wild-type protein for T308 phosphorylation by PDK1. Knockdown of raptor expression increased the phosphorylation of both S473 and T308 despite reducing Akt/PKB expression. Knockdown of rictor or mTOR expression also decreased S473 phosphorylation in HeLa and HEK-293T cells, two human cell lines that, like A549 and HT-29 cells, contain wild-type PTEN. In addition, the knockdowns also decreased S473 phosphorylation in the PTEN-null PC-3 prostate cancer cell line, a result reminiscent of that in Drosophila cells with reduced dPTEN expression. Furthermore, the knockdowns decreased S473 phosphorylation in M059J glioblastoma cells that are null for DNA-PKcs, a proposed S473K candidate. Thus, in six distinct human cell lines, rictor and mTOR but not raptor are necessary for the phosphorylation of the hydrophobic motif of Akt/PKB (Sarbassov, 2005).

Because the rictor and mTOR knockdowns inhibit phosphorylation events critical for Akt/PKB activity, they should affect Akt/PKB-regulated effectors. In HeLa cells, a reduction in the expression of rictor or mTOR but not raptor decreased phosphorylation of AFX (Foxo4a), a forkhead family transcription factor that is a direct substrate of Akt/PKB. Because the raptor-mTOR complex directly phosphorylates the hydrophobic motif site of S6K1, whether rictor-mTOR has an analogous function for Akt/PKB was determined. Rictor-mTOR complexes isolated from HEK-293T or HeLa phosphorylated S473 but not T308 of full-length, wild-type Akt/PKB in vitro. Immunoprecipitates of raptor, the ataxia telagiectasia mutated (ATM) protein, or protein kinase C α (PKCα) did not phosphorylate either site, and Akt/PKB did not autophosphorylate S473. Importantly, the raptor immunoprecipitates also contain mTOR but did not phosphorylate Akt/PKB, suggesting that for mTOR to phosphorylate Akt/PKB, it must be bound to rictor and that raptor cannot substitute. This lack of phosphorylation holds even in the raptor immunoprecipitates isolated from HEK-293T cells that contain as much mTOR as the rictor immunoprecipitates. Consistent with a key role for rictor, mTOR immunoprecipitates prepared from the rictor knockdown cells did not phosphorylate Akt/PKB despite containing a similar amount of mTOR as the controls. To verify that mTOR is the S473K in the rictor immunoprecipitates, immunoprecipitates were prepared from control cells and from two different lines of mTOR knockdown cells. Although rictor levels were equivalent in all the immunoprecipitates, only those prepared from cells expressing mTOR phosphorylated Akt/PKB in vitro. Both the LY294002 and wortmannin mTOR kinase inhibitors blocked the in vitro phosphorylation of Akt/PKB by rictor-mTOR, and LY294002 acted at concentrations that inhibit S473 phosphorylation in cells. Staurosporine, an inhibitor of Akt/PKB kinase activity, did not decrease the phosphorylation of Akt/PKB by rictor-mTOR. Thus, in vitro the rictor-mTOR complex phosphorylates S473 of Akt/PKB in a rictor- and mTOR-dependent fashion and with a drug sensitivity profile consistent with mTOR being the phosphorylating kinase (Sarbassov, 2005).

To determine whether the phosphorylation of Akt/PKB on S473 by rictor-mTOR activates Akt/PKB activity, rictor-mTOR was used to phosphorylate Akt/PKB on S473, and then PDK1 was added to the assay to phosphorylate T308. Prior phosphorylation of Akt/PKB on S473 boosted subsequent phosphorylation by PDK1 of T308, consistent with the importance of S473 phosphorylation for T308 phosphorylation and with the inhibitory effects of the rictor and mTOR knockdowns on T308 phosphorylation. After phosphorylation with rictor-mTOR and PDK1, Akt1/PKB1 had about four- to fivefold more activity than after phosphorylation with PDK1 alone, confirming the important role of S473 in fully activating Akt/PKB. Because growth factors control the phosphorylation of Akt/PKB on S473, it was determined whether the concentration of serum in the cell media regulated the in vitro kinase activity of rictor-mTOR toward Akt/PKB. Rictor-mTOR had decreased activity in HeLa cells deprived of serum and was reactivated by serum stimulation for 30 min, indicating that modulation of the intrinsic kinase activity of rictor-mTOR may be a mechanism for regulating S473 phosphorylation (Sarbassov, 2005).

These results indicate that the rictor-mTOR complex is a hydrophobic motif kinase for Akt/PKB. Rictor-TOR has essential roles in Akt/PKB hydrophobic motif site phosphorylation in Drosophila and human cells and in vitro phosphorylates full-length, wild-type Akt/PKB in a serum-sensitive fashion. No other proposed hydrophobic motif kinase has been shown to fulfill all these criteria. With hindsight, clues are seen in the literature to the important role of mTOR in Akt/PKB activation. Prolonged but not acute treatment of certain human cells with rapamycin partially inhibits Akt/PKB phosphorylation, and the current findings provide a possible rationale to explain these results. Although rapamycin does not bind to a preformed rictor-mTOR complex, during long-term rapamycin treatment the drug should eventually sequester many of the newly synthesized mTOR molecules within cells. Thus, as the rictor-mTOR complex turns over, rapamycin may interfere with its reassembly or over time become part of the new complexes. It is reasonable to expect then that prolonged rapamycin treatment may partially inhibit rictor-mTOR activity, which would explain why rapamycin is particularly effective at suppressing the proliferation of tumor cells with hyperactive Akt/PKB. The PI3K/Akt pathway is frequently deregulated in human cancers that have lost the expression of the PTEN tumor suppressor gene, and the current findings suggest that direct inhibitors of mTOR-rictor should strongly suppress Akt/PKB activity. Thus, the rictor-mTOR complex, like its raptor-mTOR sibling, may be a valuable drug target (Sarbassov, 2005).

Akt and FOXO dysregulation contribute to infection-induced wasting in Drosophila
Studies in Drosophila have taught us a great deal about how animals regulate the immediate innate immune response, but we still know little about how infections cause pathology. This study examines the pathogenesis associated with Mycobacterium marinum infection in the fly. M. marinum is closely related to M. tuberculosis, which causes tuberculosis in people. A microarray analysis shows that metabolism is profoundly affected in M. marinum-infected flies. A genetic screen identifies foxo mutants as slower-dying after infection than wild-type flies. FOXO activity is inhibited by the insulin effector kinase Akt; it was shown that Akt activation is systemically reduced as a result of M. marinum infection. Additionally, flies infected with Mycobacterium marinum undergo a process like wasting: they progressively lose metabolic stores, in the form of fat and glycogen. They also become hyperglycemic. In contrast, foxo mutants exhibit less wasting. In people, many infections—including tuberculosis—can cause wasting, much as is seen in Drosophila. This study is the first examination of the metabolic consequences of infection in a genetically tractable invertebrate and gives insight into the metabolic consequences of mycobacterial infection, implicating impaired insulin signaling as a key mediator of these events. These results suggest that the fly can be used to study more than the immediate innate immune response to infection; it can also be used to understand the physiological consequences of infection and the immune response (Dionne, 2006).

This study shows that flies infected with M. marinum undergo a process like wasting. This wasting response is in part the consequence of systemic failure of Akt activation and consequent activation of the transcription factor FOXO, resulting in a diabetes-like state. The observed failure of Akt activation is not caused by traditional insulin resistance mechanisms. Data suggest that mycobacterial infection causes a systemic reduction in Akt activation, either by reducing the level of circulating insulin or by increasing the turnover of activated Akt, or both. This results in excessive Gsk-3 and FOXO activity, which causes the progressive loss of energy stores. The study argues that the impaired insulin signaling seen in M. marinum-infected Drosophila is likely to represent a common response to many infections in many hosts, including humans, and moreover is likely a significant cause of disease morbidity (Dionne, 2006).

It was shown that foxo mutants are longer-lived when infected with M. marinum. This makes an intriguing contrast with earlier observations that flies overexpressing foxo are longer-lived than wild-type flies when uninfected. Moreover, foxo mutant flies die more rapidly than wild-type flies under conditions of oxidative stress. These observations suggest that death from old age or oxidative stress is mechanistically different from death from M. marinum infection (Dionne, 2006). 

In humans, insulin is among the most important anabolic signals, and it is also a satiety signal: type-1 diabetics (who progressively lose the insulin-producing cells of the pancreas) exhibit increases in appetite and consequently in food intake, even as they are progressively losing body mass. In fly larvae, insulin-like peptides (ILPs) appear to play a role roughly analogous to the metabolic role seen in humans: Larvae in which the insulin receptor (InR) or PI3 kinase (PI3K) are overexpressed accumulate excessive levels of fat and show reduced feeding. Conversely, larvae that lack ILP-producing cells (IPCs) in the brain become hyperglycemic (Dionne, 2006). 

In adult flies, the metabolic role of ILPs appears to be more complex. The loss of ILP signaling via IPC ablation or loss-of-function mutations in chico (the fly homolog of mammalian IRS proteins) or InR results in increased triglyceride and glycogen storage—although, in the case of IPC ablation, this increase in energy stores is still accompanied by hyperglycemia. Studies using a temperature-sensitive InR allele show that the critical period for the increase in metabolic storage is during pupariation, indicating that the storage effect should be regarded as a developmental defect rather than a physiological one. This study is the first examination of the results of insulin inhibition in wild-type adult flies without developmental perturbation. It suggests that the insulin signaling pathway acts in adult flies to drive glucose uptake and energy storage in a manner analogous to its action in mammals and larval Drosophila (Dionne, 2006). 

Immune responses pose significant costs for the host. One hypothesis suggests that the primary cost of immune responses is energetic: that metabolic energy used by the immune system is being taken from other important systems. This has been easiest to observe in cases where animals are placed under energy constraints and then forced to raise an immune response: In these situations, the induced immune responses have easily observable deleterious effects on other physiological processes. Conversely, this cost is also visible as immunosuppression in animals that are carrying out other energy-intensive activities (Dionne, 2006). 

These observations suggest that there should be mechanisms for direct control of energy allocation to the immune response. Data from this study indicate that Akt and Foxo form an important component in this regulation. The study speculates that the systemic disruption of insulin signaling may be a mechanism by which insects reduce energy allocation to nonimmune tissues; moreover, a similar mechanism might operate in mammals. The clinical literature is rich with examples of metabolic changes resulting from a variety of infections. Tuberculosis and other chronic infections can be associated with slow wasting of fatty and lean tissues and glucose intoleranc; acute bacteremia tends to be associated with rapid wasting and full-scale hyperglycemia. Resting insulin levels are an excellent predictor of survival in septic patients, and aggressive treatment of septic hyperglycemia with exogenous insulin dramatically increases survival. Wasting alone could be accounted for simply by the energetic cost of the immune response; however, infections often cause hyperglycemia as well, suggesting that systemic changes in metabolic regulation may be the underlying cause of infection-induced wasting. That is, wasting may be a pathological consequence of regulated energy reallocation (Dionne, 2006).

Other possibilities should not be overlooked: in particular, the possibility that the metabolic changes observed in the fly might be part of a pathogenic strategy on the part of the bacterium. Organisms living in the circulation can easily double their local glucose concentration simply by degrading circulating insulin. In this reading, the fact that a wide variety of infections cause hyperglycemia in mammals would be a result of the fact that this strategy is such an attractive one for a pathogen that it has been selected many times independently. However, the apparent connection between increased levels of proinflammatory cytokines and cachexia in mammals leads to the connection that infection-induced wasting is primarily a consequence of the host response—though one that is ripe for exploitation by some classes of pathogen (Dionne, 2006).

Re-evaluating AKT regulation: role of TOR complex 2 in tissue growth

Phosphatidylinositol-3-kinase (PI3K)/AKT signaling is essential for growth and metabolism and is elevated in many cancers. Enzymatic activity of AKT has been shown to depend on phosphorylation of two conserved sites by PDK1 and TOR (target of rapamycin) complex 2 (TORC2) in a PI3K-dependent manner. This study analyzed the role of TORC2-mediated AKT phosphorylation in Drosophila. Mutants removing critical TORC2 components, rictor and sin1, strongly reduced AKT hydrophobic motif (HM) phosphorylation and AKT activity, but showed only minor growth impairment. A mutant form of AKT lacking the HM phosphorylation site displayed comparable activity. In contrast to the mild effects of removing HM site phosphorylation at normal levels of PI3K activity, loss of TORC2 activity strongly inhibited hyperplasia caused by elevated pathway activity, as in mutants of the tumor suppressor PTEN. Thus, TORC2 acts as a rheostat to broaden the range of AKT signaling at the high end of its range (Hietakangas, 2007).

The PI3K/AKT signaling pathway is conserved between Drosophila and mammalian species. The lack of genetic redundancy among pathway components makes Drosophila a useful system in which to dissect the roles of the individual pathway members in vivo. Earlier analyses of other pathway members have shown that the Insulin receptor, PI3K, PDK1, and AKT are each essential for viability, and that mutant tissue displays severe undergrowth. Mutants of Drosophila insulin receptor substrate homolog, chico, are semiviable but severely growth impaired. Although individual AKT mutants are viable in mouse, the essential nature of AKT is likely to be masked by genetic redundancy among the three AKT genes. Previous studies in cultured cells have suggested that TORC2 is an important regulator of AKT phosphorylation and activity, and that this phosphorylation event is required for AKT kinase activity. It was recently shown that loss of TORC2 activity in rictor mutant mice leads to loss of AKT HM phosphorylation and to embryonic lethality, suggesting that HM phosphorylation is essential for AKT activity in the mouse. In contrast, the current findings show that TORC2-mediated phosphorylation on the HM site is not essential for AKT activity in vivo. Indeed, although AKT activity was reduced, considerable residual activity was found in flies lacking TORC2 activity. Flies expressing a mutant form of AKT lacking the HM phosphorylation site also showed considerable AKT activity in vivo. These findings indicate that the maximal level of AKT activity is limited in the absence of HM phosphorylation. Under normal physiological conditions in Drosophila, this reduced level of AKT activity is almost sufficient to support normal growth. But without HM phosphorylation, AKT cannot transduce the higher-than-normal levels of PI3K pathway activity that result from mutation of the tumor suppressor PTEN or increased insulin stimulation. When considered in this context, the lethality of rictor mutant mice could reflect a higher threshold in the requirement for AKT activity in some biological process in mouse than in fly, but the possibility of essential TORC2 targets other than AKT cannot be excluded (Hietakangas, 2007).

Perhaps the most intriguing implication of this study lies in the area of cancer biology. Elevated AKT activity is a hallmark of human cancer, with a substantial proportion of human tumors depending on AKT pathway activation, for example, due to PTEN mutations. The current findings suggest that inhibiting TORC2 activity, rather than AKT itself, may prove to be a promising strategy for cancer therapy (Hietakangas, 2007).

SREBP activity is regulated by mTORC1 and contributes to Akt-dependent cell growth

Cell growth (accumulation of mass) needs to be coordinated with metabolic processes that are required for the synthesis of macromolecules. The PI3-kinase/Akt signaling pathway induces cell growth via activation of complex 1 of the target of rapamycin (TORC1). This study shows that Akt-dependent lipogenesis requires mTORC1 activity. Furthermore, nuclear accumulation of the mature form of the sterol responsive element binding protein (SREBP1) and expression of SREBP target genes was blocked by the mTORC1 inhibitor rapamycin. Silencing of SREBP blocks Akt-dependent lipogenesis and attenuates the increase in cell size in response to Akt activation in vitro. Silencing of Drosophila SREBP (Helix loop helix protein 106) caused a reduction in cell and organ size and blocked the induction of cell growth by dPI3K. These results suggest that the PI3K/Akt/TOR pathway regulates protein and lipid biosynthesis in an orchestrated manner and that both processes are required for cell growth (Porstmann, 2008).

It was asked whether the PI3K/Akt pathway regulates the activity of SREBP in flies. Transient silencing of the catalytic subunit of PI3K, dp110, or dAkt reduced mRNA abundance of dFAS (Fatty acid synthase) and dSREBP in Kc167 cells. Conversely, silencing of dPTEN resulted in increased expression of both transcripts. Ubiquitous expression of dp110 using the da-GAL4 driver resulted in enhanced dSREBP and dFAS expression in second instar larvae compared to controls, indicating that the PI3K/Akt pathway activates dSREBP function (Porstmann, 2008).

Expression of mature dSREBP (m-dSREBP) using the MS1096-GAL4 driver resulted in significant lethality when flies were reared at 25°C (data not shown). When flies were reared at 18°C (resulting in a reduced activity of the GAL4/UAS system), severe deformation of the wing was observed. Expression of the full-length form of dSREBP (fl-dSREBP) in the wing caused less severely misshapen wings even when flies were reared at 25°C. However, coexpression of dp110 and full-length dSREBP resulted in severely deformed wings, a phenotype that is similar to that caused by expression of m-dSREBP. This result suggests that the activity of full-length dSREBP is enhanced by PI3K signaling (Porstmann, 2008).

Expression of dp110 causes an overgrowth phenotype in the wing, indicated by a 15%-20% increase in surface area of the wing. Silencing of dSREBP attenuated the increase in wing size induced by expression of dp110. Similar results were obtained using a nonoverlapping RNAi sequence targeting dSREBP or by heterozygous deletion of the dSREBP gene (Porstmann, 2008).

Expression of a kinase domain mutant of PI3K (dp110[KD]) decreases cell and organ size in Drosophila. Expression of dp110[KD] using the decapentaplegic (dpp)-GAL4 driver resulted in a 20%-30% reduction in the size of the dpp compartment. Expression of the dSREBP RNAi hairpin resulted in a small but significant decrease in wing area. However, coexpression of dSREBP RNAi with dp110KD did not further decrease the size of this compartment Taken together, these results suggest that dp110 and dSREBP are components of the same pathway in the regulation of cell growth in Drosophila (Porstmann, 2008).

Akt is negatively regulated by Hippo signaling for growth inhibition in Drosophila

Tissue growth is achieved through coordinated cellular growth, cell division and apoptosis. Hippo signaling is critical for monitoring tissue growth during animal development. Loss of Hippo signaling leads to tissue overgrowth due to continuous cell proliferation and block of apoptosis. As cells lacking Hippo signaling are similar in size compared to normal cells, cellular growth must be properly maintained in Hippo signaling-deficient cells. However, it is not clear how Hippo signaling might regulate cellular growth. This study shows that loss of Hippo signaling increases Akt (also called Protein Kinase B, PKB) expression and activity, whereas activation of Hippo signaling reduces Akt expression in developing tissues in Drosophila. While yorkie is sufficient to increase Akt expression, Akt up-regulation caused by the loss of Hippo signaling is strongly dependent on yki, indicating that Hippo signaling negatively regulates Akt expression through Yki inhibition. Consistently, genetic analysis reveals that Akt plays a critical role in facilitating growth of Hippo signaling-defective tissues. Thus, Hippo signaling not only blocks cell division and promotes apoptosis, but also regulates cellular growth by inhibiting the Akt pathway activity (Ye, 2012).

Growth inhibition mediated by Hippo signaling is essential for tissue growth and organ size control. Loss of Hippo signaling generates extra cells with their size similar to normal cells, suggesting that both cell division and cellular growth are promoted in cells lacking Hippo signaling activity. If Hippo signaling is only involved in inhibiting cell division, loss of Hippo signaling would result in extra cells that are smaller than normal cells. By altering activities of some cell-cycle regulators, it has been previously shown that an increase of cell division rate is insufficient to drive cellular growth, and therefore, cell division and cellular growth can be separately regulated. In the case of Hippo signaling, this growth-inhibitory pathway appears to play an active role to negatively control both cell division and cellular growth. This study found that akt expression is negatively regulated by Hippo signaling as a way to reduce the Akt pathway activity. Moreover, growth-promoting factor Yki is required for activating akt expression in developing tissues. The genetic evidence is also consistent with a role of akt as a critical downstream target of Hippo signaling. Thus, these results support a model in which Hippo signaling negatively regulates akt expression through Yki inhibition to coordinate cellular growth and cell division and ultimately control tissue and organ size during Drosophila development (Ye, 2012).

Because the DNA-binding protein Scalloped (Sd) interacts with Yki to regulate transcription of downstream target and three putative Sd-binding sites were found in the downstream intergenic region within a 30-kb akt genomic region, the potential enhancer activity of these elements in mediating the transcription activation property of Yki was tested. Two genomic fragments that contain these sites were tested for their potential enhancer activity to drive a GFP reporter gene expression. Neither fragment was able to respond to Yki overexpression to activate gene transcription in cultured Drosophila S2 cells, whereas a previously identified diap1 enhancer that contains Sd-binding sites was able to activate gene expression in responding to Yki. Furthermore, from a dataset generated by the Drosophila Regulatory Elements modENCODE Project, a genome-wide ChIP analysis did not detect the akt locus as an obvious target of Yki. Therefore, how Yki functions to directly or indirectly to control akt expression will need to be further investigated (Ye, 2012).

Since bantam (ban) miRNA has been shown to act autonomously to increase Akt expression in epithelial cells and non-autonomously to decrease Akt expression in neighboring neuronal cells, whether ban miRNA is involved in regulating the level of akt expression was tested by expressing ban in Drosophila S2 cells as well as larval wing discs. Preliminary data showed that ban overexpression can slightly increase the level of Akt protein. Thus, while ban is a critical downstream target of yki, ban might contribute to the upregulation of Akt expression in tissues such as larval wing discs during development (Ye, 2012).

As the Hippo pathway is highly conserved in evolution, it is possible that Akt regulation by Hippo signaling also occurs in mammalian cells. Indeed, knockdown of LATS1 in human MCF10A breast epithelial cells activated the AKT pathway as shown by the increased levels of activated AKT kinase protein, although the total Akt protein level was not. Interestingly, AKT upregulation caused by reduction of LATS1 function is critically dependent on YAP activity. However, this regulation of AKT activity is likely mediated at the post-translational level because the total AKT protein level was not changed by LATS1 knockdown or LATS1/YAP double knockdown. Although the mechanism of this AKT inhibition by Hippo signaling in mammalian cells is currently uncharacterized, clarification of how Hippo signaling can negatively regulate the AKT pathway activity would reveal mechanisms by which these two important cellular signaling pathways cross-talk for a proper developmental control of cell growth, cell division and cell death. More excitingly, this study implicates that Hippo signaling might influencing the AKT pathway activity for its nutrient control of growth, homeostasis, and longevity in animals (Ye, 2012).

Neuronal Cbl controls biosynthesis of insulin-like peptides in Drosophila melanogaster

The Cbl family proteins function as both E3 ubiquitin ligases and adaptor proteins to regulate various cellular signaling events, including the insulin/insulin-like growth factor 1 (IGF1) and epidermal growth factor (EGF) pathways. These pathways play essential roles in growth, development, metabolism, and survival. This study shows that in Drosophila Cbl (dCbl) regulates longevity and carbohydrate metabolism through downregulating the production of Drosophila insulin-like peptides (dILPs) in the brain. dCbl is highly expressed in the brain and knockdown of the expression of dCbl specifically in neurons by RNA interference increases sensitivity to oxidative stress or starvation, decreased carbohydrate levels, and shortened life span. Insulin-producing neuron-specific knockdown of dCbl results in similar phenotypes. dCbl deficiency in either the brain or insulin-producing cells upregulates the expression of dilp genes, resulting in elevated activation of the dILP pathway, including phosphorylation of Drosophila Akt and Drosophila extracellular signal-regulated kinase (dERK). Genetic interaction analyses revealed that blocking Drosophila epidermal growth factor receptor (dEGFR)-dERK signaling in pan-neurons or insulin-producing cells by overexpressing a dominant-negative form of dEGFR abolishes the effect of dCbl deficiency on the upregulation of dilp genes. Furthermore, knockdown of c-Cbl in INS-1 cells, a rat β-cell line, also increases insulin biosynthesis and glucose-stimulated secretion in an ERK-dependent manner. Collectively, these results suggest that neuronal dCbl regulates life span, stress responses, and metabolism by suppressing dILP production and the EGFR-ERK pathway mediates the dCbl action. Cbl suppression of insulin biosynthesis is evolutionarily conserved, raising the possibility that Cbl may similarly exert its physiological actions through regulating insulin production in β cells (Yu, 2012).

Genetic dissection reveals that Akt is the critical kinase downstream of LRRK2 to phosphorylate and inhibit FOXO1, and promotes neuron survival

Leucine-rich repeat kinase 2 (LRRK2) is a complex kinase and mutations in LRRK2 are perhaps the most common genetic cause of Parkinson's disease (PD). However, the identification of the normal physiological function of LRRK2 remains elusive. This study shows that LRRK2 protects neurons against apoptosis induced by the Drosophila genes grim, hid and reaper. Genetic dissection reveals that Akt is the critical downstream kinase of LRRK2 that phosphorylates and inhibits FOXO1, and thereby promotes survival. Like human LRRK2, Drosophila lrrk also promotes neuron survival; lrrk loss-of-function mutant displays reduced cell numbers, which can be rescued by LRRK2 expression. Importantly, LRRK2 G2019S and LRRK2 R1441C mutants impair the ability of LRRK2 to activate Akt, and result in a failure of preventing apoptotic death. Ectopic expression of a constitutive active form of Akt hence is sufficient to rescue this functional deficit. These data establish that LRRK2 can protect neurons from apoptotic insult through a survival pathway in which LRRK2 signals to activate Akt, and then inhibits FOXO1. These results might indicate that a therapeutic pathway to promote neuron survival and to prevent neurodegeneration in Parkinson's disease (Chuang, 2014).

Cell mixing induced by myc is required for competitive tissue invasion and destruction

Cell-cell intercalation is used in several developmental processes to shape the normal body plan. There is no clear evidence that intercalation is involved in pathologies. This study used the proto-oncogene myc to study a process analogous to early phase of tumour expansion: myc-induced cell competition. Cell competition is a conserved mechanism driving the elimination of slow-proliferating cells (so-called 'losers') by faster-proliferating neighbours (so-called 'winners') through apoptosis and is important in preventing developmental malformations and maintain tissue fitness. Using long-term live imaging of myc-driven competition in the Drosophila pupal notum and in the wing imaginal disc, this study showed that the probability of elimination of loser cells correlates with the surface of contact shared with winners. As such, modifying loser-winner interface morphology can modulate the strength of competition. Elimination of loser clones requires winner-loser cell mixing through cell-cell intercalation. Cell mixing is driven by differential growth and the high tension at winner-winner interfaces relative to winner-loser and loser-loser interfaces, which leads to a preferential stabilization of winner-loser contacts and reduction of clone compactness over time. Differences in tension are generated by a relative difference in F-actin levels between loser and winner junctions, induced by differential levels of the membrane lipid phosphatidylinositol (3,4,5)-trisphosphate. These results establish the first link between cell-cell intercalation induced by a proto-oncogene and how it promotes invasiveness and destruction of healthy tissues (Levayer, 2015).

To analyse quantitatively loser cell elimination, long-term live imaging was performed of clones showing a relative decrease of the proto-oncogene myc in the Drosophila pupal notum, a condition known to induce cell competition in the wing disc. Every loser cell delamination was counted over 10 h, and the probability of cell elimination was calculated for a given surface of contact shared with winner cells. A significant increase was observed of the proportion of delamination with winner-loser shared contact, whereas this proportion remained constant for control clones. The same correlation was observed in ex vivo culture of larval wing disc. Cell delamination in the notum was apoptosis dependent and expression of flowerlose (fwelose), a competition-specific marker for loser fate, was necessary and sufficient to drive contact-dependent delamination. Moreover it was confirmed that contact-dependent death is based on the computation of relative differences of fwelose between loser cells and their neighbours. Thus, cell delamination in the notum recapitulates features of cell competition (Levayer, 2015).

This suggests that winner-loser interface morphology could modulate the probability of eliminating loser clones. Using the wing imaginal disc, winner-loser contact was reduced by inducing adhesion- or tension-dependent cell sorting and observed a significant reduction of loser clone elimination. This rescue was not driven by a cell-autonomous effect of E-cadherin (E-cad) or active myosin II regulatory light chain (MRLC) on growth, death or cell fitness but rather by a general diminution of winner-loser contact. Competition is ineffective across the antero-posterior compartment boundary, a frontier that prevents cell mixing through high line tension. Accordingly, there was no increase in death at the antero-posterior boundary in wing discs overexpressing fweloseA in the anterior compartment. However, reducing tension by reducing levels of myosin II heavy chains was sufficient to increase the shared surface of contact between cells of the anterior and posterior compartments, and induced fwelose death at the boundary. Altogether, it is concluded that the reduction in surface contact between winners and losers is sufficient to block competition, which explains how compartment boundaries prevent competition (Levayer, 2015).

Loser clones have been reported to fragment more often than controls, whereas winner clones show convoluted morphology, suggesting that winner-loser mixing is increased during competition. This could affect the outcome of cell competition by increasing the surface shared between losers and winners. Clone splitting was used as a readout for loser–winner mixing. Two non-exclusive mechanisms can drive clone splitting: cell death followed by junction rearrangement, or junction remodelling and cell–cell intercalation independent of death. To assess the contribution of each phenomenon, the proportion of clones fragmented 48 h after clone induction (ACI) was systematically counted. A twofold increase was observed in the frequency of split clones in losers (wild type (WT) in tub-dmyc) versus WT in WT controls. Overexpressing E-cad or active myosin II was sufficient to prevent loser clone splitting, whereas blocking apoptosis or blocking loser fate by silencing fwelose did not reduce splitting. Finally, the proportion of split clones was also increased for winner clones either during myc-driven competition or during Minute-dependent competition. Altogether, this suggested that winner–loser mixing is increased independently of loser cell death or clone size by a factor upstream of fwe, and could be driven by cell–cell intercalation. Accordingly, junction remodelling events leading to disappearance of a loser–loser junction were three times more frequent at loser clone boundaries than control clone boundaries in the pupal notum. The rate of junction remodelling was higher in loser–loser junctions and in winner–winner junctions than in winner–loser junctions. The preferential stabilization of winner–loser interfaces should increase the surface of contact between winner and loser cells over time. Accordingly, loser clone compactness in the notum decreased over time whereas it remains constant on average for WT clones in WT background. Similarly, the compactness of clones in the notum also decreased over time for conditions showing high frequency of clone splitting in the wing disc, whereas clone compactness remained constant for conditions rescuing clone splitting. Altogether, it is concluded that both Minute- and myc-dependent competition increase loser–winner mixing through cell–cell intercalation (Levayer, 2015).

It was then asked what could modulate the rate of junction remodelling during competition. The rate of junction remodelling can be cell-autonomously increased by myc. Interestingly, downregulation of the tumour suppressor PTEN is also sufficient to increase the rate of junction remodelling through the upregulation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3). It was reasoned that differences in PIP3 levels could also modulate junction remodelling during competition. Using a live reporter of PIP3 that could detect modulations of PIP3 in the notum, a significant increase of PIP3 was observed in the apico-lateral membrane of tub-dmyc–tub-dmyc interfaces compared with WT–WT and WT–tub-dmyc interfaces (Fig. 3a, b). Moreover, increasing/reducing Myc levels in a full compartment of the wing disc was sufficient to increase/decrease the levels of phospho-Akt (a downstream target of PIP3, whereas fweloseA overexpression had no effect. Similarly, levels of phospho-Akt were relatively higher in WT clones than in the surrounding M-/+ cells. Thus differences in PIP3 levels might be responsible for winner–loser mixing. Accordingly, reducing PIP3 levels by overexpressing a PI3 kinase dominant negative (PI3K-DN) or increasing PIP3 levels by knocking down PTEN (UAS-pten RNAi) were both sufficient to induce a high proportion of fragmented clones and to reduce clone compactness over time in the notum , whereas increasing PIP3 in loser clones was sufficient to prevent cell mixing. Moreover, abolishing winner–loser PIP3 differences through larval starvation prevented loser clone fragmentation, the reduction of clone compactness over time in the notum and could rescue WT clone elimination in tub-dmyc background. It is therefore concluded that differences in PIP3 levels are necessary and sufficient for loser–winner mixing and required for loser cell elimination (Levayer, 2015).

It was then asked which downstream effectors of PIP3 could affect junction stability. A relative growth decrease can generate mechanical stress that can be released by cell-cell intercalation. Accordingly, growth reduction through Akt downregulation is sufficient to increase clone splitting and could contribute to loser clone splitting. However, Akt is not sufficient to explain winner-loser mixing because, unlike PIP3, increasing Akt had no effect on clone splitting. PIP3 could also modulate junction remodelling through its effect on cytoskeleton and the modulation of intercellular adhesion or tension. No obvious modifications of E-cad, MRLC or Dachs (another regulator of tension) was detected in loser cells. However, a significant reduction of F-actin levels and a reduction of actin turnover/polymerization rate were observed in loser-loser and loser-winner junctions in the notum. Similarly, modifying Myc levels in a full wing disc compartment was sufficient to modify actin levels, and F-actin levels were higher in WT clones than M-/+ cells. This prompted a test of the role of actin organization in winner-loser mixing. Downregulating the formin Diaphanous (Dia, a filamentous actin polymerization factor) by RNA interference (RNAi) or by using a hypomorphic mutant was sufficient to obtain a high proportion of fragmented clones and to reduce clone compactness over time, whereas overexpressing Dia in loser clones prevented clone splitting (UAS-dia::GFP) and compactness reduction. This effect was specific to Dia as modulating Arp2/3 complex (a regulator of dendritic actin network) had no effect on clone splitting. Thus, impaired filamentous actin organization was necessary and sufficient to drive loser-winner mixing. These actin defects were driven by the differences in PIP3 levels between losers and winners. Thus Dia could be an important regulator of competition through its effect on cell mixing. Overexpression of Dia was indeed sufficient to reduce loser clone elimination significantly (Levayer, 2015).

Filamentous actin has been associated with tension regulation. It was therefore asked whether junction tension was modified in winner and loser junctions. The maximum speed of relaxation of junction after laser nanoablation (which is proportional to tension) was significantly reduced in loser-loser and winner-loser junctions compared with winner-winner junctions. This distribution of tension has been proposed to promote cell mixing. Accordingly, decreasing PIP3 in clones reduced tension both in low-PIP3-low-PIP3 and low-PIP3-normal-PIP3 junctions, whereas overexpressing Dia in loser clones or starvation were both sufficient to abolish differences in tension, in agreement with their effect on winner-loser mixing and the distribution of F-actin. Thus the lower tension at winner-loser and loser-loser junctions is responsible for winner-loser mixing. Altogether, it is concluded that the relative PIP3 decrease in losers increases winner-loser mixing through Akt-dependent differential growth and the modulation of tension through F-actin downregulation in winner-loser and loser-loser junctions (Levayer, 2015).

Several modes of tissue invasion by cancer cells have been described, most of them relying on the departure of the tumour cells from the epithelial layer. This study suggests that some oncogenes may also drive tissue destruction and invasion by inducing ectopic cell intercalation between cancerous and healthy cells, and subsequent healthy cell elimination. myc-dependent invasion could be enhanced by other mutations further promoting intercalation (such as PTEN). Stiffness is increased in many tumours, suggesting that healthy cell-cancer cell mixing by intercalation might be a general process (Levayer, 2015).

Cyclin G functions as a positive regulator of growth and metabolism in Drosophila

In multicellular organisms, growth and proliferation is adjusted to nutritional conditions by a complex signaling network. The Insulin receptor/target of rapamycin (InR/TOR) signaling cascade plays a pivotal role in nutrient dependent growth regulation in Drosophila and mammals alike. This study identifies Cyclin G (CycG) as a regulator of growth and metabolism in during larval development in Drosophila. CycG mutants have a reduced body size and weight and show signs of starvation accompanied by a disturbed fat metabolism. InR/TOR signaling activity is impaired in cycG mutants, combined with a reduced phosphorylation status of the kinase Akt1 and the downstream factors S6-kinase and eukaryotic translation initiation factor 4E binding protein (4E-BP). Moreover, the expression and accumulation of Drosophila insulin like peptides (dILPs) is disturbed in cycG mutant brains. Using a reporter assay, it was shown that the activity of one of the first effectors of InR signaling, Phosphoinositide 3-kinase (PI3K92E), is unaffected in cycG mutants. However, the metabolic defects and weight loss in cycG mutants are rescued by overexpression of Akt1 specifically in the fat body and by mutants in widerborst (wdb), the B'-subunit of the phosphatase PP2A, known to downregulate Akt1 by dephosphorylation. Together, these data suggest that CycG acts at the level of Akt1 to regulate growth and metabolism via PP2A in Drosophila (Fischer, 2015).

This study analyzed the role of Cyclin G in growth regulation and metabolism of Drosophila. Two different cycG null mutant alleles were used, thereby allowing the following of the developmental consequences resulting from the absence of cycG gene activity instead of drawing conclusions from overexpression or RNAi experiments. Misexpression studies initially raised the assumption that CycG negatively regulated cell growth and cell proliferation in Drosophila. The current results now indicate that CycG is required for normal growth, affecting both cell size and cell number. In fact, clonal analysis revealed a cell autonomous requirement of CycG not only in the wing but also the eye anlagen. In addition, the cycG null mutants show signs of metabolic disorder. Evidence is provided that CycG facilitates InR/TORC1 mediated growth regulation via PP2A, thereby helping to sustain nutrient dependent growth in Drosophila (Fischer, 2015).

Drosophila CycG appears to have extraordinarily diverse roles. It has been involved in epigenetic regulation of homeotic gene activity, in cell cycle regulation, developmental stability and in DNA repair, and now also in metabolic homeostasis. The current work confirmed molecular interactions between CycG and Wdb proteins in vivo that had been predicted from genome-wide proteome analyses in vitro. Interestingly, similar molecular interactions have been described before for mammalian CycG1 and CycG2: both proteins interact with several B' subunits, thereby mediating the recruitment of PP2A to its different substrates. In contrast to mammals, the genetic relationship between CycG and PP2A is antagonistic in Drosophila as a reduction of PP2A activity ameliorates the consequences of CycG loss. The cycG mutation could be formally explained by a gain of PP2A activity. It is tempting to speculate that the diversity of CycG functions results from a regulation of PP2A by CycG. PP2A affects a plethora of developmental and cellular processes, hence, pleiotropy is expected in case of its misregulation. Most likely, this hypothesis is too simplified. For example, loss of cycG in the female germ line results in an increase of phosphorylated H2Av (gamma-H2Av), a known target of PP2A activity. One might have expected a reduced amount of gamma-H2Av if loss of CycG equated with a gain in PP2A activity. Instead, this study has shown that CycG is found in a protein complex together with Rad9 and BRCA2 that primarily acts in the sensing of DNA double strand breaks. The importance of Drosophila CycG in DNA double strand break repair is reminiscent of functions described for mammalian CycG proteins: albeit CycG1 and CycG2 mutant mice are viable and healthy, they are both sensitive to DNA damaging reagents. Moreover, upregulation of CycG2 was involved in the activation of Chk2 and in damage induced G2/M cell cycle arrest, i.e. in DNA damage response in mammals as well. Whether the other phenotypes and interactions reported for Drosophila CycG are linked to the regulation of PP2A remains to be addressed in more detail (Fischer, 2015).

The cycG mutants display several phenotypic characteristics of a diminished TORC1 signaling activity, including weight reduction, a reduced egg laying rate, impaired endoreplication and a general increase in lipid mobilization. Moreover, CycG activity promotes phosphorylation of the primary TORC1 targets, i.e. S6K and 4E-BP. In contrast to TOR mutants, however, cycG mutants are viable, implying that CycG facilitates InR/TOR signaling rather than being an essential factor. Overall, cycG mutant flies show typical signs of nutritional starvation distress even under normal food conditions, suggesting a problem in their capacity to take up food and/or to sense and utilize the food. This defect is not due to a general inability of the animal to grasp the feed, but instead reflects a defect in coordinating the energy status with the regulation of systemic growth. As dILP accumulation in the brain is altered in cycG mutants, it is known that the signals transmitted from the nutritional sensor fat body must be disturbed. The fact, that the growth defects of cycG mutants can be strongly ameliorated by an induction of Akt1 specifically in the fat body rules out a function of CycG in the endocrine signal emanating from the fat body. Instead, all of the data indicate that CycG acts genetically at the level of Akt1, thereby controlling TOR signaling activity (Fischer, 2015).

Akt1 is negatively regulated by PP2A, supporting a model whereby CycG exerts its positive input on Akt1 via an inhibition of PP2A. In accordance, mutations in wdb efficiently rescue the growth and metabolic defects observed in cycG mutants. Likewise a downregulation of Wdb ameliorates the weight deficits resulting from a loss of Akt1 activity. In Drosophila, Wdb acts as a tissue-specific negative regulator of Akt1: it modulates lipid metabolism in the ovary as a result of a direct interaction with Akt1, whereas no such influence was seen in eye tissue. This study has shown that Wdb-Akt1 binding in the adult head is favored in the absence of CycG, i.e. CycG is able to influence the interaction between Wdb and Akt1 presumably by its direct binding to Wdb. A consequence of CycG loss may be the enhanced binding of PP2A to Akt1 and an enforced dephosphorylation of Akt1, resulting in the inhibition of downstream TOR signaling activity and affecting lipid metabolism and growth. Moreover, the second B'-subunit of Drosophila PP2A (also called Well rounded, Wrd) is involved in the negative regulation of the S6K. Assuming a molecular interaction of Wrd and CycG, a likewise regulatory input of CycG on PP2A containing the Wrd B'-subunit is conceivable. In this case, CycG might influence S6K activity as well, having a regulatory input on InR/TOR signaling also downstream of TORC1. This scenario is complicated by the negative feed back regulation of InR signaling by S6K and of Akt1 by TORC1. Circular regulation of InR/TOR signaling has been described at several levels, implementing a tight control of dietary signals and growth but complicating genetic analyses (Fischer, 2015).

In conclusion, the identification of CycG as a novel regulator of InR/TOR signaling in Drosophila highlights the importance of studying the regulatory network at the Akt1—PP2A nexus. Based on the high conservation of the InR/TOR signaling pathway and its regulation by PP2A, mammalian fat homeostasis is likely to involve similar regulatory control mechanisms to those that have been uncovered in Drosophila. This work raises the possibility of an involvement of CycG in InR/TOR-associated diseases that might be modulated by PP2A. A better understanding of the underlying mechanisms could therefore open up avenues for new strategies to fight InR/TOR-associated disorders in the future (Fischer, 2015).

Signaling dowstream of AKT

The initiation factor 4E for eukaryotic translation (eIF4E) binds the messenger RNA 5'-cap structure and is important in the regulation of protein synthesis. Mammalian eIF4E activity is inhibited when the initiation factor binds to the translational repressors, the 4E-binding proteins (4E-BPS). The Drosophila 4E-BP (d4E-BP) is a downstream target of the phosphatidylinositol-3-OH kinase [PI(3)K] signal-transduction cascade, which affects the interaction of d4E-BP with eIF4E. Ectopic expression of a highly active d4E-BP mutant in wing-imaginal discs causes a reduction of wing size, brought about by a decrease in cell size and number. A marked reduction in cell size is also observed in post-mitotic cells. Expression of d4E-BP in the eye and wing together with PI(3)K or dAkt1, the serine/threonine kinase downstream of PI(3)K, results in suppression of the growth phenotype elicited by these kinases. These results support a role for d4E-BP as an effector of cell growth (Miron, 2001).

Drosophila 4E-BP (d4E-BP) was isolated by interaction cloning from a complementary DNA expression library using 32P-labelled deIF4EI. d4E-BP is identical to Drosophila Thor (Bernal, 2000) and homologous to 4E-BPs from other species. Phosphorylation sites in mammalian 4E-BP1 are conserved in d4E-BP, but the predicted eIF4E-binding motif in d4E-BP (YERAFMK) diverges from the canonical consensus sequence (Miron, 2001).

To examine the binding of d4E-BP to deIF4E, residues within the consensus eIF4E-binding site were mutated. Recombinant proteins were expressed in Escherichia coli, and far Western blotting was performed using 32P-labelled deIF4EI. Mutation of Tyr 54 to Ala (Y54A) or Phe (Y54F), and Met 59 to Ala (M59A) abrogates the interaction of d4E-BP with deIF4E. Mutation of Lys 60 to Ala (K60A) decreases deIF4E binding by 87%, indicating that Lys 60 contributes to deIF4E binding. However, when either Met 59 or Lys 60 are mutated to the consensus Leu, the interaction of d4E-BP with deIF4EI is 2.5-fold higher than with the wild type, and when both Met 59 and Lys 60 are so changed, deIF4E binding increases by 3.4-fold. These results indicate that d4E-BP interacts with deIF4E, albeit more weakly than previously characterized 4E-BPs, owing to its divergent eIF4E-binding motif (Miron, 2001).

4E-BP1 is hyperphosphorylated in response to insulin in many cell types. To test whether this response operates in Drosophila, Schneider-2 (S2) cells were deprived of serum and treated with insulin. Increasing levels of a slower migrating form of d4E-BP (d4E-BP) were observed consequent to insulin treatment. To determine whether the ß-form corresponds to phosphorylated d4E-BP, extracts from insulin-stimulated S2 cells were treated with either calf intestine alkaline phosphatase (CIP) or protein phosphatase 2A (PP2A). Untreated extracts (or extracts kept on ice) contain both the faster migrating alpha- and the slower migrating ß-forms. In contrast, phosphatase-treated extracts contained only the alpha-form (Miron, 2001).

LY294002 and rapamycin inhibit PI(3)K and target of rapamycin (TOR) activity, respectively, and block the insulin-induced hyperphosphorylation of 4E-BP1. Similarly, exposure of serum-deprived S2 cells to either drug before treatment with insulin, results in a dose-dependent decrease in d4E-BP phosphorylation. To determine whether phosphorylation of d4E-BP prevents its binding to deIF4E, m7GDP-agarose precipitation was performed. The alpha form is present primarily in the bound fraction, whereas the ß-form is found exclusively in the unbound fraction. These results show that d4E-BP is a downstream target of the PI(3)K pathway, and that the binding of d4E-BP to deIF4E is modulated by its phosphorylation state (Miron, 2001).

Assembly of eIF4F is essential for translational control, and overexpression of eIF4E in mammalian cells results in malignant transformation. To investigate whether eIF4F is also linked to growth control, eIF4F assembly was perturbed in Drosophila. UAS transgenic fly lines were generated that express wild-type d4E-BP or the mutant d4E-BP that binds deIF4E most strongly, d4E-BP(LL). Expression of d4E-BP was targeted to the wing-imaginal disc using MS1096-GAL4. The size and cell number of wings from males were measured. Expression of wild-type d4E-BP has no effect on wing size or pattern. However, expression of d4E-BP(LL) from one line [d4E-BP(LL)w] causes a marked reduction of wing size without affecting cell number. Another line, [d4E-BP(LL)s], which expresses d4E-BP(LL) more strongly, causes a larger reduction, which is partly due to a decrease in cell number. Since direct inhibition of cellular proliferation increases, rather than decreases, cell size, it is possible that d4E-BP(LL) also affects cell size directly, and cell proliferation as a consequence. This is supported by analysis of the effects of d4E-BP(LL) expression in larval-wing discs. Although discs from the d4E-BP(wt) and d4E-BP(LL)w lines are indistinguishable from control discs, d4E-BP(LL)s discs are 52% smaller. d4E-BP(LL)s males also required 1-2 days longer to eclose, which would account for the smaller decrease in adult wings (Miron, 2001).

Acridine-orange staining shows that d4E-BP(LL)s discs contain many apoptotic cells. Co-expression of p35, the baculovirus inhibitor of apoptosis, with d4E-BP(LL)s partially rescues the size of adult wings. To distinguish between apoptosis and decreased proliferation, cell clones expressing d4E-BP(LL), with or without p35, and co-expressing green fluorescent protein (GFP), were induced 72 h after egg deposition in developing wing discs using the flip-out technique. Clones expressing d4E-BP(LL)w contain fewer cells than wild-type clones, but co-expression of p35 with d4E-BP(LL)w does not affect the number of cells per clone, indicating that decreased proliferation, but not increased apoptosis, is the cause of reduction. Few clones expressing d4E-BP(LL)s are recovered, and they usually contain 1-2 cells. Co-expression of p35 greatly increases the number of clones recovered, but only marginally increases the number of cells per clone (1-4 cells) (Miron, 2001).

Direct interference with cell proliferation using string mutants results in increased cell size. To help distinguish effects on size from effects on proliferation, cell size was evaluated by flow cytometry (FACS). Mean forward-light scatter values for GFP-positive cells that expressed d4E-BP(LL) were reduced by 6%-8%. Because cells that expressed d4E-BP(LL) are smaller and proliferate more slowly than their wild-type counterparts, it is conceivable that d4E-BP(LL) directly affects cell growth and consequently retards proliferation, which would lead to reduced viability and ultimately apoptosis. Similar results were observed in dTOR mutants, and interpreted as a primary defect in cellular growth coupled with a consequent decrease in cell proliferation. The possibility that growth and proliferation are affected independently by d4E-BP(LL) expression cannot be excluded (Miron, 2001).

To exclude proliferation effects, the growth and viability of d4E-BP(LL) cells were examined in a post-mitotic tissue. Polyploid fat-body cells undergo successive rounds of DNA synthesis without mitoses. Cells that express d4E-BP(LL)s, induced 48 h after egg deposition in the fat body, are 45%-70% smaller than neighboring wild-type cells, but their frequency is much higher than in mitotically active tissues, such as the wing-imaginal disc. Thus, viability of cells that express d4E-BP(LL) is maintained in the absence of mitogenic signals, indicating that proliferation of wild-type neighboring cells is necessary to cause the disappearance of cells expressing d4E-BP(LL). In support of this notion is the finding that when d4E-BP(LL)s clones are induced during development of eye-imaginal discs, only the clones that are generated posterior to the morphogenetic furrow survive; the clones generated anterior to the furrow (that is, in mitotically active cells), are eliminated (Miron, 2001).

To study the possible role of d4E-BP as an effector of cell growth through the PI(3)K signaling pathway, potential interactions between d4E-BP and relevant signaling genes of this pathway were examined. Expression of wild-type d4E-BP in eye-imaginal discs, using GMR-GAL4, does not engender any discernible phenotype, whereas expression of dAkt1 results in an enlarged eye. However, co-expression of wild-type d4E-BP and dAkt1 partially suppresses the enlarged-eye phenotype, and fully suppresses the roughness induced by expression of dAkt1. Since d4E-BP by itself has no effect on eye size but is able to suppress the dAkt1 phenotype, there is a genuine epistatic relationship between d4E-BP and dAkt1 (Miron, 2001).

Other components of the PI(3)K pathway were also examined for potential epistatic interactions with d4E-BP in the wing, using dpp-GAL4 and 4E-BP(LL)s. Ectopic expression of Dp110 and dAkt1 causes an enlargement of the region encompassed by the third and fourth longitudinal veins, the anterior crossvein and wing margin. In contrast, expression of a dominant-negative mutant form of PI(3)K (Dp110D954A) or d4E-BP(LL)s results in reduction of the size of this region. Co-expression of d4E-BP(LL)s with Dp110 or dAkt1 suppresses the growth enhancement engendered by expression of these kinases, whereas co-expression of d4E-BP(LL)s with Dp110D954A results in further size reduction. Flies that lacked a copy of the gene encoding the adaptor protein p60 [the Drosophila homolog of mammalian PI(3)K subunit p85] are also reduced in size when d4E-BP(LL)s is co-expressed. These results provide genetic evidence that d4E-BP is a downstream effector of the PI(3)K pathway (Miron, 2001).

Null mutants of d4E-BP are viable and although their immune response is compromised (Bernal, 2000), they do not exhibit increased growth. Furthermore, ectopic expression of Drosophila eIF4E in a wild-type or d4E-BP null background fails to produce a growth-related phenotype. Therefore, an increase in eIF4E activity alone is not sufficient to promote cell growth in Drosophila imaginal discs. This is consistent with data in primary mouse-embryo fibroblasts, in which eIF4E overexpression leads only to oncogenic transformation when co-expressed with c-myc or E1A. Attempts were made to rescue the d4E-BP(LL)-induced growth defects in imaginal discs by co-expressing deIF4E. Unexpectedly, growth is further reduced. Thus, endogenous eIF4E expression levels are optimal for cell growth and proliferation, and in the absence of activation of the PI(3)K pathway, a further increase in eIF4E expression is either without effect or deleterious (Miron, 2001).

Many studies have shown that PI(3)K and TOR-mediated signaling is important for normal cell growth and proliferation. However, one downstream target of this pathway, S6K, regulates cell size but not proliferation. Constitutive expression of dS6K in dTOR mutants only partially suppresses the dTOR phenotype, indicating that S6K-independent targets operate downstream of dTOR. Regulation of eIF4E activity, independent of S6K, contributes to the control of cell size. In Drosophila, the activity of eIF4E is modulated through 4E-BP. Phosphorylation of eIF4E is correlated with increased translation rates. Mutation of the phosphorylation site in Drosophila eIF4E causes a cell size reduction. In summary, the results presented here show that d4E-BP acts as an important downstream effector of PI(3)K in the regulation of cell proliferation and growth, independent of S6K, and further underline the importance of translation initiation in the latter process (Miron, 2001).

Regulation of Drosophila tracheal system development by Protein kinase B

Protein kinase B (PKB, also termed Akt) is a phosphatidylinositol 3' kinase (PI3'K)-dependent enzyme implicated in survival signaling and human tumorigenesis. To identify potential targets of this protein kinase, a genetic screen was employed in Drosophila. Among several genes that genetically interacted with PKB is trachealess (trh), which encodes a bHLH-PAS domain transcription factor required for development of the trachea and other tubular organs. Trh activates expression of the fibroblast growth factor receptor Breathless, which, in turn, is required for directed migration of all tracheal branches. Using a combination of biochemical and transgenic approaches, it has been shown that direct phosphorylation of Trh by PKB at serine 665 is essential for nuclear localization and functional activation of this regulator of branching morphogenesis (Jin, 2001).

Trh has a crucial role in the internalization of the primordia to form the tracheal sacs from which the various branches of the trachea derive. Trh controls this and related processes through the transcriptional regulation of downstream target genes. The Trh transcription factor is a direct substrate for PKB/Dakt1 kinase and is selectively phosphorylated at S665. This phosphorylation event is critical for Trh nuclear localization and for its function as a transcriptional coactivator. Further, loss of function of any of Dakt1, dPTEN, or PI3'K (p60A) in Drosophila embryos results in aberrant Trh function. The PI3'K/PTEN/Dakt1 signaling pathway is therefore required for Trh activity and, consequently, tracheal development. This signaling pathway is relevant for Trh function after the initial activation of trh transcription by developmental cues governing the anterior-posterior and dorsal-ventral axes. Since the initial developmental signals regulating trh expression are transient, later stages of tracheal expression are through autoregulation. These results suggest the model whereby PKB activity, as regulated by PI3'K signaling, positively regulates the nuclear localization of Trh via phosphorylation of S665. This leads to the accumulation of Trh within the nucleus, thus promoting an autoregulatory loop, which requires phosphorylation to be maintained. PKB regulation is important for Trh function, since the effects of ectopic Trh expression are suppressed in Dakt1 mutant embryos (Jin, 2001).

To date, a few direct phosphorylation targets of PKB have been identified: Bad, GSK-3ß, and the FKHR transcription factors. Studying these substrates of PKB suggested that PKB may have evolved a substrate selection that is skewed toward motifs also bound by 14-3-3 proteins. These substrates are also negatively regulated by PKB, whereas Trh is positively regulated and does not contain 14-3-3 binding motifs. In the case of FKHR proteins, PKB phosphorylation leads to nuclear exclusion, in contrast to the case for Trh. Thus, Trh may represent another paradigm for regulation by PKB, raising the possibility of other bHLH-PAS domain proteins serving as potential substrates for PKB (Jin, 2001).

In vertebrates, branching morphogenesis is a central component of the development of tubular structures such as lungs, vasculature, kidneys, and mammary glands. Tracheal development in Drosophila has been shown to be a useful model for studying the molecular and morphological aspect of branching morphogenesis. Since PKB is involved in tracheal development through the regulation of Trh, it therefore follows that PKB may have similar role(s) during mammalian branching morphogenesis. During tumorigenesis, branching morphogenesis becomes important during the process of angiogenesis, which is a prerequisite for tumor expansion. A role for PKB/Akt in angiogenesis has been suggested. One provocative study has proposed that the loss of PTEN leads to tumor expansion through ectopic activation of PKB/Akt and hypoxia-inducible factor 1alpha (HIF-1alpha-regulated downstream target genes. Other studies have also linked PI3'K/PKB signaling to the regulation of HIF-1alpha downstream target genes. HIF-1alpha is a bHLH-PAS protein whose levels are elevated in response to hypoxic stress and is structurally similar to Trh. Since human HIF-1alpha expression can induce btl transcription and tracheal structures in Drosophila embryos, it follows that Trh and HIF-1alpha are functionally conserved. This study therefore suggests the mechanism whereby PKB/Akt regulates the expression of genes required for angiogenesis through direct phosphorylation of HIF-1alpha or a related Trh homolog. Several hypoxic response bHLH-PAS factors have been postulated to harbor PKB/Akt consensus phosphorylation sites. Identification of a human bHLH-PAS factor analogous to Trh may provide a valuable target for intervention of the angiogenic response in tumors harboring an activated PI3'K/PTEN/PKB signaling axis (Jin, 2001).

Signaling from Akt to FRAP/TOR targets both 4E-BP and S6K in Drosophila melanogaster

The eIF4E-binding proteins (4E-BPs) interact with translation initiation factor 4E to inhibit translation. Their binding to eIF4E is reversed by phosphorylation of several key Ser/Thr residues. In Drosophila, S6 kinase (dS6K) and a single 4E-BP (d4E-BP) are phosphorylated via the insulin and target of rapamycin (TOR) signaling pathways. Although S6K phosphorylation is independent of phosphoinositide 3-OH kinase (PI3K) and serine/threonine protein kinase Akt, that of 4E-BP is dependent on PI3K and Akt. This difference prompted an examination of the regulation of d4E-BP/Thor in greater detail. Analysis of d4E-BP phosphorylation using site-directed mutagenesis and isoelectric focusing-sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the regulatory interplay between Thr37 and Thr46 of d4E-BP is conserved in flies and that phosphorylation of Thr46 is the major phosphorylation event that regulates d4E-BP activity. RNA interference (RNAi) was used to target components of the PI3K, Akt, and TOR pathways. RNAi experiments directed at components of the insulin and TOR signaling cascades show that d4E-BP is phosphorylated in a PI3K- and Akt-dependent manner. Surprisingly, RNAi of dAkt also affects insulin-stimulated phosphorylation of dS6K, indicating that dAkt may also play a role in dS6K phosphorylation (Miron, 2003).

Is d4E-BP regulated by a PI3K/Akt-independent pathway similar to that described for dS6K? Analysis of signaling to d4E-BP using RNAi indicates that it is not. It is more likely that d4E-BP is a direct downstream target of the dInR-dPI3K-dPTEN-dAkt-dTSC-dTOR signaling cascade. Thus, a linear pathway from InR to Akt that is important for 4E-BP regulation is conserved between Drosophila and mammals (Miron, 2003)

dPDK1 is critical for regulating growth by phosphorylating dAkt and dS6K. RNAi of dPDK1 does not significantly affect insulin-induced phosphorylation of d4E-BP. However, consistent with the direct phosphorylation of dS6K by dPDK1, the phosphorylation of dS6K at Thr398 is completely blocked by RNAi of PDK1. Thus, the results favor a model in which d4E-BP regulation is effected through dAkt, even when dPDK1 levels are dramatically reduced, whereas dS6K requires both dAkt and dPDK1. The differential effects of dPDK1 RNAi on d4E-BP and dS6K phosphorylation can be explained as follows: dPDK1 levels may be reduced below a threshold that is required to phosphorylate dS6K but is still adequate to activate dAkt, allowing d4E-BP phosphorylation. Since dS6K requires direct phosphorylation by dPDK1, it may be more susceptible to variations in its levels. In contrast, d4E-BP, which relies on a signal relayed by dAkt, may be less affected by variations in dPDK1. In mammalian PDK1-hypomorphic mutants, a kinase activity that is 10-fold lower than normal still results in normal Akt and S6K1 activation, yet these animals are greatly reduced in size. This observation supports the notion that reduced PDK1 activity may differentially activate downstream targets (Miron, 2003).

In Drosophila, coexpression of dS6K with dPI3K does not cause additive cellular overgrowth, unlike coexpression of dAkt and dPI3K. RNAi of dPTEN in Kc 167 cells and overexpression of dPTEN in Drosophila larvae had little effect on dS6K activity. Moreover, removal of both dS6K and dPTEN in cell clones does not prevent the dPTEN-dependent overgrowth phenotype. Together, these results and the results of dPI3K and dPTEN RNAi experiments would seemingly support the notion that dS6K-dependent cell growth is not influenced by dPI3K and dPTEN. However, a different effect of dPTEN RNAi on dS6K has been reported in another study: increase in dS6K phosphorylation following RNAi of dPTEN. Consistent with this observation RNAi directed against dPI3K and dPTEN has been shown to modulate dS6K phosphorylation. A reasonable explanation for these discrepancies is that the knockdown of dPI3K and dPTEN achieved in the current experiments was not sufficient to completely deplete these proteins and affect dS6K phosphorylation (Miron, 2003 and references therein).

The role of dAkt in regulating dS6K is subject to debate. In Drosophila, Akt plays a predominant role in mediating the effects of increased PIP3 levels, and all Akt-mediated growth signals are thought to be transduced via Tsc1/2. Tsc2 is directly phosphorylated by Akt, implying that S6K is downstream of Akt in the PI3K signaling pathway. The observation that RNAi of dAkt reduces dS6K phosphorylation at Thr398 supports a direct link among dAkt, dTSC, and dS6K but contradicts the finding that TSC modulates dS6K activity in a dAkt-independent manner. Recent data also support the conclusion of a link between dAkt and dS6K. Clones of cells doubly mutant for dPTEN and dTsc1 display an additive overgrowth phenotype, suggesting that the tumor suppressors act on two independent pathways, from dPTEN to dAkt and from dTSC to dS6K. The findings demonstrate clear effects of dPTEN, dAkt, and dTSC on d4E-BP, which does not preclude the possibility that two pathways regulate d4E-BP; however, a simpler interpretation is that a single pathway is important for its regulation. A possibility is that d4E-BP requires higher dAkt activity than dS6K in order to be phosphorylated. In circumstances of low PI3K activation, low levels of PIP3 are produced, resulting in weaker dAkt activity that is sufficient for dS6K activation but not for d4E-BP phosphorylation. A differential threshold of activation could be the source of the discrepancies between the current results and those of others. This model is strongly supported by recent data showing that in cells lacking both Akt1 and Akt2 isoforms, the low level of Akt activity remaining is sufficient for robust S6K1 phosphorylation, but phosphorylation of 4E-BP1 is dramatically reduced (Miron, 2003 and references therein).

Alternatively, the results could also be explained by the existence of a negative feedback loop between dPI3K and dS6K that dampens insulin signaling by suppressing dAkt activity. This negative feedback loop has been described. Similar observations were made in mammals; insulin-induced activation of Akt is inhibited in Tsc2-deficient mouse embryonic fibroblasts. Thus, depletion of dAkt may trigger this negative feedback loop, which diminishes dS6K phosphorylation and activation. Interestingly, engagement of this feedback mechanism can also provide an explanation for the reduction in total d4E-BP levels observed in dPDK1 RNAi-treated cells. Under these conditions, the reduction of dS6K signaling is accompanied by a concomitant reduction in growth signaling on the dPI3K-dAkt branch of the pathway. Thus, a reduced level of d4E-BP is required to accommodate the reduced need for deIF4E inhibition (Miron, 2003).

The microRNA bantam, acting indirectly on Akt expression, functions in epithelial cells to regulate scaling growth of dendrite arbors in Drosophila sensory neurons

In addition to establishing dendritic coverage of the receptive field, neurons need to adjust their dendritic arbors to match changes of the receptive field. This study shows that dendrite arborization (da) sensory neurons establish dendritic coverage of the body wall early in Drosophila larval development and then grow in precise proportion to their substrate, the underlying body wall epithelium, as the larva more than triples in length. This phenomenon, referred to as scaling growth of dendrites, requires the function of the microRNA (miRNA) bantam (ban) in the epithelial cells rather than the da neurons themselves. ban in epithelial cells dampens Akt kinase activity in adjacent neurons to influence dendrite growth. This signaling between epithelial cells and neurons receiving sensory input from the body wall synchronizes their growth to ensure proper dendritic coverage of the receptive field (Parrish, 2009).

Dendrites of class IV da neurons completely and nonredundantly cover the larval body wall early in larval development, a phenomenon referred to as dendritic tiling. Once field coverage is established, dendrites continue to branch and lengthen to maintain tiling as larvae grow, providing a sensitive system for analysis of how neurons first establish and later maintain coverage of the receptive field. This study addressed the question of how late-stage dendrite growth is precisely coordinated with larval growth to maintain proper dendrite coverage of the body wall (Parrish, 2009).

To examine this process, the pickpocket-EGFP (ppk-EGFP) marker was used to monitor class IV dendrite growth before and after establishment of tiling. To quantitatively assess dendrite coverage, a metric was used that is referred to as the coverage index, the ratio of the territory covered by dendrites of a given da neuron, such as the class IV neuron ddaC, to the area of a hemisegment that harbors the da neurons. Dendrite outgrowth of class IV neurons begins at ~16 hr After Egg Laying (AEL), with class IV dendrites growing rapidly during late embryonic/early larval stages to tile the body wall between 40 and 48 hr AEL and subsequently maintaining this coverage until dendrites are pruned during metamorphosis. Between 48 hr AEL and 120 hr AEL (just prior to metamorphosis), larvae grow nearly 3-fold in length and the dorsal area of class IV receptive fields expands by more than 6-fold. Therefore, class IV dendrites grow extensively and this dendrite growth must be precisely coordinated with larval growth in order to maintain proper coverage of the receptive field (Parrish, 2009).

Class IV dendrites are located between muscle and epithelial cells. Cell divisions that give rise to larval cells are complete by mid-embryogenesis, and larval growth is achieved by increasing cell size rather than additional proliferation. Thus, all the cells that will comprise the larval body wall musculature and epithelia are in place when dendrite outgrowth begins. To simultaneously visualize growth of class IV dendrites and epithelial cells, a protein trap line was used that directs GFP expression in epithelial cells and outlines their borders (Armadillo::GFP, adherens junctions, or Neuroglian::GFP, septate junctions) in combination with ppk-GAL4 driving expression of mCD8-RFP in class IV neurons. Using these markers, growth of class IV dendrites and epithelial cells was monitored throughout embryonic/larval stages (Parrish, 2009).

Epithelial cells grow at a nearly constant rate over the time course. Likewise, the class IV neuron soma grows at a relatively constant rate. In contrast, the dendrite growth is biphasic. Initially, class IV dendrite growth outpaces growth of epithelial cells and the larva as a whole between 16 hr and 48 hr AEL, the timeframe in which class IV dendrites establish tiling. Dendrite growth slows as class IV dendrite arbors achieve complete body wall coverage, and from 48 hr to 120 hr AEL class IV dendrites grow in proportion to larval growth at a rate comparable to that of epithelial cells. This late dendrite growth will be referred to as scaling growth of dendrites (a phenomenon unrelated to synaptic scaling) to reflect the physical scaling of dendrite arbors as they grow precisely in proportion to surrounding cells and the larva as a whole in order to maintain proper coverage of the receptive field (Parrish, 2009).

To determine whether scaling growth is a general property of da neurons, dendrite growth was monitored in class I and class III da neurons, two additional morphologically distinct classes of da neurons, using the coverage index metric introduced above. Like class IV neurons, dendrites of class I and III neurons rapidly establish coverage of a characteristic region of the body wall and subsequently maintain their coverage by expanding their dendrite arbors in precise proportion to larval growth. Class III neurons cover their territory in the same timeframe as class IV neurons, first establishing receptive field coverage at about 48 hr AEL. In contrast, class I neurons covered their characteristic territory by 24 hr AEL. Thus, temporally distinct signals may regulate scaling of dendrite growth in class I and class III/IV neurons. Nevertheless, scaling growth of dendrites seems to be a general feature of da neuron development (Parrish, 2009).

Based on the fidelity of dendrite coverage in class IV neurons, a focus was placed on these neurons for studies of dendrite scaling. The finding that class IV dendrites have a rapid growth phase during establishment of tiling and a scaling phase with slower dendrite growth to maintain tiling suggests that some signal(s) attenuate dendrite growth following establishment of tiling, synchronizing growth of class IV dendritic arbors with growth of surrounding tissue. Attempts were therefore made to characterize the signaling that underlies dendrite scaling (Parrish, 2009).

To test the capacity of dendrite scaling, the effects were examined of mutations that alter the dimensions of larvae at different developmental states on class IV dendrite growth. Alleles were chosen that survive until at least the second larval instar, allowing monitoring of dendrite coverage by class IV neurons at a time when they should have already established tiling. Overall, 35 mutant alleles were screened that cause a range of defects in larval size, shape, and growth rate. Notably, class IV dendrites properly covered the receptive field in nearly all of these mutants, accommodating a broad range of receptive field areas (ranging from 10% of wild-type [WT] in chico mutants to 120% of WT in giant [gt] mutants) and shapes. Dendrites also scaled properly in mutants defective in developmental rate, for example maintaining proper receptive field coverage in b6-22 mutants that develop slowly and persist as second instar larvae or in broad (br) mutants that persist as third instar larvae for days or even weeks. Taken together, these results demonstrate the robustness of dendrite scaling growth in class IV neurons (Parrish, 2009).

Among the few mutants that had any effect on scaling growth of dendrites, the ban mutant had the most severe dendrite overgrowth phenotype observed, with the first sign of larval growth defects at 72 hr AEL. It was reasoned that ban might be required for dendrite scaling but not earlier aspects of dendrite development, and the remainder of the study focused on the role of ban in dendrite scaling. Notably, ban encodes a miRNA and might represent a regulatory node for scaling of dendrite growth since miRNAs likely regulate expression of 100 or more target genes (Parrish, 2009).

Dendrites of individual class IV neurons occupy a larger proportion of the body wall in ban mutant third instar larvae. At 96 hr AEL, ddaC class IV neurons in ban mutants have a mean coverage index of 1.22, meaning that the receptive field of the average ddaC dendrite in ban mutant larvae is 122% of the size of the dorsal hemisegment that harbors the neuron. Thus, dendrites in ban mutants promiscuously cross boundaries that are observed by dendrites of WT neurons. For example, fewer than two dendrite branches cross the midline for a given WT class IV neuron, whereas more than 18 dendrite branches cross the midline in ban mutants. The exuberant growth of dendrites in ban mutants is manifest throughout the arbor, not just at the boundaries. However, although a coverage index of >1 is seen for ban mutant, no significant tiling defect is seen because branches that cross normal boundaries still avoid dendrites of neighboring class IV neurons. In addition to these defects in dendrite coverage, class IV neurons in ban mutants show significant increases in the number of dendrites, the density of dendrites, and overall dendrite length (data not shown). However, increased terminal dendrite branching is not sufficient to increase receptive field coverage. Several other mutants have been described that increase terminal dendrite branching in class IV neurons, and none of these mutants cause an overall increase in the size of the dendritic field. For example, furry (fry) mutations cause a 100% increase in the number/density of terminal dendrites without an accompanying increase in coverage index at 96 hr AEL. Likewise, overexpression of the small GTPase Rac drastically increases terminal dendrite branching but reduces receptive field coverage (Parrish, 2009).

The dendrite growth defects in ban mutants could reflect increased dendrite growth from early stages of development or defects specific to the scaling phase of dendrite growth. To distinguish between these possibilities, dendrite growth was monitored over a developmental time course, focusing on the coverage index and midline crossing events as metrics for growth of the dendrite arbor as a whole. Importantly, class IV dendrites in ban mutants are indistinguishable from WT during the early, rapid growth phase (through 48 hr AEL) as measured by coverage index, midline crossing events, and total dendrite branch number. However, beginning at 72 hr AEL, progressively more severe defects are noted in the coverage index and a greater number of midline crossing events in ban mutants. This late-onset exuberant dendrite growth demonstrates that ban is not causing a general growth defect since ban is dispensable for establishment of dendrite coverage. Whereas a generalized defect in dendrite growth, as seen in dendritic arbor reduction (dar; mutations that lead to defective dendritic arbors but normal axonal projections), would affect both the early (isometric) and late (scaling) phases of growth, mutations that specifically affect the scaling growth of dendrites would be dispensable for the early, rapid growth of dendritic fields. This is precisely what is seen for ban mutants. Therefore, ban is specifically required for scaling of dendrite arbors, potentially by affecting growth-inhibitory signals that normally restrict dendrite growth (Parrish, 2009).

To confirm that loss of ban causes these phenotypes, the following experiments were conducted. First, whereas heterozygosity for a ban null allele or deficiencies that span the ban locus show no obvious defects in dendrite scaling, placing ban mutations in trans to a deficiency that spans the locus, but not a nearby deficiency that does not span the ban locus, recapitulates the dendrite defects described above. Second, the ban mutant dendrite defects can be fully rescued by a ban genomic rescue transgene but not a genomic transgene in which the ban locus has been deleted. Therefore, disrupting ban function is sufficient to cause defects in scaling growth of dendrites (Parrish, 2009).

Next whether ban is required for scaling growth of dendrites was tested in other classes of da neurons. Both class I and class III neurons establish proper dendrite coverage in ban mutants. However, class III dendrites are defective in scaling of dendrite growth in ban mutants, showing a significant increase in dendrite coverage after 48 hr AEL. In contrast, larval class I dendrites show no obvious defects in dendrite coverage in ban mutants, demonstrating that ban is not required for scaling in class I neurons. The onset of scaling growth of dendrites differs by 24 hr in class I and class III/IV neurons, thus different scaling signals may operate at the two time points with ban required for the scaling growth signal for class III/IV neurons that tile (Parrish, 2009).

Next, time-lapse microscopy of single neurons was conducted to characterize the cellular basis of the ban mutant phenotype. Single class IV neurons were imaged from time-matched WT or ban mutant larvae at 24 hr intervals beginning at 72 hr AEL, just after the ban phenotype is first apparent. Dynamics were monitored of every terminal dendrite that could be unambiguously followed through the time course and dendrite growth, initiation of new dendrites, dendrite retraction, and branch loss were measured. For each of these categories, ban mutants differed from WT controls, exhibiting significantly more dendrite growth and branch initiation and significantly less dendrite retraction and branch loss. Therefore, stabilization of existing dendrites, increased dendrite growth, and increased addition of new dendrites all contribute to the defect in dendrite scaling growth of the ban mutant (Parrish, 2009).

Time-lapse studies suggest that signals normally restricting dendrite growth are largely absent in ban mutants. Attempts were made to verify this hypothesis using laser ablation assays. Previous studies showed that, following embryonic ablation of a class IV neuron, dendrites of neighboring neurons grow exuberantly to invade the unoccupied territory of the ablated neuron, with the ability of dendrites to invade unoccupied territory progressively restricted in older larvae. It was therefore important to determine whether the timing of this restricted growth potential correlates with the onset of scaling of dendrite growth and whether ban is required for restriction of the dendrite growth potential (Parrish, 2009).

Consistent with prior reports, ablating a class IV neuron at 24 hr AEL led to extensive invasion by dendrites of neighboring neurons, with 55% of the unoccupied territory covered by neighboring neurons 48 hr postablation. This ability of dendrites to grow into unoccupied territory was severely attenuated 1 day later, with dendrites of neighboring neurons invading only 23% of the unoccupied territory after ablation of a class IV neuron at 48 hr AEL . The extent of invasion was even further reduced when neurons were ablated at 72 hr AEL. Therefore, the ability of dendrites to grow beyond their normal boundaries to invade unoccupied territory is severely restricted during larval development at a time coincident with the onset of scaling of dendrite growth (Parrish, 2009).

If the restriction of dendrite growth potential in larvae is caused by scaling signals that limit dendrites to growth in proportion to body wall growth, the majority of invading activity by neighboring dendrites should be present before scaling growth ensues at 48 hr AEL. To test this prediction, class IV neurons were ablated at 24 hr AEL and invasion activity was monitored at 24 hr intervals over the next 72 hr. By 48 hr AEL, dendrites of neighboring neurons had invaded unoccupied territory, and the extent of invasion was not noticeably increased at later time points. Instead, the entire dendrite arbor of class IV neurons, including the portion that invaded unoccupied territory, scaled with larval growth after 48 hr AEL. Thus, the receptive field that is established by 48 hr AEL is maintained by scaling of dendrite growth, even in cases in which dendrites establish aberrant body wall coverage. The signals responsible for dendrite scaling growth are likely distinct from the homotypic repulsion required to establish tiling as ablation of all neighboring same-type neurons does not potentiate the ability of a class IV neuron to invade unoccupied territory. Additionally, dendrites of class I da neurons, which do not rely on homotypic repulsion to establish their coverage, also exhibit scaling growth (Parrish, 2009).

As described above, dendrite coverage is properly established in ban mutants. Importantly, unlike WT controls, following ablation at 48 hr AEL, dendrites in ban mutants extensively fill unoccupied space, with dendrites in ban mutants invading unoccupied territory just as efficiently as dendrites in WT controls ablated at 24 hr AEL. Therefore, the receptive field boundaries of class IV neurons have not been fixed in ban mutants at 48 hr AEL. Dendrites in ban mutants invade unoccupied territory more efficiently than WT controls at later time points as well. Thus, either the growth-inhibitory scaling signal is lost or dendrites are refractory to the signal in ban mutants (Parrish, 2009).

To test whether machineries for dendritic tiling contribute to the progressive reduction of a dendrite's ability to invade vacant territories, mutations of fry, which encodes a gene required for establishment of dendritic tiling and of extra sex combs (esc) and salvador (sav), which function in a common pathway to regulate stability of terminal dendrites and, consequently, maintenance of dendrite coverage, were examined for effects on dendrite invasion following neuron ablation. Unlike mutations in ban, mutations in fry, esc, or sav had no effect on the ability of dendrites to invade unoccupied territory. Moreover, consistent with the scaling signal functioning in a distinct pathway, double-mutant combinations of ban with fry or esc showed additive phenotypes. Thus, ban exerts its effects on scaling of dendrite growth independently of known pathways for establishment and maintenance of dendrite coverage (Parrish, 2009).

To further characterize the signaling required for scaling growth of dendrites, it was of interest to determine where ban functions to regulate scaling. First, whether ban is expressed in neurons, surrounding cells, or both, was examined by using a miRNA activity sensor as a reporter for ban expression in third instar larvae. A control sensor directs ubiquitous expression of GFP, including robust GFP expression in muscle, epithelial cells, and sensory neurons. The ban sensor contains two ban binding sites in the 3'UTR of the transgene, hence GFP expression is attenuated in cells that express ban. Unlike the control sensor, very little, if any, GFP expression was detected in third instar muscle cells, epithelial cells, or sensory neurons using several independent transgenic fly lines with distinct insertions of the ban sensor. Significant attenuation of the ban sensor was first observ ed in larval muscle, epithelium, and PNS neurons between 48 and 72 hr AEL, precisely at the time when dendrite defects were first observed in ban mutants, suggesting that ban activity is more pronounced during this period than at earlier time points. Notably, the attenuation of the ban sensor was dependent on ban activity, as shown by the persistent, ubiquitous expression of the sensor in ban mutant larvae. Thus, ban is likely expressed in the muscle, epithelium, and PNS neurons and may be required in any of these cell types for scaling of dendrite growth (Parrish, 2009).

To determine whether ban is required cell-autonomously for dendrite scaling, MARCM was used to generate single neuron clones homozygous for a ban mutation in a heterozygous background. ban activity was effectively dampened in MARCM clones, as indicated by derepression of the ban sensor in the clones. However, loss of ban function had no significant effect on dendrite coverage of class IV neurons. Time-lapse analysis of ban mutant class IV MARCM clones revealed no defects in dendrite coverage at any time during larval development. Furthermore, ban is dispensable in other da neurons for dendrite scaling growth. Thus, ban function in sensory neurons is dispensable for scaling growth of dendrites (Parrish, 2009).

Although scaling of dendrite growth proceeds normally, there is some reduction of overall dendrite length and the number of dendrite branches in ban mutant class IV clones. Therefore, ban likely acts cell-autonomously to promote dendrite growth and nonautonomously to limit dendrite Taking advantage and ensure proper scaling (Parrish, 2009).

A genetic rescue assay was used to test the ability of transgenic expression of ban in different tissues to rescue the dendrite growth defects of ban mutants. Consistent with MARCM results, neuronal expression of ban, using either panneuronal or PNS-specific Gal4 drivers, was not sufficient to rescue the scaling growth defect of ban mutants. Thus, ban likely functions nonautonomously in nonneuronal cells to regulate scaling of da neuron dendrite growth. Moreover, expression of ban in muscle alone could not ameliorate the dendrite defects of ban mutants. Remarkably, every time ban expression was rescued in epithelial cells, significant suppression of the exuberant dendrite growth of ban mutants was found. The three epithelial Gal4 driver lines caused reductions of dendrite growth that correlated with Gal4 expression levels in epithelial cells: arm-Gal4 caused the greatest reduction in dendrite growth and had the strongest epithelial expression, whereas twi-Gal4 displayed the lowest activity and drove epithelial Gal4 expression at the lowest level. Taking advantage of the temperature-sensitive nature of Gal4 activity, rescue activity of each epithelial Gal4 line was monitored over a graded temperature series (18°C to 29°C) and it was found that, for each driver, rescue activity was directly proportional to expression level. Therefore, epithelial ban expression is sufficient to suppress the exuberant dendrite growth of ban mutants, and the extent of dendrite growth inhibition varies with the level of ban expression in epithelial cells (Parrish, 2009).

Epithelial expression of twi-Gal4 was first apparent in larval stages, suggesting that postembryonic expression of ban in epithelia is sufficient for proper scaling of dendrite growth. Given that dendrite defects in ban mutants first appear after 48 hr AEL, it was asked whether late expression of ban would suffice for dendrite scaling. To examine the temporal requirement for ban function, a heat-shock-inducible Gal4 driver was used to express ban during larval development. Indeed, inducing ban expression at 48 hr AEL was sufficient to rescue the dendrite defects of ban mutants. These findings reinforce the notion that ban is dispensable for early aspects of dendrite development (Parrish, 2009).

Resupplying ban in tissues known to regulate larval growth, such as the fat body, prothoracic (PTTH) gland, or insulin-producing cells (IPCs), had no measurable effect on dendrite growth in ban mutants. Moreover, ablation of each of these tissues mediated by a reaper transgene caused larval growth defects without obvious dendrite growth defects. Thus, ban function in the fat body, PTTH gland, or IPCs is not sufficient to modulate scaling of dendrite growth. Altogether, these results suggest that epithelial cells are likely the major functional sites for ban in regulation of PNS dendrite scaling (Parrish, 2009).

Because ban expression in epithelial cells affects scaling growth of dendrites in a dose-dependent fashion via a mechanism that likely involves growth-inhibitory signals, it was of interest to see whether ectopic epithelial expression of ban in a WT background could further inhibit dendrite growth and thus disrupt scaling of dendrite arbors. Indeed, overexpression of ban in epithelial cells resulted in a severe reduction in dendrite growth and induced striking defects in the pattern of dendrite growth over epithelial cells, with terminal dendrites appearing to wrap around epithelial cells. Consistent with ban dosage in epithelial cells regulating the strength of dendrite growth-inhibitory signals, epithelial overexpression of ban induced more robust inhibition of dendrite growth at higher temperatures (which lead to higher levels of transgene expression) (Parrish, 2009).

Since ban expression in epithelial cells is sufficient to ensure proper scaling, it was of interest to address whether ban function in epithelial cells is necessary for scaling of dendrite growth. To this end, MARCM was used to generate ban mutant epithelial cell clones. Although it was not possible to address the contribution of epithelial ban to scaling of the entire dendrite arbor using this approach (it was only possible to generate one to four cell epithelial clones), the pattern was monitored of dendrite growth over ban mutant or WT control epithelial clones. Class IV dendrites grow extensively over epithelial cells, with multiple dendrite branches often coursing over a single epithelial cell. The epithelial nucleus was used as a landmark and dendrite growth was monitored over the epithelial cell surface shadowed by the nucleus. Although the gross morphology of epithelial cells was not obviously affected in ban mutant clones, the propensity of class IV dendrites to grow into the region shadowed by the epithelial nucleus was significantly increased for ban mutant epithelial clones when compared to WT controls or ban heterozygous epithelial cells. Therefore, ban is required in epithelial cells to ensure proper dendrite growth and placement over epithelial cells (Parrish, 2009).

To gain insight into the molecular mechanism underlying dendrite scaling, a platform was developed for microarray-based expression profiling of dissociated, FACS-isolated PNS neurons or epithelial cells. Akt and numerous other candidate genes were identified that were deregulated in PNS neurons and/or epithelial cells of ban mutant larvae. Because Akt is a well-established regulator of growth, including dendrite growth in mammalian hippocampal neurons, whether ban regulates Akt as part of the scaling program was investigated (Parrish, 2009).

Microarray experiments found that Akt expression was increased in neurons but reduced in epithelial cells of ban mutants relative to WT controls. By monitoring Akt levels in lysates of larval fillets composed mostly of muscle and epithelial cells, it was found that in the absence of ban function Akt protein levels were substantially reduced. Furthermore, Akt activity was substantially reduced as shown by reductions in active, phosphorylated Akt and phosphorylated S6K, a downstream reporter of Akt activity. Therefore, Akt expression and activity are substantially reduced in ban mutant larval lysates, likely reflecting reduced Akt function in muscle/epithelia (Parrish, 2009).

Next larval fillets were immunostained to determine whether ban influences Akt protein levels in the PNS. In WT controls, Akt is detectible only at low levels in the soma or dendrites of the PNS. By contrast, in ban mutants Akt is highly expressed in the PNS and is detectible in axons, the soma, and dendrites. Similarly, phosphorylated Akt is barely detectible in the larval PNS of WT controls but is present at high levels in the PNS of ban mutants. Therefore, ban regulates Akt expression and activity in the larval PNS (Parrish, 2009).

To test whether this effect on Akt levels reflects a neuronal requirement for ban, Akt expression levels were monitored in ban mutants in which ban expression is resupplied under the control of twist-Gal4, an experimental condition that rescues both the dendrite scaling defect and larval size defect of ban mutants. It was found that ban nonautonomously regulates Akt levels in da neurons since nonneuronal expression of ban (twist-Gal4) is sufficient to dampen the ectopic neuronal Akt expression normally seen in ban mutants. Therefore, ban likely functions in epithelia to regulate signals that influence Akt expression and activity in neurons (Parrish, 2009).

Finally, it was of interest to determine whether Akt function in class IV neurons is important for scaling of dendrite growth. Based on expression data, it was predicted that increasing Akt expression/activity in class IV neurons should cause a scaling defect similar to what is seen in ban mutants. Indeed, ectopic expression of Akt, or a constitutively active form of PI3 kinase (PI3k) that leads to activation of Akt, caused a significant increase in dendrite coverage, similar to ban mutants. Conversely, antagonizing Akt activity in class IV neurons by overexpressing Pten, a PIP3 phosphatase that functions as an inhibitor of Akt activity, by knocking down Akt expression via RNAi in class IV neurons, or by generating Akt null mutant class IV neuron MARCM clones caused a significant reduction in dendrite coverage. Therefore, Akt plays a critical role in regulating dendrite coverage (Parrish, 2009).

If increased neuronal Akt activity underlies the dendrite defects in ban mutants, then antagonizing neuronal Akt activity should suppress the dendrite overgrowth in ban mutants. This hypothesis was tested with the following three experiments. First, RNAi was used to knock down Akt expression in class IV neurons of ban mutant larvae. On its own, Akt(RNAi) causes a reduction in dendrite growth and overall coverage of the receptive field, and this phenotype is epistatic to the dendrite overgrowth seen in ban mutants. Similarly, Pten was overexpressed in class IV neurons of ban mutant larvae and it was found that the Pten-mediated reduction in dendrite coverage is epistatic to the dendrite overgrowth seen in ban mutants. Finally, class IV neurons was ablated in ban mutants in the absence or presence of neuron-specific Akt RNAi and it was found that reducing neuronal Akt expression blocks the exuberant dendrite invasion activity of ban mutants. Altogether, these results strongly suggest that ban functions in epithelial cells to regulate neuronal expression/activation of Akt, and deregulation of Akt leads to the dendrite growth defects of ban mutants (Parrish, 2009).

This study has shown that nonautonomous signals coordinate growth of dendrites with the growth of their substrate and the body as a whole. Dendrites of many types of neurons cover characteristic receptive fields, and growth of the dendrite arbor in synchrony with the receptive field in a process, referred to as scaling growth of dendrites, allows a neuron to maintain proper dendrite coverage of the receptive field. Thus, scaling growth of dendrites is likely a general mechanism to ensure fidelity of dendrite coverage (Parrish, 2009).

Dendrites of class IV neurons cover their receptive field before larval growth is complete and must maintain this coverage as the larva grows. Two properties distinguish the scaling phase of dendrite growth from the early dendrite growth when the neuron establishes receptive field coverage. During the scaling phase of growth, the dendrite arbor grows precisely in proportion to receptive field expansion (which is often achieved by animal growth). Moreover, dendrite growth is constrained by boundaries delineated when the dendrite arbor first covers the receptive field. Thus, although dendrites continue to grow, growth occurs only to maintain proportional coverage of the receptive field (Parrish, 2009).

Dendrites of Drosophila da neurons exhibit a biphasic growth profile: dendrites establish coverage of their receptive field via an early, rapid growth phase and maintain this coverage via a late scaling growth phase in which dendrites grow in proportion with epithelial cells and the animal as a whole. The miRNA ban acts in the second phase to enable scaling of dendrite growth in da neurons, ensuring that dendrites maintain proper body wall coverage. Loss of ban disrupts epithelial-derived signaling that normally modulates dendrite growth, and, as a result, dendrites remain in the 'rapid growth' phase, extending beyond their normal territories. This phenotype is reminiscent of the heterochronic phenotypes of C. elegans lin-4 and let-7 mutants in that an early developmental phase is inappropriately reiterated during a later phase (Parrish, 2009).

How broadly do miRNAs regulate developmental progressions in the nervous system? Although the developmental roles of vertebrate miRNAs have been somewhat elusive because of the vast number of miRNA-encoding genes, several studies suggest that miRNAs may serve highly specialized roles in regulating developmental transitions in neuron morphogenesis. For example, completely abrogating miRNA function causes robust defects in neuron morphogenesis but not specification in zebrafish, consistent with miRNAS regulating late aspects of neuronal differentiation. Likewise, miRNAs miR9a* and miR-124 regulate the switch in subunit composition of chromatin remodeling complexes as neural progenitors differentiate into neurons in mice. Additionally, a number of miRNAs function primarily at a very late step of neuron development to regulate activity-dependent dendrite growth and synaptic plasticity. For example, neuronal activity antagonizes miR-134, which normally inhibits growth of dendritic spines, and promotes expression of mir-132, which promotes dendritic plasticity (Parrish, 2009).

Although ban is known to regulate growth in proliferating tissues in Drosophila, ban-mediated regulation of dendrite growth likely represents a distinct mode of growth control by ban for the following reasons. First, previous studies focused on autonomous regulation of tissue growth by ban. In contrast, ban acts nonautonomously to regulate scaling of dendrite growth. Second, prior studies of ban function focused on imaginal discs where growth is achieved by increasing cell number rather than cell size. By contrast, dendrite scaling involves ban-mediated regulation of growth in differentiated, postmitotic cells. Likewise, postmitotic expression of ban in the larval eye disc can also regulate cell size. Third, ban functions downstream of the tumor suppressor kinase Hippo to control proliferation, with Hippo activating the transcription factor Yorkie, which in turn activates ban expression. Whereas hippo is required cell-autonomously for establishment and maintenance of dendrite tiling, yorkie is dispensable for dendrite growth. As to the cell-nonautonomous function of ban in dendrite scaling, Hippo is not required. Although these findings suggest that ban regulates growth of proliferating and differentiated tissues by different means, it is possible that in both scenarios ban is antagonizing expression of growth-inhibitory factors, possibly even the same factors, and removing growth inhibition has different consequences on proliferative and differentiated tissues (Parrish, 2009).

It is proposed that ban positively regulates an epithelial-derived signal that modulates neuronal Akt expression and activity to influence dendrite growth. Several observations suggest that the signal acts over a short range, possibly even via direct adhesive interactions between dendrites and epithelia or the underlying matrix. First, ban overexpression in epithelial cells but not in muscle influences growth of dendrites. Second, removing ban function from epithelial cell clones influences the distribution of dendrites over the clone but not over adjacent WT epithelial cells. Third, in addition to inhibiting the overall rate of dendrite growth, overexpressing ban in epithelial cells induces exaggerated “wrapping” of epithelial cells by terminal dendrites. Since these signals appear to preferentially regulate dendrite growth during the scaling phase, ban may modulate dendrite/epithelial adhesion during the scaling phase of dendrite development (Parrish, 2009).

Morphologically distinct classes of da neurons establish type-specific dendritic coverage of the body wall and maintain this coverage by means of dendrite scaling as larvae grow. However, arbors of different da neurons develop at different rates, with class I dendrites establishing their coverage 1 day earlier than class III and class IV dendrites. Mutations in ban disrupt scaling of dendrite growth in class III and class IV but not class I neurons, suggesting that different signals regulate dendrite scaling in distinct types of neurons. Thus, temporally distinct signals or temporally restricted sensitivity to the signals may ensure that different neurons maintain appropriate coverage of their receptive field (Parrish, 2009).

Foxo is a direct target of Akt

The Drosophila Insulin receptor (InR) regulates cell growth and proliferation through the PI3K/Akt pathway, which is conserved in metazoan organisms. The Drosophila forkhead-related transcription factor Foxo is a key component of the insulin signaling cascade. Foxo is phosphorylated by Akt upon insulin treatment, leading to cytoplasmic retention and inhibition of its transcriptional activity. Mutant Foxo lacking Akt phosphorylation sites no longer responds to insulin inhibition, remains in the nucleus, and is constitutively active. Foxo activation in S2 cells induces growth arrest and activates two key players of the InR/PI3K/Akt pathway: the translational regulator d4EBP/Thor (eukaryotic initiation factor 4E binding protein) and the InR itself. Induction of d4EBP likely leads to growth inhibition by Foxo, whereas activation of InR provides a novel transcriptionally induced feedback control mechanism. Targeted expression of Foxo in fly tissues regulates organ size by specifying cell number with no effect on cell size. These results establish Foxo as a key transcriptional regulator of the insulin pathway that modulates growth and proliferation (Puig, 2003).

To establish that the slower-migrating form of Foxo induced by insulin treatment is indeed caused by Akt-catalyzed phosphorylation, a mutant form of Foxo was constructed in which all three putative dAkt phosphorylation sites (T44, S190, and S259) were mutated to alanine (FoxoA3). Both wild-type (Foxo-V5) and mutant (FoxoA3-V5) proteins were expressed in S2 cells. After transient expression, the cells were subjected to three different treatments in parallel: insulin; pretreatment with LY294002 (a specific inhibitor of PI3K that counteracts the effects of insulin) followed by insulin treatment, or no treatment control. Extracts derived from cells treated with insulin contained the slower-migrating form of wild-type Foxo when compared with control cells. Pretreatment with the PI3K inhibitor LY294002 reduced the amount of the slower-migrating form of Foxo. In contrast, no slower-migrating species was observed for the triple alanine mutant (FoxoA3) when comparing control, insulin-treated, and LY294002 + insulin-treated samples. To further confirm that the slower-migrating form of Foxo is caused by phosphorylation, cell extracts were incubated with calf intestinal phosphatase (CIP). Western blot analysis showed that the slower-migrating form of Foxo is quantitatively converted to the 113-kD form after CIP treatment. Together, these results indicate that Foxo is phosphorylated by insulin treatment and that this phosphorylation depends on the presence of the dAkt consensus residues T44, S190, and S259 (Puig, 2003).

To test how Foxo subcellular localization is affected by insulin-mediated phosphorylation, S2 cells expressing either wild-type Foxo or mutant FoxoA3 were incubated for 48 h in the absence of serum. Then insulin was added, and localization of transfected Foxo was determined by confocal microscopy after staining with the V5 antibody. When S2 cells are incubated in the absence of serum and insulin, both Drosophila Foxo and dFoxoA3 (constitutively active Drosophila Foxo in which all three putative Akt phosphorylation sites have been mutated to alanine) are found predominantly in the nucleus. After insulin treatment, Foxo is localized in the cytoplasm. In contrast, mutant FoxoA3 remains nuclear even after insulin treatment. This result is consistent with the idea that subcellular localization of Foxo is regulated by insulin (Puig, 2003).

Is Foxo phosphorylation regulated through the PI3K/Akt pathway? A constitutively active form of Drosophila Akt was used in which a myristoylation signal has been fused to the N terminus of Akt. Myr-Akt tagged with V5 epitope was cotransfected in S2 cells grown in the absence of serum and insulin with either Foxo or dFoxoA3, and the phosphorylation state of both proteins was analyzed by Western blot analysis. In the absence of Akt, both Foxo and dFoxoA3 remain unphosphorylated. When Myr-Akt is present in the cells, Foxo but not dFoxoA3 becomes phosphorylated even in the absence of insulin. This result indicates that Myr-dAkt can phosphorylate Foxo in S2 cells. To assess the effect of Foxo phosphorylation by Myr-Akt, use was made of a reporter construct containing four tandem FOXO4-binding sites upstream of the alcohol dehydrogenase distal core promoter driving the luciferase gene (pGL4xFRE). In the absence of Myr-Akt, cells cotransfected with wild-type or mutant Foxo constructs incubated without serum display comparable luciferase activity. In contrast, when Myr-Akt is present, cells cotransfected with wild-type Foxo display luciferase activity that is reduced by more than 65%, whereas activity of the mutant FoxoA3 remains essentially unchanged (Puig, 2003).

These results suggest that insulin induces Foxo phosphorylation through Akt, which leads to cytoplasmic localization and transcriptional inactivation of Foxo. To further confirm that insulin inhibits Foxo activity through Akt, RNAi experiments were performed. S2 cells transfected with either Foxo or FoxoA3 and cotransfected with the luciferase reporter pGL4xFRE were grown in the presence of insulin and treated with dsRNA directed against Drosophila Akt. As a control, dsRNA against lactose repressor (lacI) was used. As expected, Foxo activity is not inhibited by insulin when cells are depleted of Drosophila Akt by dsRNA treatment, but it is inhibited in the lacI control. These results confirm that Akt mediates insulin inhibition of Foxo (Puig, 2003).

PTEN negatively regulates Akt

Tuberous sclerosis complex (TSC) is a genetic disorder caused by mutations in one of two tumor suppressor genes, TSC1 and TSC2. Absence of Drosophila Tsc1 and/or Tsc2 (Gigas) leads to constitutive S6k activation and inhibition of PKB, the latter effect being relieved by loss of S6K. In contrast, the Pten tumor suppressor, a negative effector of PI3K, has little effect on S6k, but negatively regulates PKB (Akt1). More importantly, reducing S6k signaling rescues early larval lethality associated with loss of Tsc1/2 function, arguing that the S6k pathway is a promising target for the treatment of TSC (Radimerski, 2002b).

To determine whether loss of Tsc1/2 or Pten directly affected S6k activity, each was depleted in Drosophila Kc167 cells by dsRNAi. Quantitative Real Time PCR showed that such treatment strongly reduced levels of both transcripts. Compared with control cells, depletion of Tsc1 increases S6k activity and T398 phosphorylation, consistent with the reduced electrophoretic mobility of S6k. These results are in agreement with recent findings in TSC1 null mammalian cells (Kwiatkowski, 2002). Insulin treatment of either control cells or Tsc1-depleted cells did not significantly increase these responses beyond that of Tsc1 depletion alone, indicating that loss of Tsc function leads to full S6k activation. RAD001, a rapamycin derivative, blocks S6k activity in both control and Tsc1-depleted cells treated with insulin. However, it was consistently noted that the RAD001 block of insulin-induced S6k activation is not as strong in Tsc1-depleted cells, suggesting that not all the effects of Tsc on S6k are dependent on Tor, the Drosophila target of rapamycin. Similar results to those described here were obtained by Tsc2 depletion. In addition, the effects appear specific, since Tsc1 depletion has no effect on the basal activity of other AGC-kinase family members, such as PKB or Drosophila atypical PKC. However, insulin-induced PKB activation and S505 phosphorylation are repressed in Tsc1-depleted cells as compared with control cells, consistent with S6k acting in a negative feedback loop to dampen PKB signaling. In contrast to loss of Tsc1, depletion of Pten has little effect on S6k activity and T398 phosphorylation, whereas it leads to elevated levels of both basal and insulin-stimulated PKB activity and S505 phosphorylation. Thus, loss of Tsc1/2, but not Pten, leads to constitutive S6k activation (Radimerski, 2002b).

To determine whether the findings above could be corroborated in the animal, S6k activity was measured in extracts of Tsc1, Pten, and S6k null larvae. The results show that S6k activity in extracts derived from Tsc1 null larvae is strongly increased over that of wild-type larvae, whereas it is slightly increased in larvae lacking Pten. The opposite was found for PKB activity, which is strongly repressed in Tsc1 null larvae, and up-regulated in Pten-deficient larvae. Hence, it cannot be excluded that reduced PKB activity contributes to larval lethality of Tsc mutants. Given that loss of Tsc function leads to increased S6k activity, it was reasoned that ectopic expression of Tsc1/2, but not Pten, would inhibit S6k activity. To test this hypothesis, both tumor suppressors were expressed ubiquitously in larvae using the GAL4/UAS system, such that the GAL4 promoter chosen in each case led to developmental arrest at late larval second instar. Extracts from larvae overexpressing Tsc1/2 display strongly reduce S6k activity, whereas those from Pten overexpressing larvae have normal levels of S6k activity. In contrast, PKB activity is strongly suppressed in Pten overexpressing larvae and little affected in extracts from larvae overexpressing Tsc1/2. These data corroborate previous findings that S6k and PKB act in parallel signal transduction pathways (Radimerski, 2002a), and provide compelling evidence that they are negatively controlled by distinct tumor suppressor genes (Radimerski, 2002b).

Despite the fact that S6k and PKB act in parallel signaling pathways, loss of Tsc1/2 function leads to inhibition of PKB activity, suggesting cross-talk between the two pathways. Compatible with such a model, recent studies have shown that rapamycin treatment of adipocytes inhibits a negative feedback loop, which normally functions to dampen insulin-induced PKB activation. Since RAD001 inhibits S6k activity (Radimerski, 2002a) and increases PKB activity (Radimerski, 2002a), it raised the possibility that the effects of Tsc mutants on PKB are mediated through S6k. Consistent with this hypothesis, inhibition of PKB activity due to loss of Tsc function was relieved in the absence of S6k. Similar results were obtained by using dsRNAi in cell culture. Thus, the suppression of PKB by loss of Tsc function requires S6k (Radimerski, 2002b).

To genetically test the specificity of Tsc1/2 and Pten tumor suppressor function, either Tsc1 or Pten were removed in cells giving rise to the adult eye structure, by inducing mitotic recombination with the FLP/FRT system under the control of the eyeless promoter. In a wild-type genetic background, loss of either Tsc1 or Pten within the developing eye causes strong overgrowth of the head. Eye overgrowth by removal of Tsc1 is strongly suppressed in a genetic background null for S6k, as is ommatidia size, in agreement with a previous report analyzing double mutant clones of Tsc2 and S6k in the eye (Potter, 2001). In contrast, removal of Pten in the eyes of S6k null flies still induces overgrowth of clones with enlarged ommatidia. These findings are supported by results showing that eye overgrowth by removal of Tsc1 is still observed in clones devoid of PKB function (Potter, 2001) and overgrowth by removal of Pten is suppressed in a viable PKB mutant genetic background (Stocker, 2002). Thus, Tsc1/2 appears to be specific for the S6k-signaling pathway, whereas Pten antagonizes PI3K signaling to counteract PKB activation by decreasing PIP3 levels (Radimerski, 2002b).

Taken together, these results demonstrate that the tumor suppressor Tsc1/2 is a critical component in controlling S6k activation. Interestingly, this effect may be Tor independent, as insulin-induced S6k activation is more elevated in Tsc1/2-depleted cells pretreated with RAD001 than in control cells, and in preliminary studies, clonal overgrowth in the eye induced by loss of Tsc1 is not suppressed in a semiviable, heterorallelic Tor mutant background. Overexpression of Tsc1/2 selectively suppresses the S6k-signaling pathway, whereas Pten operates on the dPI3K-signaling pathway. Double mutations for Pten and Tsc1 are additive for clonal overgrowth, compatible with S6k and PKB independently mediating growth. Nevertheless, inhibition of PKB by loss of Tsc function shows that there is negative cross-talk between the two signaling pathways. Given this negative cross-talk, the observation that in double mutant clones growth is additive, suggests that in the absence of Pten, inhibition of PKB by loss of Tsc is circumvented. However, despite the observation that double mutations for Pten and Tsc1 are additive for clonal overgrowth, overgrowth induced by absence of Pten is suppressed in clones mutant for Tor. Since S6k does not prevent such overgrowth, it is possible that this suppression actually represents an intermediate phenotype, or that Pten negatively acts on a Tor target distinct from S6k. At this point, it is important to gain a deeper knowledge of the molecular mechanisms by which Tsc1/2 acts to suppress S6k function and how the signaling components of these two pathways cross-talk with one another (Radimerski, 2002b).

Recently, a successful Phase I clinical trial was completed for a rapamycin analog in the treatment of solid tumors. The results of the trial demonstrated that the drug was efficacious at subtoxic doses, and suggested that specific tumor types may be more sensitive to inhibition by rapamycin than others. The question that arose from the trial is, which tumors would be susceptible to rapamycin treatment? Here, it has been demonstrated for the first time in vivo that a mild reduction in S6k signaling, which alone has no blatant phenotype, is sufficient to restore viability of flies devoid of Tsc function. Thus, these findings imply that rapamycin or its derivatives might be very promising pharmaceutical agents in the treatment of tumors arising from TSC (Radimerski, 2002b).

Interaction of Akt-phosphorylated Ataxin-1 with 14-3-3 mediates neurodegeneration in a Drosophila model of spinocerebellar ataxia type 1

Spinocerebellar ataxia type 1 (SCA1) is one of several neurological disorders caused by a CAG repeat expansion. In SCA1, this expansion produces an abnormally long polyglutamine tract in the protein ataxin-1. Mutant polyglutamine proteins accumulate in neurons, inducing neurodegeneration, but an understanding of the mechanism underlying this accumulation has been unclear. The 14-3-3 protein, a multifunctional regulatory molecule, mediates the neurotoxicity of ataxin-1 by binding to and stabilizing ataxin-1, thereby slowing ataxin-1's normal degradation. The association of ataxin-1 with 14-3-3 is regulated by Akt phosphorylation, and in a Drosophila model of SCA1, both 14-3-3 and Akt modulate neurodegeneration. The finding that phosphatidylinositol 3-kinase/Akt signaling and 14-3-3 cooperate to modulate the neurotoxicity of ataxin-1 provides insight into SCA1 pathogenesis and identifies potential targets for therapeutic intervention (Chen, 2003).

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disease caused by the expansion of a CAG repeat that produces an abnormally long polyglutamine tract in the ataxin-1 protein. At least eight other inherited neurodegenerative diseases, including Huntington's disease, are caused by a similar pathogenic mechanism. In each case, the length of the CAG repeat tract correlates with disease severity: more repeats produce more severe symptoms with an earlier age of onset. The expanded polyglutamine tract appears to confer a toxic gain-of-function that intensifies with longer repeats (Chen, 2003 and references therein).

Another feature common to the polyglutamine diseases studied so far (as well as several other neurodegenerative disorders) is aberrant protein deposition: mutant polyglutamine proteins have a strong tendency to accumulate and eventually form aggregates in neurons. It has been proposed that the polyglutamine expansion alters the protein's conformation in such a way as to make the protein recalcitrant to proteasomal degradation. In the case of ataxin-1, even the unexpanded protein can produce pathology if expressed at sufficiently high levels, which suggests that wild-type ataxin-1 might have more than one stable conformation, and that one or more of these alternate conformations is toxic if it becomes abundant. Support for this idea has come from the study of alpha-synuclein, whose accumulation causes Parkinson's Disease (PD). Although rare cases of familial PD are caused by point mutations in alpha-synuclein, most PD is associated with abnormal accumulation of wild-type alpha-synuclein. These observations raise several important questions: what factors contribute to the altered protein conformation? How exactly do misfolded proteins induce neuronal dysfunction and degeneration? And what factors modulate their toxicity (Chen, 2003 and references therein)?

The subcellular localization of the polyglutamine protein, the ratio of the polyglutamine tract to the host protein, and native protein sequences flanking the CAG repeat all affect the toxicity of polyglutamine proteins. Protein modifications such as phosphorylation may also have an effect: in Alzheimer's disease (AD): for example, brain dysfunction and degeneration are linked to the accumulation of the neurofibrillary tangles that are highly enriched in the hyperphosphorylated forms of the microtubule-associated protein tau. Enhanced phosphorylation of tau by glycogen synthase kinase 3beta (GSK3beta) induces filamentous tau inclusions and accelerates tau-induced neurodegeneration in transgenic flies and mice. Given these findings, it was asked whether protein phosphorylation might play a role in SCA1 pathogenesis as well (Chen, 2003 and references therein).

Ataxin-1 is phosphorylated at serine 776 (S776) and substitution of this S776 residue with alanine (A776) greatly diminishes the ability of mutant ataxin-1 to aggregate. These results suggest that a serine at position 776 of ataxin-1 plays a role in SCA1 pathogenesis. Because this serine is normally phosphorylated, it was speculated that S776 phosphorylation might modify ataxin-1 neurotoxicity by regulating its protein-protein interactions. To test this hypothesis, attempts were made to identify proteins that interact with ataxin-1-S776 but not ataxin-1-A776, to identify the kinase that phosphorylates S776 in ataxin-1, and to examine the effects of these factors on SCA1 pathogenesis (Chen, 2003).

14-3-3 proteins bind to phosphopeptide motifs in a variety of cellular proteins to regulate diverse biological processes such as signal transduction, cell cycle control, and apoptosis. The function of 14-3-3 binding to ataxin-1 remains unclear, since the cellular function of ataxin-1 is not well understood. The present study does, however, shed light on the mechanism by which 14-3-3 renders ataxin-1 more toxic to neurons (Chen, 2003).

14-3-3 can protect its target protein from proteolysis and dephosphorylation. For example, 14-3-3 stabilizes the nicotinic receptor alpha4 subunit, elevating its steady-state protein levels. In this study, 14-3-3 was found to bind and stabilize ataxin-1 and promote its accumulation in both transfected cells and transgenic flies. The ataxin-1/14-3-3 interaction might directly stabilize a conformation of ataxin-1 that resists degradation or it might impede access to other ataxin-1-interacting proteins that would facilitate protein clearance. Note that 14-3-3 interacts not only with the expanded mutant ataxin-1 but also the unexpanded wild-type protein. It is therefore possible that 14-3-3 regulates ataxin-1's clearance under physiological conditions. This regulation becomes problematic upon CAG repeat expansion, since longer polyglutamine tracts enhance ataxin-1's interaction with 14-3-3, further stabilizing the mutant protein (Chen, 2003).

14-3-3 promotes the accumulation of ataxin-1 and also enhances aggregate formation. The finding that 14-3-3 aggravates SCA1 pathogenesis together with data showing the absence of nuclear inclusions and neuronal dysfuction in mice overexpressing ataxin-1[82Q]-A776 might resurrect the old question of whether nuclear inclusions cause SCA1 pathogenesis, but when ataxin-1 is expressed at physiologic levels, under control of endogenous promoter, neuronal dysfunction occurs in the absence of visible nuclear inclusions. The absence of nuclear inclusions in ataxin-1[82Q]-A776 mice most likely results from efficient clearance of the mutant protein due to its lack of interaction with 14-3-3 (Chen, 2003 and references therein).

To investigate the possibility that sequestration of 14-3-3 with mutant ataxin-1 interferes with the cellular functions of 14-3-3, the effects of 14-3-3 overexpression on the SCA1 phenotype were evaluated in vivo and no evidence was found that loss of 14-3-3 cellular functions plays a major role in SCA1 pathogenesis. If SCA1 pathology is caused simply by sequestration of 14-3-3 by ataxin-1, one would expect exogenous 14-3-3 to suppress the phenotype -- yet overexpression of Drosophila 14-3-3epsilon in SCA1 flies aggravates degeneration. In fact, immunolabeling of cerebellar sections from transgenic mice overexpressing ataxin-1[82Q]-S776 reveals that the distribution of 14-3-3 remains grossly unchanged without sequestration into nuclear inclusions; the colocalization of the two proteins to inclusions in cell cultures could be modulated by differences in other cellular proteins or the nature of inclusions (formed over hours in cells versus days and weeks in mice). It is likely that 14-3-3 and ataxin-1 preferentially form soluble protein complexes in vivo, whereby only a minor fraction of 14-3-3 is present in nuclear aggregates (Chen, 2003).

Consistent with the notion that polyglutamine expansion confers some toxic gain-of-function onto the host protein, larger polyglutamine expansions in ataxin-1 were found to have a higher affinity for 14-3-3. 14-3-3 is able to stabilize wild-type ataxin-1, however, and overexpression of 14-3-3 in SCA130Q flies enhances the neurotoxicity of ataxin-1[30Q]. These observations are consistent with the proposed role for 14-3-3 in stabilizing ataxin-1. The neurotoxic effects of mutant ataxin-1 are likely to be more pronounced in cells expressing high levels of 14-3-3. Many of the 14-3-3 isoforms are abundantly expressed in brain tissue, with different expression patterns for each cell-type; isoforms beta, gamma, and nu are particularly abundant in Purkinje cells, which suffer the most severe degeneration. High expression levels of certain 14-3-3 isoforms could contribute to the selective neuronal vulnerability characteristic of SCA1 (Chen, 2003).

Previous studies have found links between 14-3-3 and other human neurodegenerative disorders. The neurofibrillary tangles in AD are composed primarily of hyperphosphorylated tau proteins and contain 14-3-3, which modulates tau phosphorylation. Whether this interaction stabilizes tau remains to be determined. In PD, 14-3-3 is detectable in Lewy bodies, which accumulate alpha-synuclein. Interestingly, alpha-synuclein shares sequence homology with 14-3-3 and binds both to 14-3-3 and to some 14-3-3 binding partners. This finding suggests a possible role for either 14-3-3 or 14-3-3 binding proteins in alpha-synuclein-induced pathology. Moreover, 14-3-3 was recently found to associate with alpha-synuclein in a soluble protein complex that mediates dopamine-dependent neurotoxicity. It would be interesting to determine whether 14-3-3 plays any role in stabilizing alpha-synuclein. When searching for consensus 14-3-3 binding motifs in other polyglutamine-containing proteins, the RXXSXP motif in ataxin-2, alpha1A subunit voltage-gated calcium channel, ataxin-7, and atrophin-1 was found. Further studies are necessary to determine if there is an interaction between these proteins and 14-3-3 and whether such interactions affect the pathogenesis of SCA2, SCA6, SCA7, and DRPLA, respectively (Chen, 2003 and references therein).

Akt phosphorylates ataxin-1 and promotes its binding to 14-3-3, which in turn leads to ataxin-1 accumulation and neurodegeneration. Loss of Drosophila Akt1 function suppresses ataxin-1-induced neurodegeneration in a dosage-dependent manner. Akt is activated when recruited to the plasma membrane and phosphorylated at T308 and S473 by PDK1 and a yet-to-be identified 'S473-kinase'. That Drosophila PI3K overexpression aggravates the SCA1 phenotype more than Drosophila Akt1 overexpression is consistent with the important role of Drosophila PI3K in fully activating the signaling cascade. Because Drosophila PDK1 overexpresssion is insufficient to promote ataxin-1-induced degeneration, it is proposed that the 'S473-kinase' plays a pivotal role in activating dAkt to modulate ataxin-1's toxicity (Chen, 2003).

PI3K/Akt signaling is a major pathway mediating survival signals in neuronal cells in response to factors such as insulin-like growth factor 1. Therefore, PI3K/Akt signaling is generally considered neuroprotective, acting against stress conditions that occur during neurodegeneration. IGF-1 is known to activate PI3K/Akt signaling and to protect against neuronal death induced by amyloid-beta peptide, a toxic agent in AD. Likewise, Akt activation triggered by IGF-1 inhibits neuronal death induced by mutant huntingtin (Chen, 2003 and references therein).

It is therefore surprising to find that in SCA1 flies, PI3K/Akt promotes ataxin-1-induced neurodegeneration. It is possible that PI3K and Akt not only trigger survival signaling, as they do under other conditions, but also induce ataxin-1 phosphorylation and thus its interaction with 14-3-3. Whatever survival-promoting effect they exert may be counteracted by the greater neurotoxicity of mutant ataxin-1 accumulation in the cells. It is unlikely that Akt phosphorylation of ataxin-1 was programmed solely as a self-destruction pathway to antagonize cell survival signaling; it is more likely that the physiological activity of ataxin-1 is regulated in accordance with cell survival signaling. The differential effects of PI3K/Akt signaling upon each pathogenic protein exemplify the diversity of cellular responses in different human neurodegenerative diseases. Activation of PI3K/Akt might have beneficial effects for some neurodegenerative diseases but be deleterious for others. The availability of fly and mouse models for various neurodegenerative disorders will allow in vivo analysis of PI3K/Akt signaling as well as 14-3-3 interactions in various neurodegenerative disorders. Because 14-3-3 proteins are functionally interchangeable in different species, data obtained in model organisms are likely to prove clinically relevant (Chen, 2003).

In sum, a mechanism has been found by which PI3K/Akt signaling and 14-3-3 modulate ataxin-1 neurotoxicity. The identification of factors modulating SCA1 pathology may lead to therapeutic interventions such as interfering with ataxin-1/14-3-3 interaction using small peptides or reducing PI3K/Akt signaling by specific kinase inhibitors (Chen, 2003).

Insulin-PI3K/TOR pathway induces a HIF-dependent transcriptional response in Drosophila by promoting nuclear localization of HIF-alpha/Sima

The hypoxia-inducible factor (HIF) is a heterodimeric transcription factor composed of a constitutively expressed HIF-ß subunit and an oxygen-regulated HIF-alpha subunit. A hypoxia-inducible transcriptional response has been defined in Drosophila that is homologous to the mammalian HIF-dependent response. In Drosophila, the bHLH-PAS proteins Similar (Sima) and Tango (Tgo) are the functional homologues of the mammalian HIF-alpha and HIF-ß subunits, respectively. HIF-alpha/Sima is regulated by oxygen at several different levels that include protein stability and subcellular localization. Insulin can activate HIF-dependent transcription, both in Drosophila S2 cells and in living Drosophila embryos. Using a pharmacological approach as well as RNA interference, it has been determined that the effect of insulin on HIF-dependent transcriptional induction is mediated by PI3K-AKT and TOR pathways. Stimulation of the transcriptional response involves upregulation of Sima protein but not sima mRNA. Finally, the effect of the activation of the PI3K-AKT pathway on the subcellular localization of Sima protein was analyzed in vivo. Overexpression of dAKT and dPDK1 in normoxic embryos provokes a major increase in Sima nuclear localization, mimicking the effect of a hypoxic treatment. A similar increase in Sima nuclear localization was observed in dPTEN homozygous mutant embryos, confirming that activation of the PI3K-AKT pathway promotes nuclear accumulation of Sima protein. It is concluded that regulation of HIF-alpha/Sima by the PI3K-AKT-TOR pathway is a major conserved mode of regulation of the HIF-dependent transcriptional response in Drosophila (Dekanty, 2005).

Insulin stimulation or exposure to hypoxia can induce common target genes; such transcriptional response depends on the Drosophila HIFalpha and HIFß homologues Sima and Tango. Evidence is provided that insulin-stimulated HRE response is transduced by the PI3K-AKT pathway and, furthermore, that the effect depends on TOR and involves an increase in Sima protein levels, whereas mRNA levels are not affected. These results are in good agreement with the reported effect of the PI3K-AKT/TOR pathway on mammalian HIF, because in several cell lines activation of this pathway led to an increase in HIF protein levels or stabilization of the protein (Dekanty, 2005).

In addition, the subcellular localization of Sima depends on oxygen tension in a dose-dependent manner, and activation of the PI3K-AKT pathway also causes a major increase in Sima nuclear localization. This regulatory mechanism might represent another conserved aspect of HIF regulation, because one recent report suggests that HIF-alpha accumulates in the nucleus of retinal epithelial cells upon IGF-1alpha treatment. The molecular bases of HIFalpha nuclear accumulation upon hypoxia or PI3K activation are so far unclear. Nucleo-cytoplasmic localization of many transcription factors results from a steady-state equilibrium between nuclear import and nuclear export, and accumulation in one or the other compartment depends on the relative rate of import versus export. Whether HIF nuclear accumulation upon hypoxia or growth factor stimulation depends on regulated nuclear import or regulated nuclear export remains to be determined (Dekanty, 2005).

All major cellular features of oxygen-dependent regulation of HIF proteins are conserved in Drosophila and, thus, activation of the HRE response in Drosophila by the PI3K-AKT and TOR pathways extends even further the notion of a conserved HIF system in evolution. The functional significance of the regulation exerted by PI3K-AKT and TOR pathways over the HRE response was discussed in the context of cancer biology, because the loss of PTEN or the tuberous sclerosis complex 1 (TSC1) and TSC2 proteins is frequently associated with human tumors. What is the functional meaning of PI3K-AKT pathway regulation of the HRE response in normal cells? The PI3K-AKT and TOR pathways are regulated in part by growth factors and other endocrine signals and thus, endocrine control of the HRE response is conserved among animal species that have diverged 700 million years ago. It seems reasonable to postulate that the main physiological role of HRE induction by the PI3K-AKT pathway is the stimulation of glycolysis, but a function in the regulation of animal body size and growth control is another interesting possibility (Dekanty, 2005).

A cardinal function of the PI3K-AKT and TOR pathways throughout evolution is to regulate growth, and to determine the final size of developing organs and whole organisms. Genetic studies in Drosophila have shown that a reduction of the activity of the PI3K-AKT pathway results in flies with a reduced body size, bearing smaller cells. Likewise, a reduction in TOR signaling provokes growth decrease and, conversely, over-activation of TOR signaling due to loss-of-function of its negative regulators TSC1 and/or TSC2, leads to an increase in cell and body size. The effect of TOR on cell growth was reported to be mediated at least in part by S6K, a kinase that phosphorylates the ribosomal protein S6, leading to translational activation (Dekanty, 2005).

Besides its role in growth control, the insulin-PI3K-AKT pathway has been traditionally implicated in the regulation of circulating glucose levels and anabolic metabolism. It has been demonstrated that the cellular bases of glucose sensing and regulation of serum glucose are conserved between mammals and Drosophila, and it has been proposed that PI3K-AKT signaling in conjunction with the TOR pathway coordinates growth according to environmental conditions and the nutritional status of the organism. The mechanism involved in this coordination is still unclear. Oxygen tension is one environmental factor that has been shown to modulate growth in Drosophila, because hypoxic flies have a reduced body size (Frazier, 2001). A mechanistic explanation to this phenomenon has been provided by showing that overexpression of Sima protein causes a reduction in cell size in an autonomous manner (Centanin, 2005). Consistent with this, it has been shown that hypoxia provokes a reduction in Drosophila TOR pathway activity and that such reduction results from hyperactivation of the TSC1-TSC2 tumor suppressor complex. Similar results have been reported in mammalian cells, implying that TOR is regulated by hypoxia. Furthermore, hypoxia-dependent TSC1-TSC2 stimulation and growth inhibition are mediated by the product of a HIF/Sima-inducible gene called scylla in Drosophila and RTP801/REDD1 in mammals (Dekanty, 2005).

The results establish a direct link between pathways largely implicated in growth regulation (PI3K-AKT and TOR) and the hypoxia-responsive machinery (HIF/Sima). It is suggested that in hypoxia, HIF prolyl hydroxylase (Hph)/Fatiga activity is reduced, resulting in HIF/Sima stabilization and induction of an HRE response. One of the genes induced by hypoxia is scylla/RTP801/REDD1, which in turn activates TSC1-TSC2. Then, stimulation of the TSC complex provokes reduction of TOR activity and decreases S6K phosphorylation, resulting in growth inhibition. According to this model, PI3K-TOR activation of HIF-alpha/Sima might generate a negative feedback loop to limit or downregulate growth; in this scenario, low oxygen levels are expected to enhance Sima-dependent inhibition of growth (Dekanty, 2005).

It has been reported that mitochondrial dysfunction inhibits Hph/Fatiga activity, thereby triggering the transcriptional response to hypoxia, and also that it concomitantly provokes growth defects. It was proposed that Hph/Fatiga operates as an integration node between oxygen levels and growth regulation. The current results have shown that the effect of Hph/Fatiga on growth regulation is conveyed at least in part by Sima. Further studies will reveal whether Hph/Fatiga also plays Sima-independent roles in cell and organ size determination (Dekanty, 2005).

The protein phosphatase PP2A-B' subunit Widerborst is a negative regulator of cytoplasmic activated Akt and lipid metabolism in Drosophila

Inappropriate regulation of the PI3-kinase/PTEN/Akt kinase-signalling cassette, a key downstream target of insulin/insulin-like growth factor signalling (IIS), is associated with several major human diseases such as diabetes, obesity and cancer. In Drosophila, studies have recently revealed that different subcellular pools of activated, phosphorylated Akt can modulate different IIS-dependent processes. For example, a specific pool of activated Akt within the cytoplasm alters aspects of lipid metabolism, a process that is misregulated in both obesity and diabetes. However, it remains unclear how this pool is regulated. The protein phosphatase PP2A-B' regulatory subunit Widerborst (Wdb), which coimmunoprecipitates with Akt in vivo, selectively modulates levels of activated Akt in the cytoplasm. It alters lipid droplet size and expression of the lipid storage perilipin-like protein LSD2 in the Drosophila ovary, but not in epithelial cells of the eye imaginal discs. It is concluded that isoforms of PP2A-B' can act as subcellular-compartment-specific regulators of PI3-kinase/PTEN/Akt kinase signalling and IIS, potentially providing new targets for modulating individual subcellular pools of activated Akt in insulin-linked disease (Vereshchagina, 2008).

The signalling cassette involving Class I phosphatidylinositol 3-kinase (PI3K), phosphatase and tensin homologue on chromosome 10 (PTEN) and Akt (also known as protein kinase B or PKB) is part of a major intracellular kinase cascade that regulates multiple cellular functions including metabolism, growth, proliferation and survival. It responds to a variety of stimuli, such as insulin, other growth factors including PDGF and FGF, and attachment to the extracellular matrix. Upon activation, PI3K catalyses the formation of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] from phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. PtdIns(3,4,5)P3 is a lipid second messenger, which in turn recruits the PH-domain-containing Akt protein kinase from the cytosol to the plasma membrane. Here it is activated through phosphorylation at Thr308 by 3-phosphoinositide-dependent protein kinase 1 (PDK1) and at Ser473 (or Ser505 in the unique Drosophila Akt kinase, Akt1) by PDK2, which is thought to be the rictor-mTOR complex. Once activated, Akt subsequently phosphorylates multiple targets, leading to its numerous downstream effects (Vereshchagina, 2008).

Misregulation of Akt and its cellular targets is linked to several major human diseases. For example, cellular insulin resistance is associated with reduced signalling by the PI3K/PTEN/Akt cassette and is an important defect in individuals suffering from Type 2 diabetes. By contrast, hyperactivation of this cassette, most notably through loss-of-function mutations in the tumour suppressor PTEN, which converts PtdIns(3,4,5)P3 back to PtdIns(4,5)P2, is strongly associated with many forms of human cancer (Vereshchagina, 2008 and references therein).

Molecular genetic studies in Drosophila have resulted in several fundamental insights into the regulation and functions of the PI3K/PTEN/Akt-signalling cassette. Not only has this work highlighted the central importance of nutrient-regulated insulin/insulin-like growth factor signalling (IIS) in controlling the activity of this cassette and cell growth, but it has also revealed a critical downstream link with the nutrient-sensitive mTOR-signalling cascade, which regulates several cellular processes including protein translation and autophagy. Furthermore, studies in invertebrates have indicated roles for PI3K/PTEN/Akt and mTOR in ageing, cell polarity and neurodegeneration, functions that all appear to be conserved in mammals and which might involve a combination of cellular and metabolic defects (Vereshchagina, 2008).

If the role of PI3K/PTEN/Akt in insulin-linked diseases is to be fully understood, it is essential to determine how this single signalling cassette regulates so many different cellular functions. One important part of the explanation is presumably the existence of cell-type-specific downstream-signalling targets that perform different roles. However, recent work, much of it again initiated in flies, has indicated that Akt activity can also be differentially regulated in specific subcellular domains and that these subcellular pools of activated Akt can control different processes. For example, precise regulation of Akt activity at the apical membrane of epithelial cells by localised PTEN is required for normal apical morphology in higher eukaryotes. By contrast, cytoplasmic activated Akt appears to be required for transcription of specific IIS target genes and regulation of lipid metabolism and droplet size in nurse cells of the Drosophila female germ line (Vereshchagina, 2006). These observations have highlighted the importance of finding the molecules that regulate different pools of activated Akt in vivo, because their modulation might alter specific functions of IIS in health and disease more selectively (Vereshchagina, 2008).

In a screen for novel phosphatase regulators of IIS, Widerborst (Wdb), one of the B' regulatory subunits of the protein phosphatase PP2A, was identified as a negative regulator of the PI3K/PTEN/Akt-signalling cassette. Although wdb is essential for cell viability in some tissues, wdb mutant cells in the germ line and follicular epithelium of the ovary are viable and display phenotypes that are similar to those seen in PTEN mutant ovaries. This study shows that Wdb and Drosophila Akt1 physically interact in the ovary, and that within this tissue, Wdb regulates the subcellular pool of activated Akt1 in the cytoplasm. This study therefore highlights an important new function for PP2A-B' subunits in selectively modulating certain IIS-dependent processes by controlling signalling in a specific subcompartment of the cell (Vereshchagina, 2008).

Several lines of evidence confirm that Wdb controls IIS activity and Akt1 phosphorylation state. First, when overexpressed, wdb genetically modifies phenotypes produced by altered IIS signalling, rescuing a lethal PTEN mutant combination and modifying the effects of FOXO in the eye. Second, loss-of-function wdb mutations produce very similar phenotypes to PTEN mutations in nurse cells, elevating levels of cytoplasmic pAkt1 and LSD2 [a Perilipin/ADRP homologue that regulates lipid metabolism, and inducing an abnormal accumulation of lipid droplets. Third, although wdb mutations do not independently appear to have strong effects on growth, they do suppress growth phenotypes produced by reduced Akt1 signalling both in mutant follicle cells homozygous for the Akt11 allele and in animals carrying a hypomorphic viable combination of Akt1 alleles. Genetic interactions with the PP2A catalytic subunit Mts in the eye indicate that these effects are dependent on the PP2A regulatory activity of Wdb (Vereshchagina, 2008).

Coimmunoprecipitation experiments revealed that Akt1 and Wdb form a complex in ovaries, the tissue in which the most obvious effects of wdb on pAkt1 levels are seen. The data suggest that one isoform of Wdb affects IIS within a complex containing Akt1, presumably by directly modulating the phosphorylation state of this molecule. This regulatory interaction appears to be evolutionarily conserved, because several studies in mammalian cell culture have shown that a PP2A-type activity controls Akt phosphorylation at Ser473, the equivalent position to Ser505 in Drosophila Akt1. PP2A-B' activity has been implicated in this process. Furthermore, mammalian PP2A can dephosphorylate Akt in vitro. The phosphorylation state of Thr308 might also be affected by PP2A. However, current tools do not allow determination of the phosphorylation state of Thr342 (the equivalent position to Thr308 in mammalian Akt) in wdb mutant cells in ovaries. Nevertheless, this study adds to the current understanding of the effects of PP2A on Akt by showing for the first time that at least one PP2A-B' isoform can act as a pool-specific suppressor of activated Akt. It is thought that that this property is likely to be shared by some mammalian PP2A-B' isoforms (Vereshchagina, 2008).

Unlike several other previously characterised components of the IIS cascade, the effects of wdb mutations on IIS appear to be tissue specific. Although pAkt1 levels are strongly upregulated in wdb mutant nurse cells and follicle cells, they appear unaffected in clones within the eye. PP2A is a broad-specificity protein phosphatase, which is selectively targeted to specific signalling molecules by regulatory subunits such as Wdb. Wdb has already been shown to be involved in several signalling events, including those regulating apoptosis and the Hedgehog (Hh) pathway, pathways that might be implicated in the wdb mutant phenotype observed in the eye imaginal disc (Vereshchagina, 2008).

How can Wdb have such a central IIS-regulatory role in the ovary, but show no detectable effect on this pathway in the developing eye? It seems unlikely that wdb mutant cells in the eye die too rapidly to observe changes in Akt1 phosphorylation, because wdb clones are seen in posterior positions within eye imaginal discs, which must have formed many hours previously. The IIS cascade is active in this tissue, because mutations altering IIS produce significant effects on growth in the eye disc. However, unlike in nurse cells, activation of IIS in the developing eye primarily leads to cell surface accumulation of pAkt1, at least in pupae. Surface-localised activated Akt1 may normally be sufficient to promote eye growth, since a myristoylated membrane-anchored form of Akt1 dominantly induces overgrowth in this and other tissues. One possible explanation for these data is therefore that cytoplasmic pAkt1 levels in the eye are restricted by other unknown molecules in addition to Wdb in this tissue, so loss of wdb here has little effect, whereas increased expression can still modify the FOXO phenotype (Vereshchagina, 2008).

In this context, at least two other phosphatases might be involved in Akt1 regulation. First, there is a second isoform of PP2A-B' in flies [called PP2A-B', CG7913 or Well-rounded (Wrd); that is most closely related to mammalian PP2A-B'γ isoforms. Simian virus 40 small t antigen acts as a specific inhibitor of mammalian PP2A-B'γ, stimulating phosphorylation of Akt and other targets, and thereby promoting growth. Reduced PP2A-B'γ activity has also been linked to the establishment and progression of melanomas (Vereshchagina, 2008).

Surprisingly, a recent report suggests Wrd is nonessential. Unless it acts redundantly with Wdb, it cannot therefore play a significant role in growth regulation). Analysis of the PP2A catalytic subunit Mts, using a dominant-negative construct, indicates that this enzyme enhances the effects of FOXO and is important in normal growth regulation in the eye, perhaps consistent with the idea that the two PP2A-B' isoforms do act redundantly. Alternatively, Mts may perform some of its growth regulatory functions independently of PP2A-B' (Vereshchagina, 2008 and references therein).

A second candidate negative regulator of Akt is the novel phosphatase PHLPP, which directly dephosphorylates human Akt at Ser473 and Drosophila Akt1 at Ser505 in cell culture, a function that may be disrupted in some tumours. Drosophila PHLPP could therefore control pAkt1 accumulation at the cell surface and perhaps reduce the amount of pAkt1 that can diffuse into the cytoplasm in tissues such as the eye. Since loss of wdb in either follicle cells or nurse cells is sufficient to elevate levels of cytoplasmic pAkt1, PHLPP presumably does not play such an important role in these cell types (microarray data suggest that PHLLP is not expressed at detectable levels in the adult ovary) (Vereshchagina, 2008).

Interestingly, the data in the ovary suggest further variable tiers of pAkt1 control. In nurse cells, loss of PTEN leads to accumulation of pAkt1 and LSD2 in the cytoplasm, but most PTEN mutant follicle cell clones do not show these phenotypes, presumably because other pAkt1 regulators such as Wdb play a more dominant role in these cells. No good explanation is available for how genetically identical clones can show such phenotypic variability. There is no obvious correlation with clone size or position in the small minority of PTEN-mutant follicular clones where pAkt1 and LSD2 upregulation is observed (Vereshchagina, 2008).

Because perilipin, the mammalian LSD2 orthologue, is thought to be regulated via insulin-dependent transcriptional and post-translational mechanisms, it is proposed that the increased LSD2 expression seen in PTEN mutant nurse cell clones results from similar effects of IIS on this molecule in flies. An alternative explanation is that increased IIS promotes excess triacylglyceride (TAG) synthesis and that LSD2 is only indirectly upregulated to permit proper packaging of these triacylglycerides into lipid droplets. Analysis of wdb mutant follicle cell clones does not support this latter model, since these clones strongly upregulate LSD2 expression, but do not show obvious changes in lipid droplet accumulation (Vereshchagina, 2008).

When wdb is overexpressed in the differentiating eye, the external structure of the eye becomes more disorganised and there is a slight reduction in overall eye size. Since this effect is not noticeably suppressed by co-overexpressing Akt1, it seems unlikely to be caused by reduced IIS. Unlike PTEN mutant follicle cells, wdb mutant follicle cells are not noticeably larger than their wild-type neighbours. Furthermore, although low level constitutive expression of Wdb in a pupal-lethal PTEN mutant background can rescue these flies to viability, the rescue may be explained by altered metabolism, because the rescued flies are still larger than normal. All these observations are consistent with the model that Wdb modulates cytoplasmic pAkt1 and has less of an effect on cell surface pAkt1, which is thought to be the primary regulator of normal growth. Wdb shows a relatively strong genetic interaction with the IIS-regulated transcription factor FOXO and this is completely suppressed by Akt1, raising the possibility that low levels of pAkt1 in the cytoplasm may play an important part in controlling FOXO activity (Vereshchagina, 2008).

Although wdb does not appear to modulate growth significantly under normal IIS-signalling conditions, mutations in wdb do enhance growth when Akt1 activity is reduced. Viable Akt1 mutant animals are larger in the presence of a heterozygous wdb mutation, while the Akt11 recessive growth phenotype in follicle cells is strongly suppressed by wdb. Interestingly, it has been reported that mutations in foxo have no effect on growth in otherwise normal animals, but that when IIS is reduced in chico mutants, which produce small adults, this phenotype is partially suppressed by loss of foxo function. The current data are consistent with this result, and may indicate that growth regulation in chico flies relies more on cytoplasmic pAkt1 and its effects on downstream targets like FOXO than it does in normal flies (Vereshchagina, 2008).

In conclusion, the identification of a PP2A-B' subunit as a novel cell-type-specific regulator of IIS within a specific subcellular compartment highlights the importance of studying the subcellular control of this signalling pathway in multiple cell types in vivo. Akt activation also promotes lipid synthesis and droplet formation in many mammalian cell types. This is likely to involve similar regulatory control mechanisms for cytoplasmic pAkt to those uncovered in flies. This work therefore raises new issues concerning the underlying causes of IIS-associated disease. For example, excess accumulation of lipid and obesity could be linked to selective changes in cytoplasmic pAkt control and might therefore be modulated by specific PP2A-B' subunits. Developing a better understanding of this form of regulation could therefore suggest new strategies for disease-specific treatments of IIS-linked disorders in the future (Vereshchagina, 2008).

FOXO-independent suppression of programmed cell death by the PI3K/Akt signaling pathway in Drosophila

Signaling through the PI3K/Akt/FOXO pathway plays an important role in vertebrates in protecting cells from programmed cell death. PI3K and Akt have been similarly shown to be involved in survival signaling in Drosophila. However, it is not known whether PI3K and Akt execute this function by controlling a pro-apoptotic activity of Drosophila FOXO. This study shows that elevated signaling through PI3K and Akt can prevent developmentally controlled death in the salivary glands of the fruit fly. Drosophila FOXO is not required for normal salivary gland death and the rescue of salivary gland death by PI3K occurs independent of FOXO. These results give support to the notion that FOXOs have acquired pro-apoptotic functions after separation of the vertebrate and invertebrate lineages (Liu, 2006).

To determine whether elevated signaling through PI3K can rescue normal salivary gland death, the catalytic subunit of PI3K, Dp110, was expressed in late-prepupal glands using P{UAS-Dp110} and a heat-shock GAL4 driver. Most of the pupae expressing the subunit still possessed intact salivary glands 20 h APF, i.e., ~6 h after the glands are normally destroyed. This result shows that a high level of PI3K activity can overcome the stimuli that normally lead to the destruction of the salivary glands in early pupae. Moreover, it suggests that the PI3K pathway is normally inactive or, at least, strongly downregulated in dying salivary glands (Liu, 2006).

If the effect of PI3K is mediated by the canonical PI3K/Akt pathway, an elevated activity of Akt should have the same or a similar effect as an elevated PI3K activity. To test this prediction, both wild-type Akt and a constitutively active form of Drosophila Akt, Daktmyr, were expressed in late-prepupal salivary glands. Daktmyr carries a myristoylated amino terminus that targets the protein to the cell membrane. Under normal conditions, Akt is recruited to the membrane by the PI3K product PIP3 and subsequently activated by phosphorylation through PDK1. The expression of both UAS-Dakt myr and UAS-akt was driven by heat-shock GAL4. The constitutively active Daktmyr led to a complete rescue of salivary gland death, whereas unmodified Akt had no effect. It is concluded that only membrane-associated Akt can rescue salivary gland death. This is consistent with the normal mechanism of Akt activation that requires PI3K-induced recruitment of Akt to the cell membrane. The inhibition of death by active Akt, but not by inactive Akt, confirms that PI3K activity is limited in late-prepupal salivary glands and underscores the specificity of the observed effect (Liu, 2006).

In summary, the results show that the survival function of PI3K/Akt does not depend on the inactivation of dFOXO and that dFOXO has no apparent role in the activation of PCD in the salivary glands. Moreover, they indicate that an intact PI3K/Akt signaling pathway is not required for salivary gland survival. However, downregulation of the pathway may be required for salivary gland death, because elevated signaling through the pathway can rescue the salivary glands. Importantly, this study on the role of PI3K/Akt/dFOXO signaling in salivary gland death did not reveal a pro-apoptotic role of dFOXO, further strengthening the assumption that the functions of FOXOs in apoptosis are a late evolutionary acquisition in the vertebrate lineage (Liu, 2006).

Reduction of Lobe leads to TORC1 hypoactivation that induces ectopic Jak/STAT signaling to impair Drosophila eye development

The TOR and Jak/STAT signal pathways are highly conserved from Drosophila to mammals, but it is unclear whether they interact during development. The proline-rich Akt substrate of 40 kDa (PRAS40) mediates the TOR signal pathway through regulation of TORC1 activity, but its functions in TOR complex 1 (TORC1, a rapamycin-sensitive form of Tor in mice that consists of mTOR, raptor, and mLST8) proved in cultured cells are controversial. The Drosophila gene Lobe (L) encodes the PRAS40 ortholog required for eye cell survival. L mutants exhibit apoptosis and eye-reduction phenotypes. It is unknown whether L regulates eye development via regulation of TORC1 activity. This study found that reducing the L level, by hypomorphic L mutation or heterozygosity of the null L mutation, resulted in ectopic expression of unpaired (upd), which is known to act through the Jak/STAT signal pathway to promote proliferation during eye development. Unexpectedly, when L was reduced, decreasing Jak/STAT restored the eye size, whereas increasing Jak/STAT prevented eye formation. Ectopic Jak/STAT signaling and apoptosis are mutually dependent in L mutants, indicating that L reduction makes Jak/STAT signaling harmful to eye development. In addition, genetic data suggest that TORC1 signaling is downregulated upon L reduction, supporting the idea that L regulates eye development through regulation of TORC1 activity. Similar to L reduction, decreasing TORC1 signaling by dTOR overexpression results in ectopic upd expression and apoptosis. A novel finding from these data is that dysregulated TORC1 signaling regulates the expression of upd and the function of the Jak/STAT signal pathway in Drosophila eye development (Wang, 2009).

The target of rapamycin (TOR) and Jak/STAT signal pathways are highly conserved in animals and important in many developmental processes. Dysregulation of these pathways can lead to cancer formation. This study presents data showing that TOR regulates the function of Jak/STAT signaling during Drosophila eye development (Wang, 2009).

The gene unpaired (upd) encodes a ligand that activates Drosophila Jak/STAT signaling. It is expressed in the posterior margin of the dorsal/ventral (D/V) boundary, the posterior center (PC), in the larval eye imaginal disc at second and early third instar stages. Notch at the D/V boundary activates the transcription of eye gone (eyg), which activates upd expression at the PC. Expression of upd is also regulated by Hedgehog (Hh) signaling. The cells of Drosophila compound eyes are derived from the eye-antennal disc, which develops from ectoderm of the embryo and grows inside the larva. These cells proliferate rapidly during the first and second instar stage. In early third instar larvae, morphogenetic furrow (MF) that arise at the posterior margin progresses in a wave-like manner toward the anterior margin of the eye disc. Jak/STAT signaling is known to promote proliferation during eye development, and is required for MF initiation; a loss of Jak/STAT function results in reduced eyes. Therefore, Jak/STAT signaling is regulated by Notch/Eyg and the Hh signaling pathways, and plays positive roles in eye development (Wang, 2009).

TOR signaling is one of the downstream branches of insulin signal pathway. Insulin and insulin-like growth factor elicit a signal cascade involving phosphatidyl-inositol 3-kinase (PI3K) that stimulates PDK-mediated Akt phosphorylation. Phosphorylated Akt can activate TOR, which nucleates the TOR complex 1 (TORC1), allowing it to phosphorylate the downstream targets, the translational repressor eukaryotic initiation factor (4EBP) and the ribosomal protein S6 kinase (S6k). Phosphorylation of 4EBP and S6K promotes CAP-dependent translation and thereby increases protein synthesis. In addition, activation of TOR can also promote ribosome biogenesis via Myc. Loss of the Drosophila TOR (dTOR) function reduces eye size, indicating that TOR signaling is required for eye development (Wang, 2009).

PRAS40 mediates the insulin signal pathway from Akt to TORC1. Upon insulin stimulation, activated Akt phosphorylates PRAS40 and causes it to dissociate from TORC1, allowing TORC1 signaling to proceed. Thus, PRAS40 can apparently act as an inhibitor of TORC1. However, it has been reported that PRAS40 is required for TORC1 activity, and thus the interactions of PRAS40 with TORC1, based on studies in cultured cells are controversial. The effect of PRAS40 on TORC1 signaling in vivo is still unclear (Wang, 2009).

The Drosophila Lobe (L) protein shares high sequence conservation with PRAS40. L mutants have reduced adult eyes and exhibit ectopic apoptosis during eye development, indicating that L is required for eye development. But whether it regulates eye development via regulation of TORC1 activity is unknown (Wang, 2009 and references therein).

This study identified a new L allele, Lfee. Quantitative RT-PCR and genetic analysis revealed that Lfee is a hypomorphic allele. The eye defect was mediated by ectopic Jak/STAT signaling and cell apoptosis. In L mutants, the ectopic Jak/STAT signaling had a negative effect on eye development, but not a positive one as previously reported. It was also found that TORC1 signaling was hypoactivated in L mutants, suggesting that, like PRAS40, L is required for TORC1 activity. This study suggests that hypoactivated TORC1 signaling in L mutants result in ectopic Jak/STAT signaling and apoptosis, impairing eye development (Wang, 2009).

The spontaneous mutant fly, freaky eye (fee), is homozygously viable and has abnormal adult eyes. The eyes of most fee flies are smaller than those of wild type flies because of a nick at the anterior border of the eye. At the nicked region, extra hairs and/or rod-like tissues are usually present. Overgrowth of eye tissue occasionally occurs, resulting in eye enlargement. The eyes of fee flies were categorized into six classes depending on their size relative to the eyes of the wild type. The various eye-reduction phenotypes of fee flies were similar to those of L mutants. For example, the Lsi heterozygote exhibits slightly reduced eyes that are nicked near the anterior D/V boundary, similar to the major fee phenotype. In the Lsi homozygote, the ventral eye is absent, which is also reminiscent of the fee phenotype (Wang, 2009).

Whether fee is a mutant of L was investigated; the trans-heterozygotes for fee and the null mutant Lrev6-3 had smaller eyes than fee flies. In addition, fee could to be recombined with Lrev6-3, suggesting that fee is allelic to L. Quantitative RT-PCR showed that the L mRNA levels were highly reduced in fee flies, suggesting that fee is a L hypomorphic mutant; therefore these were designated Lfee (Wang, 2009).

This study shows that reduction of L phenocopies overexpression of dTOR. Overexpression of dTOR has been reported to produce phenotypes similar to that of loss of dTOR, because excess dTOR may titrate cofactors and thereby decrease TOR activity. This suggests that TOR signaling is downregulated by L reduction. Consistent with this, genetic analysis of L mutants and TOR signal pathway component suggest TORC1 hypoactivity in L mutants. PRAS40 has been proposed to function in the assembly of TORC1. It is possible that, in similar way to dTOR overexpression, reducing L impairs TORC1 assembly, thus decreasing TORC1 signaling. Reduction of L may disrupt eye development through downregulation of TORC1 signaling, supporting the idea that PRAS40 is required for TORC1 activity (Wang, 2009).

Drosophila eye development requires the TOR and Jak/STAT signal pathways, but it is not know whether an interaction between these two signal pathways occurs. Endogenous upd expression is present in the posterior center (PC) of the eye disc, but not in the interior eye disc. This study demonstrated that L reduction can induce ectopic upd expression in the interior eye disc, indicating that L is a negative regulator for upd expression. The data show that L reduction-mediated eye disruption is due to hypoactivation of TORC1 signaling, suggesting that hypoactivity of TORC1 is responsible for inducing upd expression (Wang, 2009).

Ectopic upd expression is induced by reduction of L (Lfee and Lrev/+), but not by its complete loss (Lrev homozygous clones), suggesting that different L levels may cause distinct effects. As PRAS40 acts to transmit the Akt signal to TORC1, complete loss, but not reduction, of L could result in an uncoupling between Akt and TORC1. This would release the Akt-mediated inhibition of TORC1, resulting in increased TORC1 activity. Thus, complete loss of L or PRAS40 may increase TORC1 activity. It is possible that the opposite functions of PRAS40 reported in cultured cells could be due to different PRAS40 levels remaining after knockdown. Whether complete loss of L function inhibits or promotes TORC1 signaling in Drosophila eyes remains to be investigated (Wang, 2009).

Mosaic analysis data showed that dTOR homozygote clones did not induce ectopic upd expression, suggesting that complete loss of dTOR function has a different effect from that of L reduction. Overexpression of dMyc can completely restore the eye size in the Lfee flies, but only partially represses the eye defect of dTOR overexpression. These data support the idea that L reduction may not equate to loss of dTOR. It was reasoned that as TOR is involved in TORC1 and TORC2, its loss should eliminate the functions of both TORC1 and TORC2. Because L participates only in TORC1 signaling, reduction of L would affect TORC1 signaling only. The regulation of TORC1 and TORC2 signaling by L needs further investigation (Wang, 2009).

It was found that suppressing apoptosis can decrease ectopic upd expression upon L reduction, suggesting that apoptosis is a cause of ectopic upd expression. It has been reported that apoptosis can activate ectopic upd expression and Jak/STAT signaling via Notch signaling in apoptosis-induced compensatory proliferation. However, ectopic upd expression on L reduction is not likely to be mediated by Notch activity, and no ectopic proliferation occurs. Thus, apoptosis due to L reduction is different from apoptosis-induced compensatory proliferation. Further, TOR hypoactivation may trigger ectopic upd expression independent of apoptosis; suppression of apoptosis did not eliminate all ectopic upd expression. Further investigation of how hypoactivated TORC1 regulates upd expression is needed (Wang, 2009).

The Drosophila Upd acts through Jak/STAT signaling to promote proliferation during eye development. However, this study found that on L reduction, decreasing Jak/STAT signaling could restore the eye defect, whereas increasing the upd expression level could completely abolish eye development. Thus, an unexpected finding was that ectopic Jak/STAT signaling in L mutants is harmful to eye development (Wang, 2009).

The fact that decreasing Jak/STAT signaling can reduce apoptosis in L mutants indicates that the induction of ectopic Jak/STAT signaling is required for apoptosis. It was reasoned that the apoptosis-promoting ability of Jak/STAT is possibly due to its repression of Serrate (Ser) expression. Ser expression is inhibited by L mutation, and loss of Ser function during eye development causes apoptosis. The current data showed that heterozygosity for Ser can reduce eye size in Lfee heterozygotes, but not in the wild type, suggesting that decreased Ser expression may play a role in eye reduction. Whether Ser repression mediates the apoptosis remains to be investigated. In addition, because inhibition of apoptosis does not strongly restore the L eye defect, but decreasing Jak/STAT activity fully restores it (comparing ey > p35 and Stat92Ets), there is the possibility that the ectopic Jak/STAT activity affects eye development via an apoptosis-independent mechanism. Thus, a novel finding from the data is that Jak/STAT signaling can negatively regulate eye development (Wang, 2009).

An important issue is the control over the positive and negative roles of Jak/STAT signaling during eye development. Overexpression of upd driven by ey-GAL4 in the wild type produces adult with enlarged eyes, but it eliminates eye formation in L mutants. Because L reduction exhibits hypoactivation of TORC1 signaling, it is speculated that TORC1 signaling plays a role in controlling the balance between the opposing functions of Jak/STAT signaling (Wang, 2009).

In summary, reduction of the Drosophila PRAS40 L results in hypoactivation of TORC1 signaling. This leads to apoptosis and ectopic Jak/STAT activation, both of contribute to disruption of eye development. The data indicate that TORC1 signaling is able to regulate the expression and functions of the Jak/STAT signal pathway during eye development. Further studies using L mutants may uncover the mechanisms by which L regulates TORC1 signaling, and how TOR controls the Jak/STAT signal pathways. Also noteworthy is the report that decreasing PRAS40 can increase apoptosis of tumor cells, and it is therefore of interest to investigate whether PRAS40 and TORC1 can regulate the Jak/STAT signal pathway in tumors (Wang, 2009).

AKT and TOR signaling set the pace of the circadian pacemaker

The circadian clock coordinates cellular and organismal energy metabolism. The importance of this circadian timing system is underscored by findings that defects in the clock cause deregulation of metabolic physiology and result in metabolic disorders. On the other hand, metabolism also influences the circadian clock, such that circadian gene expression in peripheral tissues is affected in mammalian models of obesity and diabetes. However, to date there is little to no information on the effect of metabolic genes on the central brain pacemaker which drives behavioral rhythms. This study found that the AKT and TOR-S6K pathways, which are major regulators of nutrient metabolism, cell growth, and senescence, impact the brain circadian clock that drives behavioral rhythms in Drosophila. Elevated AKT or TOR activity lengthens circadian period, whereas reduced AKT signaling shortens it. Effects of TOR-S6K appear to be mediated by SGG/GSK3beta, a known kinase involved in clock regulation. Like SGG, TOR signaling affects the timing of nuclear accumulation of the circadian clock protein Timeless. Given that activities of AKT and TOR pathways are affected by nutrient/energy levels and endocrine signaling, these data suggest that metabolic disorders caused by nutrient and energy imbalance are associated with altered rest:activity behavior (Zheng, 2010).

There are several possible mechanisms by which nutrient and energy metabolism could affect peripheral clocks. Local physiological factors dependent on metabolic activity could influence the expression of core clock components and of nuclear receptors that regulate clock gene expression. Indeed, cellular redox state, AMPK activity, NAD+ levels, and SIRT1 activities appear to feed into the circadian clock in peripheral tissues such as the liver. AMPK, which acts upstream of TSC in mammals, directly phosphorylates Cryptochrome in peripheral tissues. However, prior to this work, there was no known mechanism for the modulation of the central pacemaker by nutrient-sensing pathways. This study identifies such a mechanism by demonstrating that metabolic genes such as AKT and TOR-S6K act in the central pacemaker cells in the brain. The lengthened circadian period caused by high-fat diet in mammals is likely mediated by these molecules. This conclusion is further supported by a recent cell-culture-based genome-wide RNAi study that implicated the PI3K-TOR pathway in the regulation of circadian period. In addition, another ribosomal S6 kinase (S6KII) was found to influence the circadian clock through its interaction with casein kinase 2β. Importantly, daily fasting:feeding cycles driven by the central clock regulate circadian gene transcription in the liver, whereas clock function in the liver contributes to energy homeostasis. It is speculated that metabolic stress or energy imbalance affects AKT and TOR-S6K signaling, resulting in general circadian disruption, which in turn exacerbates metabolic deregulation and, consequently, facilitates the development of metabolic syndromes prevalent in modern society (Zheng, 2010).

The SCFSlimb E3 ligase complex regulates asymmetric division to inhibit neuroblast overgrowth

Drosophila larval brain neuroblasts divide asymmetrically to balance between self-renewal and differentiation. This study demonstrates that the SCFSlimb E3 ubiquitin ligase complex, which is composed of Cul1, SkpA, Roc1a and the F-box protein Supernumerary limbs (Slimb), inhibits ectopic neuroblast formation and regulates asymmetric division of neuroblasts. Hyperactivation of Akt leads to similar neuroblast overgrowth and defects in asymmetric division. Slimb associates with Akt in a protein complex, and SCFSlimb acts through SAK and Akt to inhibit neuroblast overgrowth. Moreover, Beta-transducin repeat containing, the human ortholog of Slimb, is frequently deleted in highly aggressive gliomas, suggesting a conserved tumor suppressor-like function (Li, 2014).

It has been shown that Akt associates with Slimb and can be ubiquitinated by Slimb. It was determined which domain of Akt is important for its association with Slimb. The central region of Akt, which contains its protein kinase domain (T2), but not the N-terminus (T1, containing a pleckstrin homology domain) or the C-terminus (T3, containing an AGC kinase domain), interacted with Slimb in S2 cells by co-immunoprecipitation. Taken together, these biochemical and genetic data suggest that the SCFSlimb complex inhibits ectopic NB formation in part through an association with Akt (Li, 2014).

Beta-transducin repeat containing (BTRC/β-TrCP; human homologue of Slimb) showed a significant loss in 72.5% of glioma patients. Its copy number was an independent predictor of prognosis in a multivariate analysis. BTRC copy loss was observed in patients with glioblastoma (82%) and oligodendroglioma (68%). It was also observed in patients with mesenchymal and proliferative (85 and 88%, respectively), which are frequently associated with activated AKT signaling, a central oncogenic pathway regulating glioblastoma (GBM) growth and survival. The BTRC functional module was derived by mining mRNA expression databases and mapped to 544 mRNA transcripts. This module was able to stratify patients into two subgroups and was significantly associated with survival. A multivariate Cox Regression model confirmed that the BTRC functional subgroup was independently associated with survival. Furthermore, BTRC copy changes have a significant inverse correlation with the gene expression of AKT2 and PIK3CD (Li, 2014).

β-TRCP, the mammalian Slimb homolog, is speculated to play a greater role as an oncogene than as a tumor suppressor. This study has demonstrated that the SCFSlimb complex plays an important role in NB self-renewal and asymmetric division. It acts at least in part through the oncogenic protein Akt, a key player that regulates NB self-renewal and asymmetric division. The activation of Akt in NBs appears to be independent of Phosphatase and tensin homologue (PTEN), a negative regulator of PI3K/Akt signaling, because no NB overgrowth were observed in two loss-of-function alleles, PTENC494 and PTEN1. A recent report suggested a role for Target of rapamycin (TOR) signaling in restraining tumorigenesis in larval brains. PI3K/TOR also interacts genetically with Pins in the suppression of tumor growth in larval brains, suggesting the complex role of PI3K/Akt and the interconnected TOR pathway in NB homeostasis (Li, 2014).

Drosophila Tribbles antagonizes insulin signaling-mediated growth and metabolism via interactions with Akt kinase

Drosophila Tribbles (Trbl) is the founding member of the Trib family of kinase-like docking proteins that modulate cell signaling during proliferation, migration and growth. In a wing misexpression screen for Trbl interacting proteins, the Ser/Thr protein kinase Akt1. Given the central role of Akt1 in insulin signaling, the function of Trbl was tested in larval fat body, a tissue where rapid increases in size are exquisitely sensitive to insulin/insulin-like growth factor levels. Consistent with a role in antagonizing insulin-mediated growth, trbl RNAi knockdown in the fat body increased cell size, advanced the timing of pupation and increased levels of circulating triglyceride. Complementarily, overexpression of Trbl reduced fat body cell size, decreased overall larval size, delayed maturation and lowered levels of triglycerides, while circulating glucose levels increased. The conserved Trbl kinase domain is required for function in vivo and for interaction with Akt in a yeast two-hybrid assay. Consistent with direct regulation of Akt, overexpression of Trbl in the fat body decreased levels of activated Akt (pSer505-Akt) while misexpression of trbl RNAi increased phospho-Akt levels, and neither treatment affected total Akt levels. Trbl misexpression effectively suppressed Akt-mediated wing and muscle cell size increases and reduced phosphorylation of the Akt target FoxO (pSer256-FoxO). Taken together, these data show that Drosophila Trbl has a conserved role to bind Akt and block Akt-mediated insulin signaling, and implicate Trib proteins as novel sites of signaling pathway integration that link nutrient availability with cell growth and proliferation (Das, 2014: 25329475).

TORC2 mediates the heat stress response in Drosophila by promoting the formation of stress granules

The kinase TOR is found in two complexes, TORC1, involved in growth control, and TORC2 with less well defined roles. This study asked whether TORC2, disrupted by use of Rictor mutant flies, has a role in sustaining cellular stress. TORC2 inhibition in Drosophila was shown to lead to a reduced tolerance to heat stress. Accordingly, upon heat stress, both in the animal and Drosophila cultured S2 cells, TORC2 is activated and is required for the stability of its known target Akt/PKB. The phosphorylation of the stress activated protein kinases is not modulated by TORC2, nor is the heat-induced upregulation of heat shock proteins. Instead, it was shown, both in vivo and in cultured cells, that TORC2 is required for the assembly of heat-induced cytoprotective ribonucleoprotein particles, the pro-survival stress granules. These granules are formed in response to protein translation inhibition imposed by heat stress that appears less efficient in the absence of TORC2 function. It is proposed that TORC2 mediates heat resistance in Drosophila by promoting the cell autonomous formation of stress granules (Jevtov, 2015).

TOR (Target of rapamycin) is a conserved serine/threonine kinase of the PI3K-related kinase family, and functions in two distinct complexes, TOR complex 1 (TORC1) and TOR complex 2 (TORC2). Each complex comprises the kinase along with specific regulatory subunits that give the kinase its functional specificity and structural distinction. The core adaptor proteins of TORC1 are Raptor and LST8, whereas next to LST8, Rictor and Sin1 are the conserved components of TORC2. Removing either of the proteins from a cell destabilizes the TORC2 complex and inhibits its kinase activity. Since its original discovery in screens for rapamycin suppressors, TOR has been extensively studied in the context of TORC1, and has been shown to stimulate key anabolic cellular processes and inhibit the degradative pathway of autophagy with crucial roles in metabolic diseases, cancer and aging. TORC1 is widely regarded as the central node in cell growth control; its activity is dependent on growth factors and nutrient availability, and it is generally shut down in times of stress (Jevtov, 2015).

Unlike TORC1, TORC2 is less well understood and knowledge on upstream cues regulating its activity is scarce. Its role in growth under normal conditions is minor. In lower eukaryotes, TORC2 is activated upon nitrogen starvation, osmotic, heat and oxidative stress and DNA damage, and the TORC2 response to these environmental stresses is related to its likely ancient role in cellular signalling. TORC2 also has a role in actin cytoskeleton rearrangement. Recently, it has also been implicated in gluconeogenesis and sphingolipid metabolism, as well as apoptosis. Protein kinase B (PKB), also known as Akt, a membrane-associated kinase from the family of AGC kinases, with well described roles in cell growth, metabolism and stress, is one of the best characterized downstream targets of TORC2. In vitro, TORC2 has been shown to directly phosphorylate the hydrophobic loop of Akt (S473 in mammals or S505 in Drosophila), thereby increasing its kinase activity (Jevtov, 2015).

There are three well-studied stress response mechanisms in cells. The first is mediated via the stress-activated protein kinases (SAPKs), p38, JNK and Erk, either to protect the cell or to prime it for apoptosis. The second response is the rapid upregulation of transcription of genes encoding heat shock proteins (HSPs) that act as chaperones for cellular proteins to protect them against misfolding and aggregation in stressful conditions. The third includes branches that regulate translation and mRNA turnover. It is well established that heat exposure, oxidative stress and starvation induce the attenuation of bulk protein translation, polysome disassembly and accumulation of untranslated mRNAs. These are stored in cytoplasmic RNA-protein particles (RNP) known as stress granules along with translation initiation factors and RNA-binding proteins. From stress granules, stalled mRNAs can also be transported to the P-bodies (a different type of RNPs that contain RNA decay machinery) for degradation, or upon stress relief, transferred back to polysomes for translation re-initiation. Besides serving as transient protective storage of translation initiation components, the stress granules have also been suggested to serve as a transient station of the SAPKs and other pro-apoptotic kinases under stress, which is regarded to be a protective cellular mechanism against apoptosis. Whether TORC2 acts on these pathways in stress is not known (Jevtov, 2015).

This study shows that TORC2 is specifically required for heat resistance in vivo as Drosophila melanogaster mutants for TORC2 components are selectively sensitive to heat stress. This sensitivity is accompanied by the reduced phosphorylation of Akt due to the loss of the protein itself. Conversely, Akt phosphorylation is enhanced by heat in wild-type Drosophila larvae and cultured cells, showing that TORC2 is activated. Whereas the stress kinase and the HSP branches of the stress response are not affected, we show that the heat-induced stress granule formation is significantly delayed upon loss of TORC2 function, both in cells and in the animals and an reduction of translation inhibition imposed by heat stress might be a cause for this delay. Taken together, it is proposed that under heat stress conditions, TORC2 promotes survival by enabling stress granule assembly (Jevtov, 2015).

The results show that one key branch of the response to heat stress, the formation of stress granules, is delayed by the loss of TORC2 function both in Drosophila tissues and cultured cells. TORC2 is activated upon heat stress and mediates the formation of stress granules, likely required for heat resistance at the cellular level in Drosophila (Jevtov, 2015).

How TORC2 mediates stress granule formation is not clear. Heat stress is known to stimulate the inhibitory phosphorylation of the initiation factor eIF2 αresulting in protein translation stalling. However, this phosphorylation is not required for stress granule formation in Drosophila upon heat stress, so it is unlikely that TORC2 modulates this event (Jevtov, 2015).

This study shows that stress granule formation is delayed by loss of TORC2 function and it is suggested that this is due to a lift on the overall translation inhibition imposed by heat stress, but also under basal conditions. Depletion of TORC2 components appears to stimulate protein translation. This is in accordance with the observations that depletion of either Rictor or Sin1 from Drosophila S2 cultured cells causes their increased proliferation (115%) and cell diameter, respectively. This activation of translation upon loss of TORC2 function could be due to activation of TORC1, as observed previously in Kc cells, another Drosophila cultured cell line. There, depletion of Rictor elevates levels of the phosphorylated 4E-BP, a known target of TORC (Jevtov, 2015).

However, Rictor and Sin1 mutant flies are smaller in size than control animals, suggesting that this translation activation potentially leading to an increase in cell growth and proliferation might be the tissue specific. This might mirror the tissue-specific response in stress granule formation that we report here (Jevtov, 2015).

Such stimulated translation, even upon heat stress might delay or impair stress granule assembly. However, Sin1 depletion has a much stronger effect on translation than Rictor depletion, yet stress granule assembly is inhibited to the same extent in both backgrounds. So whether this lift in translation inhibition is the sole parameter impairing stress granule formation remains to be further investigated. In this regard, Rictor is found at the ribosomes interacting with RACK1, a selective mediator in stress granule function. Thus, it remains to be determined whether TORC2 senses ribosomal activity and mediates the stress granule assembly on its own, rather than indirectly, by providing balance to TORC1 (Jevtov, 2015).

Interestingly, the ribosomal localization of Rictor activates Akt, the TORC2 downstream kinase and this study shows that Akt is activated upon heat stress both in animals and cell lines, in line with mammalian studies. This heat-mediated activation is in line with the finding that S. pombe mutants for Tor1 (kinase of TORC2), Sin1 and Gad8 (Akt ortholog) are also sensitive to heat stress. This suggests that TORC2 - Akt signalling axis represents an ancient and conserved cellular mechanism to cope with heat stress (Jevtov, 2015).

Surprisingly, however, this study found that TORC2 function not only modulates Akt phosphorylation but also its stability. Strikingly, the absence of TORC2 function both in cells and larvae rapidly and significantly obliterates Akt, probably through increased degradation. It is likely not due to a lower translation during stress since translation is less inhibited in heat stress in the absence of TORC2 components. This correlates well with studies in mammalian cells, where PKCalpha, a second known downstream target of TORC2, and a small fraction of Akt are degraded by the proteasome ubiquitin pathway in cells depleted for TORC2 components. This is due to the lack of phosphorylation by TORC2 that primes PKCalpha and Akt for ubiquitination. Whether and how Akt plays a role in stress granule formation in Drosophila remains to be investigated (Jevtov, 2015).

The TORC2 based mechanism that this study proposes is different from the one described in mammalian cells (especially cancer cell lines) where TORC1 is a key player. Indeed, depletion of TORC1 components impairs stress granule assembly by reducing the phosphorylation of 4E-BP, subsequently preventing the formation of eIF4E-eIF4GI cap-dependent translational initiation complexes. In S2 cells, however, this study did not observe a direct role for TORC1 in stress granule formation. Neither Raptor depletion nor rapamycin treatment impairs stress granule formation upon heat stress. Whether this differential involvement of TORC1 and TORC2 in stress granule formation is cell- and tissue- dependent and acts via different pathways remains to be tested. Alternatively, the different mechanisms may suggest that mammals have evolved more sophisticated mechanisms to cope with stress. TORC1 has also been shown to be sequestered in stress granules during heat and other stresses where it suppresses its own apoptotic activity, corroborating its role in stress granule function (Jevtov, 2015).

The importance of studying environmental effects on signalling pathways, like the TOR pathway, is illustrated by the central role of these pathways for progression of diseases, such as metabolic and neurological diseases or cancer. Elucidating the modulation of such pathways under different environmental conditions can potentially identify new targets and processes playing roles in the physiological or pathological regulation of cell survival (Jevtov, 2015).

p53 and PI3K/AKT signalings are up-regulated in flies with defects in the THO complex

The THO complex (THO; see Thoc5) is an evolutionary conserved protein required for the formation of export-competent mRNP. The growing evidence indicates that the metazoan THO plays important roles in cell differentiation and cellular stress response. But the underlying mechanisms are poorly understood. This study examined the relevance of THO to cellular signaling pathways involved in cell differentiation and cellular stress response. When the endogenous p53 level was examined in the testis, it was found to be sustained much longer during spermatogenesis in the THO mutant compared to that of wild-type. In flies with impaired THO, overexpression of p53 by eye-specific GAL4 not only enhanced p53-mediated retinal degeneration, but p53 level was also elevated compared to the control flies. Since the body size of the THO mutant flies was significantly larger than control flies, whether the PI3K/AKT signaling is enhanced in the mutant flies was also examined. The results showed that the endogenous level of phosphorylated AKT, which is the active form, was highly elevated in the THO mutants. Taken together, these results suggested that both p53 and PI3K/AKT signalings are up-regulated in the flies with impaired THO (Moon, 2013).

A previous report showed the reduced life span and the increased susceptibility to the environmental stresses in mutant flies for Drosophila THO subunits. To understand the underlying mechanisms of these defects, this study investigated genetic interactions of THO with 2 cellular signaling pathways, p53 and PI3K/AKT pathways (Moon, 2013).

The following evidence suggests that defects in the function of THO cause up-regulation of p53 in a cell autonomous manner. First, endogenous level of p53 was sustained much longer during spermatogenesis in the male germline lacking THO compared to the control germline. FRT/FLP-based clonal analysis showed that p53 level was cell-autonomously sustained in the mutant clone. Second, mutations in the THO subunit genes elevated the level of overexpressed p53 in the eye, showing an increased sensitivity to p53-mediated apoptosis. Third, the sensitivity to p53-mediated apoptosis was directly correlated with the genetic background; the more severe defect in THO the genetic background had, the greater the sensitive to p53-mediated apoptosis was (Moon, 2013).

Why p53 is up-regulated in the flies with impaired THO? The fact that the nucleolar integrity was severely disrupted in flies lacking THO let to a postulate that disruption of nucleolus might be a good candidate to answer for this question. It has been known that disruption of nucleolus mediated stabilization of p53 in response to DNA damage and other stresses in mammalian cells. In addition, it has been reported that genetic disruption of nucleolus by knocking out murine TIF-1A gene caused p53 to be stabilized by dissociating it from MDM2. However, it is doubtful that the same is true in the Drosophila model. First, mutations in THO subunits caused nucleolar disruption only in certain types of cells including male germline and salivary gland cells. Moreover, this study failed to find any signs of nucleolar disruption in the THO-deficient eyes which were sensitized to p53-mediated apoptosis. Second, it has recently been reported that p53 level was not significantly increased in the eyes of viriato mutant in which nucleolar architecture was severely compromised. Finally, the Drosophila homolog of MDM2, which plays a key role in nucleolar disruption-mediated p53 stabilization in mammalian cells, has not been found to date. Consistent with these facts, in Drosophila, it has been shown that posttranslational modification rather than abundance was sufficient to activate p53 signaling in response to DNA damage. For these reasons, it is speculated that nucleolar disruption is not directly involved in the up-regulation of p53 in the THO mutant flies. An alternative possibility is that the phenotype of condensed chromatin structure in thoc5 might represent genomic instability, and the genomic instability could lead to activation of MNK, Drosophila homolog of CHK2, which activates p53 following DNA damage. But the fact that DNA damage activates p53 without significant changes in protein level is inconsistent with the current findings which show obvious changes of p53 level in the testis. Another alternative explanation for these ambiguities is that upregulation of p53 in the THO mutant may be restricted to certain limited types of cells, and the mechanisms underlying this may also be different depending on cell types. To clarify these issues further studies are certainly required (Moon, 2013).

In addition to p53 signaling, PI3K/AKT signaling was examined in the THO mutant flies. It has been well established that PI3K/AKT signaling pathway is a key player in regulating life span as well as body size in Drosophila. Combined with the previously reported lifespan reduction, increased body size in the THO mutant flies compared to control is well matched with the known phenotypes of mutant flies with defects in PI3K/AKT signaling pathway. Although no global increase in PI3K/AKT signaling was detected in the THO mutant flies, a cell-autonomous elevation of endogenous p-AKT level in the mutant male germline provided a piece of evidence for the relevance of Drosophila THO with PI3K/AKT signaling (Moon, 2013).

Another interesting finding in this report is that the levels of both p53 and p-AKT are very high in the wild-type male germ germline. If p53 is important for spermatogenesis, why p53-null flies are not sterile? With regard to the female germline, it has recently been reported that DNA double strand breaks formed during meiotic recombination provoked activation of p53, and unrepaired DNA breaks during meiotic recombination led to sustained p53 activity. But it has been known that meiotic recombination is very rare in the Drosophila male germline, and this study showed that p53 was detected only in the pre-meiotic germline. Thus, it is unlikely that the role of p53 in male germline is similar to that in female germline. Certainly these issues will be a good topic for future study (Moon, 2013).

Taken together, this study found the significant genetic interactions of THO with 2 cellular signaling pathways, p53 and PI3K/AKT signaling pathways. Both signalings were up-regulated by THO dysfunction in a cell autonomous manner. However, it seems unlikely that THO generally plays a major role in regulating these signaling pathways, because not only Western blot analysis of whole-fly extract, but also FRT/FLP-mediated clonal analysis in the imaginal discs showed no significant changes in the endogenous levels of both p53 and p-AKT in the THO mutants. It seems rather likely that the effect of THO dysfunction on these two signaling pathways is different depending on cell types; it might be generally mild in most cells except certain types of cells such as germline (Moon, 2013).


The temporal and spatial expression of the DRAC-PK/Akt1 transcripts during development has been examined. For Northern blot analysis two probes were used: the cDNA DRAC 7 and a probe from the 3`-untranslated region located between the first polyadenylation signal (nucleotides 4374-4379) and a polyadenylation signal at nucleotides 5013-5018 in the DRAC-PK genomic sequence. Whereas DRAC 7 recognizes all classes of transcripts, the 3'-end probe would detect only those that use the second polyadenylation signal. Northern analysis of total RNA isolated from staged collections of embryos and larvae and from adult female flies using DRAC 7 as a probe revealed the presence of two major transcripts of 2.7 and 3.9 kb. The 2.7-kb transcript is expressed at high levels in 0-3-h embryos and is also expressed in adult females, indicating that this is a maternally regulated mRNA. The 3.9-kb transcript is expressed throughout embryogenesis and larval stages and is also detected in early and late pupal stages. This transcript is also expressed in the adult female flies, indicating both maternal and zygotic regulation of gene expression. When the 3'-probe was used, only the larger transcript could be detected, suggesting that the 2.7-kb transcript is generated by use of the first polyadenylation signal. This indicates that the cDNA SDE-RAC 109 is derived from the 2.7-kb maternal transcript (Andjelkovic, 1995).

In situ hybridization to whole-mount embryos and dissected ovaries, using DRAC 7 as a probe, demonstrates that maternally provided DRAC-PK transcripts are synthesized in the nurse cells of the ovaries. During oocyte maturation there is no apparent localization of the mRNA. No variation in expression could be detected throughout embryogenesis. Before cellularization, after cellularization, and throughout gastrulation, germ band extension, and retraction, DRAC-PK/Akt1 transcripts remain uniformly distributed and are expressed at a high level (Andjelkovic, 1995).

Northern analysis demonstrates that the DRAC-PK gene expression is both maternally and zygotically regulated. Also Western blot analysis reveals differences in DRAC-PK protein forms and abundance between embryos and adult flies. The expression of the DRAC-PK gene protein products during the Drosophila life cycle was examined. The major form detected throughout development is DRAC-PK66. The highest level of expression (arbitrarily taken as 100%) is found in 0-3-h embryos and subsequently declines to 60% during late embryogenesis. A further decline in DRAC-PK66 levels is observed during larval development (20% in the third instar larvae), followed by a sharp increase in the early pupae (80%). The protein levels are 30% in adult flies. DRAC-PK85 follows the expression of DRAC-PK66, except in the three larval stages where it could not be detected. There is no difference in the expression of DRAC-PK66 and -85 between female and male flies. p120 is detected during embryogenesis, where its expression follows the pattern of DRAC-PK66 and -85. It is found to be significantly higher in early than in late pupae, which is not the case with DRAC-PK66 and -85. These results suggest the involvement of DRAC-PK and the p120 protein in embryogenesis, as well as postembryonically. The spatial expression of the DRAC-PK proteins during embryogenesis was analyzed using the affinity-purified antirecombinant antibody No. 36. Proteins are uniformly distributed in all embryonic stages, which is in good correlation with the transcript localization. Also, during the syncytial blastoderm stage, specific staining is detected in the cytoplasm surrounding dividing nuclei and is always excluded from the chromatin (Andjelkovic, 1995).

Cytoplasmic activated protein kinase Akt regulates lipid-droplet accumulation in Drosophila nurse cells

The insulin/insulin-like growth factor signalling (IIS) cascade performs a broad range of evolutionarily conserved functions, including the regulation of growth, developmental timing and lifespan, and the control of sugar, protein and lipid metabolism. Recently, these functions have been genetically dissected in Drosophila, revealing a crucial role for cell-surface activation of the downstream effector kinase Akt in many of these processes. However, the mechanisms regulating lipid metabolism and the storage of lipid during development are less well characterized. The nutrient-storing nurse cells of the fly ovary were used to study the cellular effects of intracellular IIS components on lipid accumulation. These cells normally store lipid in a perinuclear pool of small neutral triglyceride-containing droplets. Loss of the IIS signalling antagonist PTEN, which stimulates cell growth in most developing tissues, produces a very different phenotype in nurse cells, inducing formation of highly enlarged lipid droplets. Furthermore, the accumulation of activated Akt in the cytoplasm is responsible for this phenotype and leads to a much higher expression of LSD2, the fly homologue of the vertebrate lipid-storage protein perilipin. This work therefore reveals a signalling mechanism by which the effect of insulin on lipid metabolism could be regulated independently of some of its other functions during development and adulthood. It is speculated that this mechanism could be important in explaining the well-established link between obesity and insulin resistance that is observed in Type 2 diabetes (Vereshchagina, 2006).

These data suggest that the effect of cytoplasmic P-Akt on lipid storage may be cell type-specific. Increasing IIS throughout the whole organism in viable Pten mutants surprisingly reduces total lipid content, whereas decreasing IIS elevates lipid levels both in flies and mice. It has not been possible to show biochemically whether triglycerides are increased rather than merely redistributed in Pten mutant ovaries, because only a small minority of the nurse cells is mutant. However, in some late-stage clones, in which cytoplasmic volumes are decreasing, lipid levels often appear elevated in mutant cells. Insulin-stimulated increases in lipid-droplet number and size have been observed in mammals, and, interestingly, when tumours are induced in mouse liver by raising IIS, lipid-storage mechanisms are also activated. It is proposed that these cell type-specific responses are due to selective accumulation of an IIS-modulated, cytoplasmic activated Akt pool that must, to some extent, be controlled independently of cell-surface P-Akt. Indeed, a Drosophila phosphatase has been identified that regulates levels of cytoplasmic P-Akt in nurse cells, and it has been shown that mutations in this gene produce a very similar enlarged lipid-droplet phenotype, confirming this hypothesis (Vereshchagina, 2006).

How could elevated cytoplasmic P-Akt induce such a dramatic lipid-droplet phenotype in nurse cells? In mammals, Akt can promote the transcription of genes involved in lipid biosynthesis and storage pathways. The data indicate LSD2/perilipin is one of these targets. IIS also post-translationally upregulates the activity of mammalian perilipin. Interestingly, ovaries mutant for Lsd2 show altered lipid accumulation, but droplets are still formed. Therefore, LSD2 is almost certainly one, but not the only, target for IIS in the control of lipid-droplet accumulation in nurse cells. In this context, it is interesting to note that, in addition to its proposed role in coating lipid droplets, LSD2 has recently been shown to regulate microtubule-dependent trafficking of these organelles. Since cytoplasmic P-Akt could still be associated with intracellular membranes or the droplet surface, it may be well positioned to modulate this transport process (Vereshchagina, 2006).

Obesity is a well-established predisposing factor in the acquisition of cellular insulin resistance and Type 2 diabetes. Increased levels of circulating free fatty acids (FFAs) associated with obesity appear to be important in this link. However, it is unclear whether other mechanisms are also involved or how reduced insulin sensitivity ultimately impacts on lipid storage. Molecules downstream of Akt are known to regulate cell-surface IIS through at least two negative-feedback loops involving downstream S6 kinase and the transcription factor FOXO. This work therefore raises the possibility that any predisposition towards increased cytoplasmic P-Akt could specifically promote lipid storage and also selectively suppress insulin-dependent events at the cell surface. It will be interesting to investigate further the molecules involved in controlling this P-Akt pool and whether the feedback mechanisms have any role to play in linking obesity and insulin resistance in Type 2 diabetes (Vereshchagina, 2006).

Phosphatidylinositol 3-kinase and Akt nonautonomously promote perineurial glial growth in Drosophila peripheral nerves

Drosophila peripheral nerves, structured similarly to their mammalian counterparts, comprise a layer of motor and sensory axons wrapped by an inner peripheral glia (analogous to the mammalian Schwann cell) and an outer perineurial glia (analogous to the mammalian perineurium). Growth and proliferation within mammalian peripheral nerves are increased by Ras pathway activation: loss-of-function mutations in Nf1, which encodes the Ras inhibitor neurofibromin, cause the human genetic disorder neurofibromatosis, which is characterized by formation of neurofibromas (tumors of peripheral nerves). However, the signaling pathways that control nerve growth downstream of Ras remain incompletely characterized. This study shows that expression specifically within the Drosophila peripheral glia of the constitutively active RasV12 increases perineurial glial thickness. Using chromosomal loss-of-function mutations and transgenes encoding dominant-negative and constitutively active proteins, it was shown that this nonautonomous effect of RasV12 is mediated by the Ras effector phosphatidylinositol 3-kinase (PI3K) and its downstream kinase Akt. The nonautonomous, growth-promoting effects of activated PI3K are suppressed by coexpression within the peripheral glia of FOXO, a transcription factor inhibited by Akt-dependent phosphorylation. It is suggested that Ras-PI3K-Akt activity in the peripheral glia promotes growth of the perineurial glia by inhibiting FOXO. In mammalian peripheral nerves, the Schwann cell releases several growth factors that affect the proliferative properties of neighbors. Some of these factors are oversecreted in Nf1 mutants. These results raise the possibility that neurofibroma formation in individuals with neurofibromatosis might result in part from a Ras-PI3K-Akt-dependent inhibition of FOXO within Schwann cells (Lavery, 2007).

Activating Ras specifically within the peripheral glia was sufficient to increase growth of the perineurial glia. In addition, activating the Ras effector PI3K, but not Raf or Ral, within the peripheral glia was sufficient to increase perineurial glial growth, and inhibiting PI3K activity in the peripheral glia, but not perineurial glia, suppressed the growth-promoting effects of activated Ras. It was also found that activity within the peripheral glia of the PI3K-activated kinase Akt was both necessary and sufficient to mediate the growth-promoting effects of PI3K. Finally, it was found that overexpression within the peripheral glia of FOXO, the forkhead-box transcription factor that is phosphorylated and inactivated by Akt-dependent phosphorylation, was sufficient to suppress the growth-promoting effects of PI3K. Together, these results suggest that Ras activity in the peripheral glia activates nonautonomous growth via the PI3K and Akt-dependent inhibition of FOXO. This observation is consistent with the previous observations that Nf1 mouse Schwann cells oversecrete growth factor(s) that cause increased recruitment of mast cells into the peripheral nerve and is consistent in part with the observation that the proliferation defects of Nf1 mutant mouse or human cells requires hyperactivation of Tor in a PI3K- and Akt-dependent manner (Lavery, 2007).

Perineurial glial growth in Drosophila peripheral nerves is regulated by several genes. These genes include Nf1, which is the Drosophila ortholog of human Nf1, push, which is thought to encode an E3 ubiquitin ligase and two genes implicated in neurotransmitter signaling: amnesiac, which is thought to encode a neuropeptide similar to vertebrate pituitary adenylate cyclase-activating polypeptide, and inebriated (ine), which encodes a member of the Na+/Cl-dependent neurotransmitter transporter family. Some of these genes might regulate perineurial glial growth via the activity of Ras or PI3K within peripheral glia. For example, mutations in push, but not ine, enhance the perineurial glial growth phenotype of RasV12 expressed in peripheral glia. These observations are consistent with the possibility that the activity of ine regulates Ras-GTP levels within peripheral glia. In contrast, push might regulate PI3K in a Ras-independent manner or act in the perineurial glia to regulate sensitivity to peripheral glial growth factors. Additional experiments will be required to distinguish between these possibilities (Lavery, 2007).

There are several lines of evidence from mice and humans suggesting that cell nonautonomous growth regulation, as a consequence of intercellular signaling, underlies neurofibroma formation. First, although neurofibromas arise in individuals heterozygous for Nf1 after loss of Nf1+ from cell(s) within peripheral nerves, neurofibromas are heterogeneous and contain cells that are not clonally related, such as Schwann cells, perineurial cells, and fibroblasts. These observations suggest that neurofibromas arise when a core of Nf1 cells cause overproliferation of their heterozygous neighbors via nonautonomous means. Second, neurofibroma formation in mice and humans requires a homozygous Nf1 mutant genotype in Schwann cells but not other cells within the tumor. Third, Ras-GTP levels in Schwann cells from the mouse Nf1 knock-out mutant are uniformly elevated. In contrast, only a subset of Schwann cells from human neurofibromas exhibit elevated Ras- GTP levels; the possibility has been raised that this subset, but not other Schwann cells from the tumor, is homozygous for Nf1. In this view, these Nf1 cells recruited neighboring Schwann cells that were heterozygous for Nf1 into the tumor by nonautonomous means, such as by the excessive release of one or more growth factors. Fourth, it has been demonstrated that Nf1 Schwann cells oversecrete the ligand for the c-Kit receptor. This oversecretion increased migration of mast cells into peripheral nerves and might be an essential step in neurofibroma formation. These Schwann cells also oversecrete additional factors whose physiological role remains unclear. The molecular mechanisms by which neurofibromin regulates the synthesis or release of these molecules remain incompletely understood. The current observations that Ras activity in the peripheral glia promotes growth nonautonomously via the PI3K- and Akt-dependent inhibition of FOXO might provide insights into the mechanisms by which peripheral nerve growth is regulated nonautonomously by the mammalian Schwann cell (Lavery, 2007).

By hyperactivating Ras, Nf1 mutations could in principle cause tumors via any of several Ras effector pathways. In addition, the diverse types of tumors observed in individuals with neurofibromatosis could result from hyperactivation of distinct Ras effector pathways. The Raf pathway has been viewed as a more relevant effector pathway than the PI3K pathway, mostly because the importance of Ras in the activation of PI3K under physiological conditions remains controversial. In particular, although it is clear that the oncogenic RasV12 mutant is sufficient to activate PI3K, it has sometimes been difficult to demonstrate that wild-type Ras is necessary for PI3K activation. Presumably, this difficulty reflects the fact that PI3K can be activated by Ras-independent as well as Ras-dependent mechanisms, such as direct activation by activated receptor tyrosine kinases or by PIKE-L (phosphatidylinositol kinase enhancer). However, more recently, it has been demonstrated that PI3K and Akt are hyperactivated in several Nf1 mutant cell types and that this hyperactivation is Ras dependent. Furthermore, PI3K activation plays an essential functional role in Nf1-mediated growth defects, as is demonstrated by the observation that PI3K- and Akt-dependent Tor activation is necessary for the proliferation defects of Nf1 mutants to occur: application of rapamycin, a Tor inhibitor, attenuates the ability of Nf1 mutant cells to proliferate. These observations demonstrate that PI3K and Akt play key roles in at least some aspects of Nf1-induced tumor growth (Lavery, 2007).

The results are consistent with these observations. By comparing the effects on perineurial glial growth of peripheral–glial expression of activated Raf, PI3K, or Ral, it was possible to demonstrate that activation of PI3K, not Raf or Ral, is sufficient to promote perineurial glial growth and that PI3K activity in the peripheral glia is necessary to observe the nonautonomous effect of activated Ras on perineurial glial growth. It was similarly shown that Akt activity os necessary and sufficient to mediate the growth-promoting effects of PI3K. However, previous studies have observed that Tor activation is critical for the PI3K- and Akt-dependent growth regulation of Nf1 mutant cells, this study observed a critical role for the PI3K- and Akt-dependent inhibition of the transcription factor FOXO. It is possible that the phenotype observed in previous studies reflects the well characterized ability of PI3K–Tor to activate growth cell autonomously, whereas the phenotype reported in this study reflects nonautonomous growth regulation. In this view, PI3K and Akt regulate autonomous and nonautonomous growth via the Tor and FOXO pathways, respectively (Lavery, 2007).

FOXO presumably inhibits the growth-promoting effects of PI3K and Akt by transcriptional regulation of target genes. Candidate FOXO target genes include those encoding the molecules oversecreted by Nf1 Schwann cells, whereas other targets might be represented in the distinct transcript profiles exhibited by Nf1 Schwann cells or malignant peripheral nerve sheath tumors compared with wild-type Schwann cells. For example, Schwann cells from neurofibromas, but not normal Schwann cells, express the epidermal growth factor (EGF) receptor. Other potential targets include genes encoding growth factors, although ectopic expression within the peripheral glia of two candidate genes, Hedgehog and the EGF ligands spitz and gurken, failed to induce perineurial glial growth. Additional experiments will be required to identify the FOXO targets that regulate nonautonomous growth in peripheral nerves (Lavery, 2007).

Nutrition-responsive glia control exit of neural stem cells from quiescence

The systemic regulation of stem cells ensures that they meet the needs of the organism during growth and in response to injury. A key point of regulation is the decision between quiescence and proliferation. During development, Drosophila neural stem cells (neuroblasts) transit through a period of quiescence separating distinct embryonic and postembryonic phases of proliferation. It is known that neuroblasts exit quiescence via a hitherto unknown pathway in response to a nutrition-dependent signal from the fat body. This study has identified a population of glial cells that produce insulin/IGF-like peptides in response to nutrition, and shows that the insulin/IGF receptor pathway is necessary for neuroblasts to exit quiescence. The forced expression of insulin/IGF-like peptides in glia, or activation of PI3K/Akt signaling in neuroblasts, can drive neuroblast growth and proliferation in the absence of dietary protein and thus uncouple neuroblasts from systemic control (Chell, 2010).

A transcriptome analysis comparing VNCs from newly hatched larvae and VNCs from larvae at the end of the first instar suggested that the expression of dILP6 and dILP2 increases in the VNC during neuroblast reactivation. The seven dILPs are expressed in distinct spatiotemporal patterns during development. dILP6 is reported to be expressed in the larval gut and the pupal fat body , whereas dILP2 is known to be expressed in the IPC neurons of the brain (along with dilps 1, 3, and 5). To determine whether dILP6 is also expressed in the CNS, a dilp6-GAL4 line was generated. dilp6-GAL4 drives expression in a subset of the surface glia that wraps the CNS. Strong expression was evident by mid first instar and was maintained throughout neuroblast reactivation. The expression of dILP2 was assayed by immunohistochemistry; it too was expressed in the same surface glial population. The glial cells labeled by dilp6-GAL4 are located above the neuroblasts and underneath the surrounding basement membrane. They are stellate in appearance, with several processes radiating from the central cell body. Thus, dILPs, expressed by glial cells, are ideally positioned to activate the dInR pathway in neuroblasts during reactivation (Chell, 2010).

Drosophila Akt is a key transducer of increased PIP3 levels, such as those seen in response to dInR/PI3K activation. Following recruitment to the cell membrane, Akt is activated by PDK1-mediated phosphorylation. This study found that, when PI3K activity was increased in neuroblasts by expression of dp110CAAX (a membrane-targeted, constitutively active, version of the PI3K catalytic subunit), the levels of phosphorylated Akt (pAkt) were concomitantly increased. To test whether Akt activation is sufficient for the exit from quiescence, a membrane-targeted form of Akt (myr-Akt) was expressed in neuroblasts of larvae reared on a sucrose-only diet. myr-Akt expression was sufficient to drive both growth and cell-cycle re-entry (as evidenced by extensive pH3 labeling) in quiescent neuroblasts in the absence of the nutritional stimulus. Indeed, expression of myr-Akt was more potent than dp110CAAX, as all grh-GAL4- positive neuroblasts reactivated. The difference in the number of neuroblasts that reactivated in response to dp110CAAX (4%–12%) and myr-Akt (100%) may reflect a differential sensitivity to negative feedback regulation in the pathway. Myr-Akt may escape negative control more readily than wild-type Akt that has been activated by dp110CAAX (Chell, 2010).

Once neuroblast reactivation has been ectopically triggered by either PI3K or Akt, then neuroblast proliferation occurs at approximately the same rate. When reactivated neuroblasts were assayed at 24 hr, they had generated on average six or seven daughter cells under either condition. For dp110CAAX, the daughter cells of 29 reactivated neuroblasts from 10 tVNCs were counted; on average, each neuroblast had 6.76 daughter cells. For myr-Akt, the daughter cells of 40 reactivated neuroblasts from four tVNCs were counted; on average, each neuroblast had 6.65 daughter cells. Thus, dInR/PI3K appear to act via their canonical downstream pathway, and when activated in neuroblasts, this pathway is sufficient for reactivation (Chell, 2010).

Drosophila neuroblasts in the central brain and thoracic ventral nerve cord (tVNC) are quiescent for 24 hours between their embryonic and larval phases of proliferation. Quiescent neuroblasts are easily identifiable and are amenable to genetic manipulation, making them a potentially powerful model with which to study the transition between quiescence and proliferation. However, the mechanisms regulating the exit from quiescence, either intrinsic or extrinsic, are not well established. Genetic studies found that Drosophila FGF, in concert with Drosophila Perlecan, promotes the neuroblast transition from quiescence to proliferation, but this effect is indirect (Barrett, 2008). Exit from quiescence is physiologically coupled to larval growth and development via a nutritional stimulus (Britton, 1998). The Drosophila fat body performs many of the storage and endocrine functions of the vertebrate liver and acts as a sensor, coupling nutritional state to organismal growth. In response to dietary amino acids, the fat body secretes a mitogen that acts on the CNS to bring about neuroblast proliferation (Britton, 1998). This fat body-derived mitogen (FBDM) initiates cell growth in quiescent neuroblasts and promotes (or at least permits) cell-cycle re-entry (Britton, 1998). Yet the identity of the FBDM, the cell type upon which it acts, and the downstream pathway activated in neuroblasts have remained unknown (Chell, 2010).

Neuroblast entry into quiescence is governed intrinsically by the same transcription factor cascade that controls neuroblast temporal identity. This study has identified a population of surface glial cells that respond to the nutrition-dependent stimulus by expressing dILPs, and showns that the dInR/PI3K pathway is required by neuroblasts to exit quiescence in response to nutrition. Forced expression of dILPs in glia or activation of PI3K/Akt signaling in neuroblasts can drive neuroblast growth and proliferation in the absence of dietary protein and thus uncouple neuroblast reactivation from systemic nutritional control (Chell, 2010).

Cell growth and division are not strictly coupled in neuroblasts. In Drosophila Perlecan (dPerlecan) loss-of-function mutants, the majority of neuroblasts appear to increase in size but then remain G1 arrested. This suggested that a dedicated mitogen might exist to promote cell-cycle progression. Drosophila Activin-like peptides (ALPs; Zhu, 2008) are required for normal levels of neuroblast division in the larval brain and appear to be one such dedicated mitogen (Chell, 2010).

Perlecan is expressed by glia and forms part of the basement membrane that enwraps the CNS. Perlecan was proposed to modulate Drosophila FGF [Branchless (Bnl)], allowing it to act as a mitogen for neuroblasts. However, it now appears that the action of Bnl is indirect via a still to be identified cell type (Barrett, 2008). One possibility is that Bnl acts on glia to modulate the expression of other proteins, such as dILPs or ALPs, which then in turn act on neuroblasts directly. This study shows that expression of dILPs by glia leads to neuroblast reactivation in the absence of dietary protein; however, the number of mitoses falls short of that seen under normal dietary conditions. This could be explained by the absence of another nutritionally dependent mitogen. It will be of interest to see whether the glial expression of ALPs, like that of dILPs, relies on dietary protein (Chell, 2010).

In the larval CNS, neuroblasts and their progeny are completely surrounded by glial cell processes. If the interaction between neuroblasts and surrounding glia is disrupted by expression of a dominant-negative form of DE-cadherin, the mitotic activity of neuroblasts is severely reduced (Dumstrei, 2003). In the mammalian brain, glial cells are involved in a wide variety of processes, including axon guidance, synapse formation, and neuronal specification. Glial cells, with the extracellular matrix and vasculature, also make up the adult neural stem cell niche. Astrocytes have been shown to promote neural stem cell proliferation in culture and can express proproliferative factors such as FGF-2 and IGF-I. Thus, astrocytes are thought to be a key component of the niches that dynamically regulate neural stem cell proliferation in the adult brain (Chell, 2010).

This study has shown that Drosophila surface glia can transduce systemic signals and, by expressing dILP2 and dILP6, control neuroblast exit from quiescence. Glial cells also express dPerlecan and ana and are the source of the Activin-like peptides that have a direct mitogenic effect on neuroblasts. Thus, much like mammalian glial cells, Drosophila glial cells perform a number of the functions that define a niche and control the proliferation of neural stem cells (Chell, 2010).

Recent results suggest a role for IGF-1 in the control of neural stem cell division (Mairet-Coello et al., 2009). IGF-1 injection into rat embryonic brain results in a 28% increase in DNA content postnatally as a consequence of increased DNA synthesis and entry into S phase. Conversely, DNA synthesis and entry into S phase are decreased when the PI3K/Akt pathway is blocked. Furthermore, the loss of PTEN, the tumor suppressor and PI3K antagonist, enhances the exit from G0 of neural stem cells cultured from mouse embryonic cortex. It was suggested that a concomitant increase in cell size may push the cells to enter G1 (Chell, 2010).

This study shows, in vivo, that glial expression of insulin-like peptides activates the dInR/PI3K/Akt pathway in Drosophila neural stem cells and is responsible for their exit from quiescence. This pathway promotes cell growth and the transition from G0 to G1 and is also sufficient to promote G1-S and mitosis. Given that IGF-1 and the PI3K/Akt pathway can promote cell-cycle progression in vertebrate neural stem cells, this same pathway may regulate vertebrate neural stem cell reactivation in the same way as has been shown in this study for Drosophila (Chell, 2010).

The identity of the proposed FBDM, secreted by the fat body in response to dietary protein, remains unknown. However, explant CNS culture experiments demonstrated that the FBDM can act directly on the CNS to bring about neuroblast reactivation (Britton, 1998). This study has identified the surface glia as a key relay in the nutritional control of neuroblast proliferation. If the receptor protein(s) that controls glial dILP expression/secretion can be identified, then, by extension, it might be possible to identify the FBDM and approach a comprehensive understanding of how neural stem cell proliferation is coupled to nutrition and organism-wide growth (Chell, 2010).

Fragile X Protein is required for inhibition of insulin signaling and regulates glial-dependent neuroblast reactivation in the developing brain

Fragile X syndrome (FXS) (FXS) is the most common form of inherited mental disability and known cause of autism. It is caused by loss of function for the RNA binding protein FMRP, which has been demonstrated to regulate several aspects of RNA metabolism including transport, stability and translation at synapses. Recently, FMRP has been implicated in neural stem cell proliferation and differentiation both in cultured neurospheres as well as in vivo mouse and fly models of FXS. Previous studies have shown that FMRP deficient Drosophila neuroblasts upregulate Cyclin E, prematurely exit quiescence, and overproliferate to generate on average 16% more neurons. This study further investigated FMRP's role during early development using the Drosophila larval brain as a model. Using tissue specific RNAi it was found that FMRP is required sequentially, first in neuroblasts and then in glia, to regulate exit from quiescence as measured by Cyclin E expression in the brain. Furthermore, the hypothesis was tested that FMRP controls brain development by regulating the insulin signaling pathway, which has been recently shown to regulate neuroblast exit from quiescence. The data indicate that phosphoAkt, a readout of insulin signaling, is upregulated in dFmr1 brains at the time when FMRP is required in glia for neuroblast reactivation. In addition, dFmr1 interacts genetically with dFoxO, a transcriptional regulator of insulin signaling. These results provide the first evidence that FMRP is required in vivo, in glia for neuroblast reactivation and suggest that it may do so by regulating the output of the insulin signaling pathway (Callan, 2012).

Although there is a clear role for FMRP in the glia, its precise function in these cells remains poorly understood. It was previously shown that FMRP is expressed in glia during embryonic development but appears downregulated postnatally in the mouse brain. Co-culture of glia and hippocampal neurons demonstrated that glial cells contribute to the neuroanatomical defects found in the Fragile X brain, albeit the factors responsible are yet to be identified. This work provides the first in vivo evidence for FMRP's requirement in glial cells, and future work will focus on using more restricted glial Gal4 drivers to dissect the contribution of different glial types to regulating neuroblast reactivation in the dFmr1 mutant brains (Callan, 2012).

Several mRNA targets have been predicted or confirmed for FMRP. Given FMRP's complex tissue specific and temporal requirements during development it is likely that more remain to be identified and confirmed in vivo. Some clues as to the possible pathways and targets regulated by FMRP in the developing brain come from recent reports that glia can provide cues (in the form of secreted dILPs) to neighboring neuroblasts awaiting a reactivation signal. These findings together with the current data showing that FMRP is required sequentially in neuroblasts, then in glia, for proper neuroblast reactivation suggest a model whereby FMRP may control the timing and/or levels of insulin signaling in the brain by acting in different tissues at different times during development. While more work is needed to fully validate this model and to identify the direct mRNA targets of FMRP during brain development, initial tests of the model were carried out by evaluating the levels of pAkt, a readout of insulin signaling in the brain. This work shows that indeed, at the time when FMRP is required in glia (12-18 h ALH), more cells belonging to neuroblast lineages express pAkt. Coupled with the genetic interaction discovered between dFmr1 and dFoxO, a downstream effector as well as inhibitor of insulin signaling, it is suggested that FMRP controls neural stem cell behavior by directly regulating components of insulin signaling. Notably, PI3K, an upstream activator of Akt has been previously shown to be a target of FMRP in the context of mGluR signaling at synapses. Thus for its autonomous function in neuroblasts, FMRP could regulate PI3K directly, while later, in glia, for its nonautonomous function, FMRP could control (directly or indirectly) the expression of the dILPs secreted by glial cells. Notably, in vertebrates, Insulin Growth Factor-1 (IGF-1) and PI3K/Akt can also promote cell-cycle progression in neural stem cells, thus raising the possibility that the current findings in the fly model may be highly relevant to the molecular mechanisms underlying FXS. While more work is needed to elucidate FMRP's role in the communication between the glial niche and neuroblasts, it is tempting to speculate that FMRP may regulate similar signaling cassettes and molecules (i.e., PI3K) in different developmental contexts. The Drosophila model offers unique opportunities to dissect tissue specific regulation such as glial versus neuroblast specific mRNA targets in future experiments (Callan, 2012).

Regeneration of Drosophila sensory neuron axons and dendrites is regulated by the Akt pathway involving Pten and microRNA bantam

Both cell-intrinsic and extrinsic pathways govern axon regeneration, but only a limited number of factors have been identified and it is not clear to what extent axon regeneration is evolutionarily conserved. Whether dendrites also regenerate is unknown. This study reports that, like the axons of mammalian sensory neurons, the axons of certain Drosophila dendritic arborization (da) neurons are capable of substantial regeneration in the periphery but not in the CNS, and activating the Akt pathway enhances axon regeneration in the CNS. Moreover, those da neurons capable of axon regeneration also display dendrite regeneration, which is cell type-specific, developmentally regulated, and associated with microtubule polarity reversal. Dendrite regeneration is restrained via inhibition of the Akt pathway in da neurons by the epithelial cell-derived microRNA bantam but is facilitated by cell-autonomous activation of the Akt pathway. This study begins to reveal mechanisms for dendrite regeneration, which depends on both extrinsic and intrinsic factors, including the PTEN-Akt pathway that is also important for axon regeneration. This study has thus established an important new model system -- the fly da neuron regeneration model that resembles the mammalian injury model -- with which to study and gain novel insights into the regeneration machinery (Song, 2012).

The present study shows that Drosophila sensory neuron dendrites and axons are capable of regeneration in a cell type-specific manner. While dendrites and axons share the same cell type specificity in their capacity for regeneration, they differ in their developmental regulation, with axons but not dendrites retaining the regeneration ability throughout larval development. It was further shown that the evolutionarily conserved PTEN-Akt signaling pathway is important for the regeneration of dendrites as well as axons and that both axon regeneration and dendrite regeneration are accompanied by the reversal of microtubule polarity (Song, 2012).

It is known that Drosophila larval axons undergo a scaling process in which axons substantially increase their length in accordance with the growth of the organism. Thus, it raises an important issue of whether larval axons regenerate or simply scale after axotomy. This question may be addressed with the following two considerations. First, larval axons scale while maintaining their neural connections; da neuron axons have already formed synaptic connections with neurons in the VNC, and these axon projections are not significantly altered as larvae grow in size. Therefore, this increase of axon length does not involve bona fide axon pathfinding or synaptogenesis. Thus, axon scaling differs from the developmental axon outgrowth before synapse formation and is different from axon regeneration, which involves reinitiation of the developmental program for severed axons to generate growth cones or growth cone-like structures and pathfind to reach their targets. In the larval injury model, the axon is severed and therefore develops a new growing tip, reroutes following the presumptive trajectory by active or passive cues, and may or may not eventually establish synaptic contacts with their right targets in the CNS. This process resembles the regeneration program rather than axon scaling. Second, all of the da neurons, including class I, class III, and class IV, show similar axon scaling during larval stages. However, class IV but not class I or class III da neurons displayed axon regrowth after axotomy. The fact that only class IV da neurons are capable of regrowth, although all of these different types of da neurons undergo scaling, strongly suggests that class IV da neurons possess a unique regrowth potential that allows their severed axons to reinitiate the developmental program for axon outgrowth. For these reasons, it is believed that a subset of the larval axons can regenerate after injury, although the possibility cannot be excluded that the ability of these axons to scale contributes to their regeneration potential. While this regeneration process may or may not fully recapitulate the regeneration program in adults, understanding how this process takes place in larvae will provide invaluable insights into the axon regeneration machinery (Song, 2012).

The ability of class IV but not class I or class III da neurons to readily regenerate their axons and dendrites could conceivably reflect cell type-specific features, including the transcription programs. One interesting question is whether the same program that governs the cell type morphology may also influence their regeneration capacity. For dendrite regeneration of class IV da neurons, either the regenerated dendrite or the neighboring dendritic branch continues to grow to fill the available space. Thus, they may possess a persistent growing potential that is inhibited by neighboring branches nearby so that the branches might overgrow if those inhibitory signals are removed. Therefore, with some branches removed due to injury, the remaining branches will regrow to take over the vacant space. Since class I and class III da neuron dendrites show very limited space-filling ability, these dendrites may lack the growth potential required for regeneration (Song, 2012).

In response to injury, class IV da neurons regenerate their axons substantially, while class I da neurons partially reverse the microtubule polarity of nearby dendrites and convert one of these dendrites into a pseudo-axon (Stone, 2010). Taken together with the current finding that class IV but not class I or class III da neurons are able to regenerate their dendrites, which are also associated with the reversal of microtubule polarity, these observations raise the question of whether pathways controlling neuronal polarity and/or cytoskeletal rearrangement may influence dendrite and axon regeneration (Song, 2012).

Several lines of evidence suggest that dendrite regeneration depends on a balance of influences. First, there may be competition between de novo dendrite regeneration and invasion of neighboring branches. Successful regeneration prevents invasion and vice versa. Second, there could be a balance of extrinsic inhibitory cues, as in the form of the ban miRNA in epithelial cells, and intrinsic growth-promoting signals, as conveyed by the activation of the Akt pathway (Song, 2012).

Moreover, given that activation of the Akt pathway at later stages of development is not sufficient to elevate the extent of dendrite regeneration to that during early larval development, it seems likely that either factors downstream from Akt are developmentally regulated to turn off the regeneration program at later stages or, alternatively, other pathways may contribute to this inhibition (Song, 2012).

Whereas dendrite and axon regeneration display differences with respect to developmental regulation, the PTEN-Akt pathway is important for regeneration of axons as well as dendrites. This pathway not only regulates the extent of dendrite growth of class IV da neurons during development, but also affects their dendrite regeneration and axon regeneration in the CNS. Together with previous work (Park, 2008; Liu, 2010), these results support the notion that modulating neuronal intrinsic PTEN and Akt activity is a potential therapeutic strategy for promoting axon regeneration and functional repair after CNS trauma (Song, 2012).

This work focuses on class IV da neurons, which behave very differently from class I da neurons in regeneration. In particular, unlike class I da neurons, the class IV da neuron is capable of regenerating its axon in the periphery but not inside the CNS, thereby providing the first example of this phenomenon in invertebrates. A recent study of Caenorhabditis elegans PLM neurons, a type of mechanosensory neurons that consistently regrow their axons upon laser-mediated axotomy, has identified multiple genes important for axon regeneration (Chen, 2011), illustrating the power of the genetic approach. The injury model involving Drosophila class IV da neuron axotomy in the CNS (VNC) in the current study has the additional feature that it resembles the injury model involving mammalian DRG neuron axotomy in the CNS (spinal cord) at the cellular and molecular level: Both da neuron and DRG neuron axons regenerate poorly in the CNS even though they display robust regeneration in the periphery, and in both cases, axon regeneration in the CNS is enhanced by activation of the PTEN-Akt pathway. Importantly, while the PTEN-Akt pathway has been shown to be critical for mammalian axon regeneration in the CNS, this has not been shown in invertebrate models; for example, in C. elegans, PTEN (DAF-18) has no effect on axon regrowth. The current finding underscores the usefulness of the Drosophila system that this study developed as a model to uncover evolutionarily conserved mechanisms for CNS axon regeneration. Moreover, the elaborate and stereotyped dendritic branching pattern of da neurons provides a sensitive assay system to begin studying the injury responses and regeneration of dendrites, which may yield clues to facilitate studies of mammalian neuronal dendrites and identify novel approaches to promote dendrite recovery for the treatment of nervous system trauma (Song, 2012).

Roles of PINK1, mTORC2, and mitochondria in preserving brain tumor-forming stem cells in a noncanonical Notch signaling pathway

The self-renewal versus differentiation choice of Drosophila and mammalian neural stem cells (NSCs) requires Notch (N) signaling. How N regulates NSC behavior is not well understood. This study shows that canonical N signaling cooperates with a noncanonical N signaling pathway to mediate N-directed NSC regulation. In the noncanonical pathway, N interacts with PTEN-induced kinase 1 (PINK1) to influence mitochondrial function, activating mechanistic target of rapamycin complex 2 (mTORC2)/AKT signaling. Importantly, attenuating noncanonical N signaling preferentially impairs the maintenance of Drosophila and human cancer stem cell-like tumor-forming cells. These results emphasize the importance of mitochondria to N and NSC biology, with important implications for diseases associated with aberrant N signaling (Lee, 2013).

These results uncover a novel mechanism of N in regulating NSC self-renewal and maintenance through a noncanonical signaling pathway involving PINK1, mTORC2, and AKT. A central feature of this noncanonical N signaling pathway is specific mitochondrial roles of N in regulating respiratory chain complex (RCC) function through direct interactions with PINK1 and select RCC subunits and in activating mTORC2. N could act through a number of possible mechanisms, such as facilitating the import or assembly of RCC components, as suggested by its interaction with complex I subunits, or directing the quality control of mitochondria, as has been implicated for PINK1. Future studies will determine the exact domains of N involved in PINK1 interaction and whether noncanonical N signaling is ligand-dependent. Although the exact mechanism remains to be determined, the results will help in understanding earlier observations in Drosophila that mutations in N affected mitochondrial respiration and data from mammalian systems implicating N in mitochondrial and metabolic regulation. The physiological significance of this newly defined noncanonical N pathway is also underscored by the phenotypes of various diseases associated with N dysregulation. For example, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a disease caused by mutations in NOTCH3, is associated with mitochondrial impairment. On the other hand, GOF mutations of Notch1 are implicated in over half of human T-cell acute lymphoblastic leukemia (T-ALL), in which a pathogenic role of mTORC2 has been proposed, although how N impinges on mTORC2 in this setting is unknown. Mammalian N has been shown to act through AKT and mitochondria to promote T-cell survival, although the mechanism is distinct from the one uncovered in this study. Finally, mTORC2 was shown to be required for the self-renewal and maintenance of cancer stem cells but dispensable in normal stem cells. The findings that cancer stem cell-like brain tumor-forming cells are particularly dependent on the noncanonical N pathway in flies and humans identify the newly discovered noncanonical N signaling pathway as a potential target for disease intervention (Lee, 2013).

Insulin signaling regulates neurite growth during metamorphic neuronal remodeling

Although the growth capacity of mature neurons is often limited, some neurons can shift through largely unknown mechanisms from stable maintenance growth to dynamic, organizational growth (e.g. to repair injury, or during development transitions). During insect metamorphosis, many terminally differentiated larval neurons undergo extensive remodeling, involving elimination of larval neurites and outgrowth and elaboration of adult-specific projections. This study shows in the fruit fly that a metamorphosis-specific increase in insulin signaling promotes neuronal growth and axon branching after prolonged stability during the larval stages. FOXO, a negative effector in the insulin signaling pathway, blocks metamorphic growth of peptidergic neurons that secrete the neuropeptides CCAP and bursicon. RNA interference and CCAP/bursicon cell-targeted expression of dominant-negative constructs for other components of the insulin signaling pathway (InR, Pi3K92E, Akt1, S6K) also partially suppresses the growth of the CCAP/bursicon neuron somata and neurite arbor. In contrast, expression of wild-type or constitutively active forms of InR, Pi3K92E, Akt1, Rheb, and TOR, as well as RNA interference for negative regulators of insulin signaling (PTEN, FOXO), stimulate overgrowth. Interestingly, InR displays little effect on larval CCAP/bursicon neuron growth, in contrast to its strong effects during metamorphosis. Manipulations of insulin signaling in many other peptidergic neurons revealed generalized growth stimulation during metamorphosis, but not during larval development. These findings reveal a fundamental shift in growth control mechanisms when mature, differentiated neurons enter a new phase of organizational growth. Moreover, they highlight strong evolutionarily conservation of insulin signaling in neuronal growth regulation (Gu, 2014).

It is well established that insulin/insulin-like signaling (IIS) is crucial for regulating cell growth and division in response to nutritional conditions in Drosophila. However, most studies have focused on growth of the body or individual organs, and comparatively little is known about the roles of IIS during neuronal development, particularly in later developmental stages. Drosophila InR transcripts are ubiquitously expressed throughout embryogenesis, but are concentrated in the nervous system after mid-embryogenesis and remain at high levels there through the adult stage. This suggests that IIS plays important roles in the post-embryonic nervous system. Recently, analysis of Drosophila motorneurons, mushroom body neurons, and IPCs has revealed important roles of PI3K and Rheb in synapse growth or axon branching. These studies revealed some growth regulatory functions of IIS in the CNS, but they have not explored whether the control of neuronal growth by IIS is temporally regulated (Gu, 2014).

This study has shown that IIS strongly stimulates organizational growth of neurons during metamorphosis, whereas the effects of IIS on larval neurons are comparatively modest. Recently, similar results have been reported in mushroom body neurons, in which the TOR pathway strongly promoted axon outgrowth of γ-neurons after metamorphic pruning. Expression of FOXO or reduction of InR function had no significant effect on larval growth of the CCAP/bursicon neurons, or on the soma size of many other larval neurons. Thus, while IIS has been shown to regulate motorneuron synapse expansion in larvae, the current findings indicate that IIS may not play a major role in regulating structural growth in many larval neurons. This is consistent with a recent report that concluded that the Drosophila larval CNS is insensitive to changes in IIS (Gu, 2014).

When InRact was used to activate IIS without ligand, a modest but significant increase was seen in the soma size of the more anterior CCAP/bursicon neurons during larval development. This result indicates that the IIS pathway is present and functional in these larval neurons, but the ligand for InR is either absent or inactive. During metamorphosis, unlike in larvae, downregulation of IIS by altering the level of InR or downstream components of the pathway significantly reduced CCAP/bursicon neuron growth. Thus, the results suggest that IIS is strongly upregulated during metamorphosis to support post-embryonic, organizational growth of diverse peptidergic neurons, and this activation may at least in part be due to the presence of as yet unidentified InR ligands during metamorphosis (Gu, 2014).

Attempts were to identify this proposed InR ligand source by eliminating, in turn, most of the known sources of systemic DILPs. None of these manipulations had any effect on metamorphic growth of the CCAP/bursicon neurons. These results are consistent with three possible mechanisms. First, there may be a compensatory IIS response to loss of some dilp genes. For example, a compensatory increase in fat body DILP expression has been observed in response to ablation of brain dilp genes. Second, the growth may be regulated by another systemic hormone (e.g. DILP8) that was not tested, or by residual DILP peptides in the RNAi knockdown animals. Third, a local insulin source may be responsible for stimulating metamorphic outgrowth of the CCAP/bursicon neurons. Consistent with this view, a recent report showed that DILPs secreted from glial cells were sufficient to reactivate neuroblasts during nutrient restriction without affecting body growth, while overexpression of seven dilp genes (dilp1-7) in the IPCs had no effect on neuroblast reactivation under the same conditions. It seems likely that glia or other local DILP sources play an important role in regulating metamorphic neuron growth, but further experiments will be needed to test this model (Gu, 2014).

When IIS was manipulated in the CCAP/bursicon neurons, changes were observed in cell body size (and sometimes shape) and in the extent of branching in the peripheral axon arbor. Although this study focused analysis of neurite growth on the peripheral axons, which are easily resolved in fillet preparations, corresponding changes were also observed in the size and complexity of the central CCAP/bursicon neuron arbor. These IIS manipulations (both upregulation or downregulation) resulted in the above structural changes as well as wing expansion defects, suggesting that the normal connectivity of the CCAP/bursicon neurons was required for proper functioning of this cellular network. This model is consistent with the observation of two subsets of morphologically distinct bursicon-expressing neurons (the BSEG and BAG neurons), which are activated sequentially to control central and peripheral aspects of wing expansion. The BSEG neurons project widely within the CNS to trigger wing expansion behavior as well as secretion of bursicon by the BAG neurons. In turn, the BAG neurons send axons into the periphery to secrete bursicon into the hemolymph to control the process of tanning in the external cuticle. Therefore, manipulation of IIS within these neurons, and the changes in morphology that result, may disrupt the wiring and function of this network. However, because the possibility cannot be ruled out that these IIS manipulations also altered neuronal excitability, synaptic transmission, or neuropeptide secretion, this study relied on measurements of cellular properties (and not wing expansion rates) when assessing the relative effects of different IIS manipulations on cell growth (Gu, 2014).

The results indicate that IIS is critical for organizational growth, which occurs during insect metamorphosis but is also seen during neuronal regeneration in other systems. However, the regenerative ability of many neurons is age-dependent and context-dependent; immature neurons possess a more robust regenerative capacity, while the regenerative potential of many mature neurons is largely reduced. In particular, the adult vertebrate CNS displays very limited regeneration, in marked contrast to the regeneration abilities displayed by the peripheral nervous system. Recent studies in mice suggest that age-dependent inactivation of mTOR contributes to the reduced regenerative capacity of adult corticospinal neurons, and activation of mTOR activity through PTEN deletion promoted robust growth of corticospinal tract axons in injured adult mice. The current genetic experiments demonstrate a requirement for activity of TOR, as well as several other IIS pathway components both upstream and downstream of TOR, in controlling organizational growth of many peptidergic neurons. This suggests that under certain conditions, the activation of IIS may be a crucial component of the conversion of mature neurons to a more embryonic-like state, in which reorganizational growth either after injury or as a function of developmental stage is possible. Given the strong evolutionary conservation of these systems and the powerful genetic tools available to identify novel regulatory interactions in Drosophila, studies on the control of organizational growth in this species hold great promise for revealing factors that are crucial for CNS regeneration (Gu, 2014).

Effects of Mutation or Deletion

The decision between survival and death is an important aspect of cellular regulation during development and malignancy. Central to this regulation is the process of apoptosis, which is conserved in multicellular organisms. A variety of signaling cascades have been implicated in the modulation of apoptosis, including the phosphatidylinositol (Pl) 3-kinase pathway. Activation of Pl 3-kinase is protective, and inhibition of this lipid kinase enhances cell death under several conditions, including deregulated expression of c-Myc, neurotrophin withdrawal and anoikis. Recently, the protective effects of Pl 3-kinase have been linked to its activation of the pleckstrin homology (PH)-domain-containing protein kinase B (PKB or AKT). PKB/AKT was identified from an oncogene, v-akt, found in a rodent T-cell lymphoma. To initiate a genetic analysis of PKB, a Drosophila PKB/AKT mutant (termed Dakt1) was isolated and characterized. It exhibits ectopic apoptosis during embryogenesis as judged by induction of membrane blebbing, DNA fragmentation and macrophage infiltration. These data implicate Dakt1 as a cell survival gene in Drosophila, consistent with cell protection studies in mammals (Staveley, 1998).

A single amino-acid change, F327I, was found to be a kinase dead mutant form of Akt1. This phenylalanine (F) residue is a core residue in subdomain VII of the kinase catalytic domain, forming the 'DFG' motif which is highly conserved among protein kinases including mammalian PKB proteins. Since Dakt1 is a maternally expressed gene, the maternal contribution of Dakt1 was tested using germline clone (GLC) analysis. GLC q females, containing clones of cells bearing the F327I mutation, produce embryos that lack various portions of larval cuticle by the end of embryogenesis. The range of phenotypes depends on the level of zygotic Dakt1 activity in the embryo. Without any zygotic expression of Dakt1, q GLC embryos produce only a few scraps of cuticle. When q GLC embryos express some zygotic Dakt1, some ventral cuticle is produced. This phenotype is extensively suppressed by expression of Dakt1 using a heat-shock inducible hs-Dakt1 transgene. Since mammalian PKB/AKT has been implicated in anti-apoptotic activity, a test was performed of Dakt1 GLC embryos for evidence of apoptosis. Acridine orange (AO) staining has been shown to be a good indicator of apoptosis (and not necrosis) in Drosophila, detecting cellular events such as membrane blebbing. AO staining of Dakt1 GLC embryos shows extensive apoptosis compared to wild-type embryos. Confirmation of an effect on apoptosis was performed using a TUNEL assay in Dakt1 embryos to detect the incidence of DNA fragmentation. Dakt1 embryos shows extensive DNA breakage as assayed by TUNEL in situ. Incidence of TUNEL signal in Dakt1 GLC embryos precedes the initiation of the signal in wild-type embryos. TUNEL signal accumulates during development of Dakt1 embryos to engulf the majority of the embryo. During apoptosis, macrophages converge to engulf cellular fragments by phagocytosis. Antibody to Croquemort (Crq), the Drosophila homolog of CD36, was used to detect macrophages in embryos. Dakt1 embryos exhibit a significant increase in Crq expression, as compared to wild-type embryos. This expression is at the cell surface and focuses on the apoptotic cells. It appears, therefore, that loss of Dakt1 activity results in premature and ectopic apoptosis with the characteristics of membrane blebbing, DNA fragmentation and macrophage-mediated endocytosis (Staveley, 1998).

The deficiency of the reaper (rpr), grim and hid genes [Df(3L)H99] blocks apoptosis in Drosophila. Overexpression of any of these three genes results in ectopic apoptosis in embryos. It was therefore of interest to see whether Dakt1 mutants result in apoptosis through the mis-expression of rpr, grim or hid. This was found not to be the case, since Dakt1 mutant embryos do not show overexpression of these genes. Loss of rpr, grim and hid in H99 do not suppress the phenotype of Dakt1 GLC embryos. These results suggest that Dakt1 and H99 modulate apoptosis via distinct mechanisms. To test the involvement of caspases in Dakt1-mediated apoptosis, the baculoviral caspase-inhibitory protein p35 was expressed in Dakt1 embryos. Ectopic p35 has been shown to block caspase activity and suppress apoptosis in Dakt1 embryos, and hs-p35 effectively blocks apoptosis in Dakt1 embryos, demonstrating the requirement for caspase activity in this process (Staveley, 1998).

These epistasis tests suggest that Dakt1 does not function upstream of the rpr, grim and hid gene functions in the embryo. It is possible, though, that Dakt1 might be regulated by the rpr, grim and hid genes (at the H99 locus) and in fact act downstream of these genes. This presents two possibilities: (1) Dakt1 and the H99 locus represent independent pathways; (2) the H99 locus might repress Dakt1 function. This study thus provides the first genetic evidence implicating PKB as an anti-apoptotic factor (Staveley, 1998).

To determine whether Akt1 transduces growth-related signals, Akt1-deficient somatic clones were generated in the developing eye by mitotic recombination. Adult eyes exhibit a reduction in size in Akt1-deficient rhabdomeres, which, in some instances, co-exist with normal-sized heterozygous cells in the same ommatidium. Akt1 mutant clones are rare and small and are obtained only after heat-shock during the third instar larval stage. These observations indicate that the lack of Akt1 clones in the adult retina following induction at early larval stages may have resulted from cell competition, by which the Akt1-deficient cells would be eliminated and replaced by the surrounding wild-type sister cells. The phenotype of the Akt1-deficient rhabdomeres may have resulted from perturbations of cell growth or proliferation. The smaller size of these rhabdomeres shows that Akt1 is essential for normal cell growth, but dispensable for cell-fate determination. Moreover, the co-existence of Akt1 mutant rhabdomeres with wild-type twin-spot rhabdomeres in the same ommatidium suggests a cell-autonomous control of cell growth by Akt1 (Verdu, 1999).

The small size of the clones of Akt1-deficient cells could be the result of an impairment in the proliferation, survival, or both, of homozygous null cells. To evaluate more specifically the effects of Akt1 on proliferation or growth in vivo, upstream activation sequence (UAS)-Akt1 lines were generated with which to investigate the effects of altering the amount of Akt1 during Drosophila eye and wing imaginal disc development. The gmrGAL4 transgene targets expression of Akt1 to cells posterior to the morphogenetic furrow, producing flies exhibiting enlarged and bulging eyes with a mild disruption of the regular, external lattice. Similar, but less pronounced, effects are observed with a sevGAL4 transgene. Quantitative analysis reveals that the Akt1-induced increase in the size of the eye is caused by an increase in the size but not in the number of ommatidia. To determine the extent of this phenotype, tangential sections of these eyes were examined. In spite of the rough appearance of the adult compound eye, Akt1 expression does not affect the normal process of photoreceptor cell-fate determination in these larger ommatidia (Verdu, 1999).

Akt plays a central part in promoting the survival of a wide range of cell types in mammalian systems and in Drosophila embryos. However, overexpression of Akt1 does not alter the normal rate of apoptosis in the eye, as shown by equivalent acridine orange staining in control and gmrGAL4/UAS-Akt1 eye imaginal discs. Hence, overexpression of Akt1 affects neither the normal processes of cell-fate determination nor apoptosis in the developing retina (Verdu, 1999).

Pten, a Drosophila homolog of the mammalian PTEN tumor suppressor gene, plays an essential role in the control of cell size, cell number, and organ size. In mosaic animals, Pten minus cells proliferate faster than their heterozygous siblings, show an autonomous increase in cell size, and form organs of increased size, whereas overexpression of Pten results in opposite phenotypes. The loss-of-function phenotypes of Pten are suppressed by mutations in the PI3K target Dakt1 and the translational initiation factor eif4A, suggesting that Pten acts through the PI3K signaling pathway to regulate translation. Although activation of PI3K and Akt has been reported to increase rates of cellular growth but not proliferation, loss of Pten stimulates both of these processes, suggesting that PTEN regulates overall growth through PI3K/Akt-dependent and -independent pathways. Furthermore, Pten does not play a major role in cell survival during Drosophila development. These results provide a potential explanation for the high frequency of PTEN mutation in human cancer (Gao, 2000).

Previous studies have shown that loss of PTEN function promotes cell survival in mammals through activation of Akt. In addition, PTEN acts through Akt in metabolic and longevity control in C. elegans. Dakt1, a Drosophila homolog of Akt, has been suggested to play a role in cell survival in embryogenesis (Staveley, 1998) and cell size control (Verdu, 1999). A hypomorphic allele of Dakt1 has been identified in the large-scale gene disruption project carried out by the Berkeley Drosophila Genome Project. This allele is semilethal, and homozygous survivors show reduced body size and cell size, consistent with a role of Dakt1 in growth control. To examine whether Pten controls cell size through regulating Akt activity, Pten mutant clones were generated in Dakt1 mutant animals. Dakt1 mutation completely suppressed the increase of cell size associated with the Pten mutation. This result provides strong in vivo evidence that Dakt1 functions downstream of (or in parallel to) Pten in the control of cell size. Taken together, these genetic interactions suggest that the role of Pten in opposing signaling through the PI3K/Akt pathway is conserved between flies and vertebrates (Gao, 2000).

A mutation has been isolated in the Drosophila homolog of TSC1 (Tsc1). Cells mutant for Tsc1 are dramatically increased in size yet differentiate normally. Organ size is also increased in tissues that contain a majority of mutant cells. Clones of Tsc1 mutant cells in the imaginal discs undergo additional divisions but retain normal ploidy. Flow cytometry analysis indicates that the increase in cell size is not due to endoreplication. Tsc1 protein is shown to bind to Drosophila Tsc2 in vitro. Overexpression of Tsc1 or Tsc2 alone in the wing and eye has no effect, but co-overexpression leads to a decrease in cell size, cell number, and organ size. Genetic epistasis data are consistent with a model that Tsc1 and Tsc2 function together in the insulin signaling pathway (Potter, 2001).

Recent work has demonstrated that the insulin signaling pathway plays an important role in the regulation of cell size, cell number, and organ size. Mutations of Drosophila PTEN (dPTEN), which functions as a negative regulator of insulin signaling, result in phenotypes that resemble the effects of Tsc1 and Tsc2 mutations. Therefore, genetic epistasis experiments were performed to test whether Tsc1 or Tsc2 might also function to negatively regulate insulin signaling. Overexpression of Drosophila insulin receptor (dinr) using the eyGAL4 driver leads to lethality at 25°C and a dramatic increase in ommatidia number in escapers at room temperature. Co-overexpression of Tsc1 and Tsc2 (but not either Tsc1 or Tsc2 alone) rescues both the lethality and the extra ommatidia phenotype caused by dinr overexpression. Furthermore, overexpression of dinr using the pGMR-GAL4 driver leads to an increase in ommatidium size, which is also suppressed by co-overexpression of Tsc1 and Tsc2. Clones of dinr mutant ommatidia are smaller in size than wild-type. Ommatidia that are mutant for both dinr and Tsc1, however, exhibit the Tsc1 mutant phenotype of increased ommatidium size (Potter, 2001).

Overexpression of dPTEN using the pGMR-GAL4 driver leads to eyes with a decreased ommatidium size. However, overexpression of dPTEN is unable to suppress the clonal Tsc1 mutant phenotype. Similar to dinr, clones of dAkt mutant ommatidia are smaller in size. Ommatidia mutant for both dAkt and Tsc1 display the Tsc1 phenotype. Similarly, ommatidia that contained Tsc2 mutant clones in a dAkt mutant background exhibit the Tsc2 mutant phenotype. These results suggest that in the eye, Tsc1 and Tsc2 function genetically epistatic to (downstream of) dinr, dPTEN, and dAkt (Potter, 2001).

Genetic analyses suggest that the TSC genes act in a parallel pathway that converges on the insulin pathway downstream from Akt. The most convincing evidence for a functional link between the TSC genes and insulin signaling comes from the observation that heterozygosity of TSC1 or TSC2 is sufficient to rescue the lethality of loss-of-function InR mutants. This argues that the TSC genes are intimately linked to insulin signaling, rather than functioning in a totally independent cell-growth pathway. These results suggest that the TSC tumor suppressor genes are novel negative regulators of insulin signaling, and modulating the activities of the TSC genes might provide a potential way to correct insulin signaling defects in certain diseases such as diabetes and obesity (Gao, 2001).

Previous studies have shown that loss of inr or Akt leads to decreased cell size. To investigate the relationship between inr, Akt, and the TSC genes, TSC1;Akt and TSC1;inr double-mutant clones were studied. Cells homozygous for a strong allele of inr, or a null allele of Akt are smaller, and are rarely recovered in adult eye clones because of cell competition during development. However, TSC1;inror TSC1;Akt1 double-mutant cells showed a similar cell size increase as that observed in TSC1- cells. Furthermore, the competitive disadvantage of inr and Akt mutant cells is also rescued in the TSC1;inr or TSC1;Akt1 double-mutant clones, resulting in larger clones that contained more cells. This result suggests that TSC1 acts genetically downstream from Akt. This observation is compatible with either TSC1 acting molecularly downstream from Akt in the linear InR-PI3K-Akt pathway, or TSC1 acting in a parallel pathway that converges on the insulin pathway downstream from Akt (Gao, 2001).

The Drosophila gene chico encodes an insulin receptor substrate that functions in an insulin/insulin-like growth factor (IGF) signaling pathway. In the nematode C. elegans, insulin/IGF signaling regulates adult longevity. Mutation of chico extends fruit fly median life-span by up to 48% in homozygotes and 36% in heterozygotes. Extension of life-span is not a result of impaired oogenesis in chico females, nor is it consistently correlated with increased stress resistance. The dwarf phenotype of chico homozygotes was also unnecessary for extension of life-span. The role of insulin/IGF signaling in regulating animal aging is therefore evolutionarily conserved (Clancy, 2001).

In Drosophila, the insulin/IGF receptor INR, the insulin receptor substrate Chico, the phosphatidylinositol 3-kinase (PI3K) Dp110/p60, and the PI3K target protein kinase B (PKB, also known as DAkt1) form a signaling pathway that regulates growth and size. The effects on aging of hypomorphic mutations in Inr (equivalent to daf-2) and PKB, and null mutations in chico and the catalytic (Dp110, equivalent to age-1) and adapter (p60) PI3K subunits were examined. All mutants were tested as heterozygotes. chico1 and PKB3 homozygotes and InrGC25/InrE19 transheterozygotes, which form viable dwarf adults, were also examined. The remaining mutations were homozygous lethal (Clancy, 2001).

Most mutants tested had normal or significantly decreased life-span. For example, PKB3 homozygotes and InrGC25/InrE19 flies are short-lived. By contrast, chico1 extends life-span. Homozygous chico1 females exhibit an increase of median and maximum life-span of up to 48% and 41%, respectively. chico1 heterozygotes also exhibit increases in median life-span of up to 36% and 13% in females and males, respectively. Homozygous males, however, are slightly short-lived (Clancy, 2001).

Of the mutations tested, only chico1 increases life-span. This may be because the effect of reduced IIS on life-span depends on the degree to which signaling is reduced. Unlike the other null mutations in IIS genes tested, chico1 is not homozygous lethal, presumably because the INR receptor can signal to PI3K directly, as well as indirectly via Chico. Thus, chico1 mutants may be long-lived because of the relatively mild reduction in pathway activity that they bring about. Notably, severe IIS mutations in C. elegans can cause premature mortality in some adults, although the maximum life-span of populations is invariably increased. This is probably why InrGC25/InrE19 flies are short-lived: Demographic analysis indicates that a reduction in the age-specific mortality rate acceleration occurs, whose effect on survival is masked by an elevated rate of age-independent mortality. Furthermore, a different heteroallelic Drosophila Inr mutant to that tested here exhibits an 85% increase in female life-span. By contrast, in short-lived PKB3 populations, no reduction in mortality rate acceleration is seen. This raises the possibility that a second pathway downstream of chico might regulate aging in Drosophila. Interestingly, Chico contains potential binding sites for the Drk/Grb2 docking protein, consistent with signaling via Ras/mitogen-activated protein kinase (Clancy, 2001).

Analysis of Drosophila Foxo indicates that it is a critical PKB target, but that it mediates only one aspect of PKB function. Several lines of evidence support this model. (1) The effects of ectopic overexpression of Foxo and the human homolog hFOXO3a in the developing Drosophila eye are altered by Dp110 and PKB signaling as well as by nutrient levels. Under conditions of lowered insulin signaling, the phenotypes resulting from expression of foxo and hFOXO3a are dramatically enhanced. This situation was mimicked by expressing a PKB-insensitive phosphorylation mutant, suggesting that endogenous PKB signaling is required to mitigate the effects of ectopically expressed Foxo and hFOXO3a. (2) The physiological relevance of Foxo in PKB signaling is most vividly demonstrated by the observation that the larval lethality associated with the complete loss of PKB is rescued by foxo mutations to the extent that some flies develop to pharate adults. The lethality associated with loss of PKB function is therefore to a large extent due to the hyperactivation of Foxo. (3) Loss of Foxo function suppresses the effects of insulin-signaling mutations only partially; Foxo mediates a reduction in cell number but not in cell size in response to reduced insulin signaling (Jünger, 2003).

Genetic analysis of the control of body size in Drosophila has revealed two classes of mutations. Flies carrying mutations in chico or viable allelic combinations of Inr, Dp110, and PKB are reduced in body size by up to 50% owing to a reduction in both cell size and cell number. Conversely, flies mutant for S6K exhibit a more moderate reduction in body size, caused almost exclusively by a reduction in cell size. This suggests that the pathways controlling cell number and cell size bifurcate at or below PKB. Although foxo single mutants have no obvious size phenotype, loss of foxo substantially suppresses the cell-number reduction observed in insulin-signaling mutants. It appears that Foxo mediates the repression of proliferation in flies mutant for Inr, chico, Dp110, and PKB without being required for the reduction in cell size. Chico-Foxo double mutant flies even have slightly smaller cells than chico mutants, suggesting that removal of Foxo permits cell-cycle acceleration under conditions of impaired insulin signaling. The pathway controlling body size in response to insulin therefore bifurcates at the level of PKB: PKB controls cell number by inhibiting Foxo function and PKB controls cell size, at least under some conditions, by regulating S6K activity by phosphorylation of TSC2 (Jünger, 2003).

The signaling systems controlling cell size and cell number are tightly interconnected. Genetic and biochemical analyses have revealed five different links between the TSC-TOR-S6K pathway and the Inr-PKB-Foxo pathway. (1) Under conditions of unnaturally high insulin-signaling activity (that is, following the oncogenic activation of PKB) PKB phosphorylates and inactivates TSC2, resulting in increased activation of S6K. Under normal culture conditions this regulation does not seem critical, however, loss of dPKB function does not lower dS6K activity in larval extracts. (2) Under physiological conditions, PDK1 regulates PKB as well as S6K. (3) S6K itself downregulates dPKB activity in a negative feedback loop. (4) Under severe starvation conditions, nuclear Foxo presumably activates target genes that reduce cell proliferation. One of these target genes is 4E-BP, which encodes an inhibitor of translation initiation. When conditions improve, the insulin and TOR signaling pathways can stimulate translation by disrupting the 4E-BP/eIF4E complex via phosphorylation of 4E-BP, and in parallel by repressing FOXO-dependent 4E-BP expression. (5) Under even more severe starvation or stress conditions, full activation of Foxo upregulates expression of the insulin receptor itself, thus rendering the cell hypersensitive to low insulin levels. These multiple positive and negative interactions ensure a continuous fine adjustment of the growth rate to changing environmental conditions (Jünger, 2003).

Scylla acts downstream of PKB but upstream of TSC

Diverse extrinsic and intrinsic cues must be integrated within a developing organism to ensure appropriate growth at the cellular and organismal level. In Drosopohila, the insulin receptor/TOR/S6K signaling network plays a fundamental role in the control of metabolism and cell growth. scylla and charybdis (a. k. a. charybde), two homologous genes identified as growth suppressors in an EP (enhancer/promoter) overexpression screen, act as negative regulators of growth. The genes are named after mythological monsters that lived in the Strait of Messina between Sicily and Italy, posing a threat to the passage of ships. The simultaneous loss of both genes generates flies that are more susceptible to reduced oxygen concentrations (hypoxia) and that show mild overgrowth phenotypes. Conversely, either scylla or charybdis overactivation reduces growth. Growth inhibition is associated with a reduction in S6K but not PKB/Akt activity. Together, genetic and biochemical analysis places Scylla/Charybdis downstream of PKB and upstream of TSC1. Furthermore, scylla and charybdis are induced under hypoxic conditions and scylla is a target of Drosopohila HIF-1 (hypoxia-inducible factor-1: Similar) like its mammalian counterpart RTP801/REDD1, thus establishing a potential cross-talk between growth and oxygen sensing (Reiling, 2004).

Although loss of Scylla function does not produce a mutant phenotype on its own, whether it would alter the PKB/PDK1 overexpression eye phenotype was tested. Indeed, loss of Scylla function enhances the PKB/PDK1 overgrowth phenotype. Thus, Scylla is essential for attenuating the increased growth in response to hyperactivation of the Inr pathway. Furthermore, loss of Scylla partially suppresses the growth reduction associated with reduced PKB function as assessed by comparing weights of PKB3 single mutants to scy31 PKB3 double mutants. In contrast, complete loss of Scylla in a heteroallelic S6K combination does not rescue the S6K single mutant phenotype indicating that S6K is epistatic over scylla (Reiling, 2004).

Moreover, verexpression of scylla and charybdis not only suppresses the growth phenotype caused by over-activation of the Inr pathway in the eye but to a certain extent also rescues the lethality associated with the ubiquitous increase in Inr pathway activity due to either overexpression of PKB or loss of PTEN. scylla rescues the male-specific lethality caused by ubiquitous expression of PKB and organismal lethality associated with the partial but not complete loss of PTEN function. Similarly, PKB-associated male lethality is also rescued by charybdis overexpression. This indicates that scylla and charybdis have the capacity to act as potent negative regulators of insulin signaling downstream of PKB and PDK1 (Reiling, 2004).

Several lines of evidence suggest that Scylla and Charybdis act upstream of TSC and Rheb. Tsc1/2 mutant flies can be rescued to adulthood by reducing S6K signaling, and a mere reduction of one TOR copy in a Tsc1 mutant context results in a rescue to the pupal stage. Whether ubiquitous scylla overexpression could rescue the larval lethality of heteroallelic Tsc1/2 mutant combinations (Tsc12G3/Tsc1Q87X and Tsc256/Tsc2192) was examined using the da-Gal4 or Act5C-Gal4 drivers in combination with a UAS-scy transgene or EPscy at 18°C, 25°C, and 29°C. Ubiquitous overexpression of scylla/charybdis in a Tsc1/2 mutant background did in no case extend larval development beyond first/second instar, and these larvae died at the same time as Tsc1/2 mutants. Moreover, the big head phenotype of Tsc2192 (and Tsc256) induced by the eyflp/FRT system was not further enhanced in scyEP9.85 char180 Tsc2 triple-mutant heads. It has been shown that heads composed almost entirely of scylla charybdis double-mutant cells are enlarged. Conversely, GMRGal4-driven co-overexpression of Tsc1, Tsc2, and scylla or charybdis in the eye does not further reduce the small eye phenotype induced by coexpression of Tsc1 and Tsc2 on their own. The absence of an additive growth effect upon loss of Tsc2, scylla, and charybdis or overexpression of Tsc1/2 and scylla or charybdis suggests that they function in the same pathway. These results are consistent with the idea that Scylla and Charybdis act upstream of the TSC complex. This conclusion is further supported by the fact that neither a Rheb-dependent bulging eye phenotype nor organismal lethality could be suppressed by scylla/charybdis coexpression (Reiling, 2004).

Insulin signaling mediates sexual attractiveness in Drosophila

Sexually attractive characteristics are often thought to reflect an individual's condition or reproductive potential, but the underlying molecular mechanisms through which they do so are generally unknown. Insulin/insulin-like growth factor signaling (IIS) is known to modulate aging, reproduction, and stress resistance in several species and to contribute to variability of these traits in natural populations. This study shows that IIS determines sexual attractiveness in Drosophila through transcriptional regulation of genes involved in the production of cuticular hydrocarbons (CHC), many of which function as pheromones. Using traditional gas chromatography/mass spectrometry (GC/MS) together with newly introduced laser desorption/ionization orthogonal time-of-flight mass spectrometry (LDI-MS), it was established that CHC profiles are significantly affected by genetic manipulations that target IIS. Manipulations that reduce IIS also reduce attractiveness, while females with increased IIS are significantly more attractive than wild-type animals. IIS effects on attractiveness are mediated by changes in CHC profiles. Insulin signaling influences CHC through pathways that are likely independent of dFOXO and that may involve the nutrient-sensing Target of Rapamycin (TOR) pathway. These results suggest that the activity of conserved molecular regulators of longevity and reproductive output may manifest in different species as external characteristics that are perceived as honest indicators of fitness potential (Kuo, 2012).


C. elegans SGK-1 is the critical component in the Akt/PKB kinase complex to control stress response and life span

The DAF-2 insulin receptor-like signaling pathway controls metabolism, development, longevity, and stress response in C. elegans. SGK-1, the C. elegans homolog of the serum- and glucocorticoid-inducible kinase SGK, acts in parallel to the AKT kinases to mediate DAF-2 signaling. Loss of sgk-1 results in defective egg-laying, extended generation time, increased stress resistance, and an extension of life span. SGK-1 forms a protein complex with the AKT kinases, and is activated by and strictly depends on PI3K-like dependent kinase (PDK-1; Protein kinase 61C in Drosophila, the central mediator of cell signaling between phosphoinositide 3-kinase and various intracellular serine/threonine kinases including Akt). All three kinases of this complex are able to directly phosphorylate DAF-16/FKHRL1, yet have different functions in DAF-2 signaling. Whereas AKT-1 and AKT-2 are more important for regulating dauer formation, SGK-1 is the crucial factor for the control of development, stress response, and longevity. These data also suggest the existence of a second pathway from DAF-2 to DAF-16 that does not depend on AKT-1, AKT-2, and SGK-1 (Hertweck, 2004).

The mammalian insulin signaling pathway affects multiple downstream targets mediating a variety of cellular responses to insulin. Genetic studies have shown that a similar pathway exists in C. elegans that involves insulin-like ligands, the DAF-2 insulin/IGF-I receptor-like protein, the AAP-1 PI3K-like adaptor subunit, the AGE-1 PI3K-like catalytic subunit, the DAF-18 PTEN lipid phosphatase, and the PI3K-like dependent kinase PDK-1, which activates the Akt/PKB-like serine/threonine (S/T) protein kinases AKT-1 and AKT-2. AKT-1 and AKT-2 are candidates to antagonize the forkhead transcription factor DAF-16 to repress genes that regulate diapause, longevity, stress response, and energy storage, and to activate genes necessary for metabolism and reproductive growth. Reduction of daf-2 gene activity or other genes that are positively regulated by daf-2, like aap-1, age-1, pdk-1, or akt-1/-2, cause a constitutive developmental arrest at the dauer larval stage (Daf-c). Reduction-of-function mutations in genes that antagonize daf-2 signaling, such as daf-18 and daf-16, suppress the dauer constitutive phenotype of daf-2 and the other Daf-c mutants (Hertweck, 2004 and references therein).

According to the current model of Akt/PKB activation in mammalian cells, phosphatidylinositol-3,4-diphosphate (PIP2) and/or phosphatidylinositol-3,4,5-triphosphate (PIP3) bind to the amino-terminal pleckstrin homology (PH) domain of Akt/PKB, resulting in the recruitment of Akt to the plasma membrane. PDK1 colocalizes with Akt/PKB to the cell membrane and activates it by phosphorylation. Activated Akt/PKB is capable of promoting cell survival by phosphorylating and inactivating the mammalian DAF-16 homolog FKHRL1. However, in C. elegans, RNAi depletion of the Akt/PKB homologs akt-1 and akt-2 is not sufficient to shut down the DAF-2 insulin signaling pathway entirely. Moreover, inactivation of the Akt/PKB consensus phosphorylation sites in DAF-16 was not sufficient to induce dauer formation and life span extension. Therefore, other, yet-unidentified components have to exist downstream of pdk-1 to essentially control metabolism, development, longevity, and stress response (Hertweck, 2004 and references therein).

Serum- and glucocorticoid-inducible kinases (SGKs) are S/T protein kinases with approximately 55% sequence similarity to Akt/PKB. SGK expression is regulated by a variety of external stimuli, including growth factors like TGF-β and IGF-I, steroid or peptide hormones like insulin, cytokines, alterations in cell volume, osmotic changes, and cortical brain injury. Recent studies have revealed a role for SGK in cell survival signaling depending on PI3K. SGK is thought to be activated by PDK1 at sites that are equivalent to the Akt/PKB phosphorylation sites. PDK1 uses its hydrophobic PIF binding pocket to interact with and activate SGK. SGK may promote cell survival in parallel or complementary to Akt/PKB, since it participates in phosphorylating and inactivating FKHRL1 at sites different from those modified by Akt/PKB (Hertweck, 2004 and references therein).

Using reverse genetic approaches and biochemical analyses, the C. elegans homolog of the serum- and glucocorticoid-inducible kinase, SGK-1 is shown to function in parallel to AKT-1 and AKT-2 to mediate DAF-2 insulin receptor-like signaling. SGK-1 is activated by and strictly depends on PDK-1, and binds to AKT-1 and AKT-2 to form a multimeric protein complex. Direct evidence is provided that the AKT-1/AKT-2/SGK-1 complex transduces AGE-1/PI3K signals via PDK-1 to control the intracellular localization and activation of DAF-16/FKHRL1 by phosphorylation. In this respect, AKT-1/AKT-2/SGK-1 compete with a parallel branch within the DAF-2 pathway. Moreover, inactivation of sgk-1, but not of akt-1 or akt-2, results in a retarded postembryonic development, defective egg-laying, extended life span, and increased stress tolerance. It is concluded that SGK-1 is the critical kinase of the AKT-1/AKT-2/SGK-1 regulatory complex that controls aspects of development, longevity, and stress response (Hertweck, 2004).

Rictor/TORC2 regulates fat metabolism, feeding, growth, and life span in Caenorhabditis elegans

Rictor is a component of the target of rapamycin complex 2 (TORC2). While TORC2 has been implicated in insulin and other growth factor signaling pathways, the key inputs and outputs of this kinase complex remain unknown. Mutations have been identified in the C. elegans homolog of rictor in a forward genetic screen for increased body fat. Despite high body fat, rictor mutants are developmentally delayed, small in body size, lay an attenuated brood, and are short-lived, indicating that Rictor plays a critical role in appropriately partitioning calories between long-term energy stores and vital organismal processes. Rictor is also necessary to maintain normal feeding on nutrient-rich food sources. In contrast to wild-type animals, which grow more rapidly on nutrient-rich bacterial strains, rictor mutants display even slower growth, a further reduced body size, decreased energy expenditure, and a dramatically extended life span, apparently through inappropriate, decreased consumption of nutrient-rich food. Rictor acts directly in the intestine to regulate fat mass and whole-animal growth. Further, the high-fat phenotype of rictor mutants is genetically dependent on akt-1, akt-2, and serum and glucocorticoid-induced kinase-1 (sgk-1). Alternatively, the life span, growth, and reproductive phenotypes of rictor mutants are mediated predominantly by sgk-1. These data indicate that Rictor/TORC2 is a nutrient-sensitive complex with outputs to AKT and SGK to modulate the assessment of food quality and signal to fat metabolism, growth, feeding behavior, reproduction, and life span (Soukas, 2009).

A PP2A regulatory subunit regulates C. elegans insulin/IGF-1 signaling by modulating AKT-1 phosphorylation

The C. elegans insulin/IGF-1 signaling (IIS) cascade plays a central role in regulating life span, dauer, metabolism, and stress. The major regulatory control of IIS is through phosphorylation of its components by serine/threonine-specific protein kinases. An RNAi screen for serine/threonine protein phosphatases that counterbalance the effect of the kinases in the IIS pathway identified pptr-1, a B56 regulatory subunit of the PP2A holoenzyme. Modulation of pptr-1 affects IIS pathway-associated phenotypes including life span, dauer, stress resistance, and fat storage. PPTR-1 functions by regulating worm AKT-1 phosphorylation at Thr 350. With striking conservation, mammalian B56beta regulates Akt phosphorylation at Thr 308 in 3T3-L1 adipocytes. In C. elegans, this ultimately leads to changes in subcellular localization and transcriptional activity of the forkhead transcription factor DAF-16. This study reveals a conserved role for the B56 regulatory subunit in regulating insulin signaling through AKT dephosphorylation, thereby having widespread implications in cancer and diabetes research (Padmanabhan, 2009).

AKT-1 regulates DNA-damage-induced germline apoptosis in C. elegans

The cellular response to genotoxic stress involves the integration of multiple prosurvival and proapoptotic signals that dictate whether a cell lives or dies. In mammals, AKT/PKB regulates cell survival by modulating the activity of several apoptotic proteins, including p53. In Caenorhabditis elegans, akt-1 and akt-2 regulate development in response to environmental cues by controlling the FOXO transcription factor daf-16, but the role of these genes in regulating p53-dependent apoptosis is not known. In this study, it was shown that akt-1 and akt-2 negatively regulate DNA-damage-induced apoptosis in the C. elegans germline. The antiapoptotic activity of akt-1 is independent of its target gene daf-16 but dependent on cep-1/p53. Although only akt-1 regulates the apoptotic activity of cep-1, both akt-1 and akt-2 modulate the intensity of the apoptotic response independently of the transcriptional activity of CEP-1. Finally, it was shown that AKT-1 regulates apoptosis but not cell-cycle progression downstream of the HUS-1/MRT-2 branch of the DNA damage checkpoint (Quevedo, 2007).

In C. elegans, mrt-2, hus-1, and clk-2 encode checkpoint proteins that transmit DNA-damage signals to the core apoptotic pathway through CEP-1/p53. HUS-1 and MRT-2 form part of the 9:1:1 complex, whereas CLK-2 functions in parallel to the 9:1:1 complex. In addition to activating apoptosis, these checkpoint genes also promote cell-cycle arrest in the mitotic region of the germline in response to DNA damage independently of cep-1. Checkpoint mutants also produce inviable embryos after treatment with IR because they are unable to repair damaged DNA. Because AKT-1 appears to act upstream of CEP-1/p53, it was asked whether akt-1 also has a role in the checkpoint response. It was found that germline cell-cycle arrest was not altered in either akt-1 gain-of-function or loss-of-function mutants, and the survival of progeny from akt-1(mg144) and akt-1(ok525) worms were no more sensitive to IR than wild-type worms. Therefore, these results indicate that AKT-1 does not act as a checkpoint protein but likely lies downstream of the DNA damage checkpoint to regulate the apoptotic activity of CEP-1/p53. To test this, double mutants were generated between akt-1(ok525) and loss-of-function alleles in the clk-2, mrt-2, and hus-1 checkpoint genes. It was found that clk-2(qm37);akt-1(ok525) double mutants were as resistant to damage-induced apoptosis as clk-2(qm37) single mutants, indicating that akt-1 does not act downstream of clk-2. However, irradiated mrt-2(e2663);akt-1(ok525) or hus-1(op244);akt-1(ok525) double mutants exhibited similar levels of apoptosis as irradiated wild-type controls, indicating that AKT-1 acts downstream of, or in parallel to, the 9:1:1 checkpoint. This suggests that inhibition of AKT-1 is part of the mechanism by which the HUS-1/MRT-2 complex signals to activate CEP-1/p53-dependent apoptosis in response to DNA damage. To assess this, CEP-1/p53 transcriptional activity was measured in hus-1(op244);akt-1(ok525) and mrt-2(e2663);akt-1(ok525) double-mutant animals. Although CEP-1/p53 is modestly activated in hus-1(op244) and mrt-2(e2663) single mutants treated with IR, presumably because the CLK-2 checkpoint is active, this activation was not enhanced by the akt-1(ok525) allele. Therefore, the increased germ-cell apoptosis observed in mrt-2(e2663);akt-1(ok525) and hus-1(op244);akt-1(ok525) double mutants treated with IR was not due to an increase in CEP-1/p53 transcriptional activity. Because AKT-2 is able to regulate apoptosis without affecting CEP-1/p53 transcriptional activity, hus-1(op244);akt-2(ok393) double mutants were created and similar levels of apoptosis were observed in these mutants as with the hus-1(op244);akt-1(ok525) strain. Because the increased IR-induced apoptosis observed in akt-1(ok525) mutants requires functional cep-1, these results suggest that CEP-1 may also regulate apoptosis independently of its transcriptional activity, as described in mammalian cells. The possibility that CEP-1 regulates the transcription of genes, other than egl-1, that also regulate germline apoptosis cannot be ruled out. A third possibility is that AKT-1/2 can modulate the magnitude of the apoptotic response independently of CEP-1, perhaps by regulating components of the core apoptotic pathway (Quevedo, 2007).

Signaling upstream of AKT

TRANCE, a TNF family member, and its receptor, TRANCE-R, are critical regulators of dendritic cell and osteoclast function. TRANCE activates the antiapoptotic serine/threonine kinase Akt/PKB through a signaling complex involving c-Src and TRAF6. A deficiency in c-Src or the addition of Src family kinase inhibitors blocks TRANCE-mediated PKB activation in osteoclasts. c-Src and TRAF6 interact with each other and with TRANCE-R upon receptor engagement. TRAF6, in turn, enhances the kinase activity of c-Src leading to tyrosine phosphorylation of downstream signaling molecules such as c-Cbl. These results define a mechanism by which TRANCE activates Src family kinases and PKB and provide evidence of cross-talk between TRAF proteins and Src family kinases (Wong, 1999).

The serine/threonine kinase Akt (also known as protein kinase B) (Akt/PKB) is activated upon T-cell antigen receptor (TCR) engagement or upon expression of an active form of phosphatidylinositide (PI) 3-kinase in T lymphocytes. The small GTPase Rac1 is implicated in this pathway, connecting the receptor with the lipid kinase. In Jurkat cells, activated forms of Rac1 or Cdc42, but not Rho, stimulate an increase in Akt/PKB activity. TCR-induced Akt/PKB activation is inhibited either by PI 3-kinase inhibitors (LY294002 and wortmannin) or by overexpression of a dominant negative mutant of Rac1 but not Cdc42. Accordingly, triggering of the TCR rapidly stimulates a transient increase in GTP-Rac content in these cells. Similar to TCR stimulation, L61Rac-induced Akt/PKB kinase activity is also LY294002 and wortmannin sensitive. However, induction of Akt/PKB activity by constitutively active PI 3-kinase is unaffected when dominant negative Rac1 is coexpressed, placing Rac1 upstream of PI 3-kinase in the signaling pathway. When analyzing the signaling hierarchy in the pathway leading to cytoskeleton rearrangements, it was found that Rac1 acts downstream of PI 3-kinase, a finding that is in accordance with numerous studies in fibroblasts. These results reveal a previously unrecognized role of the GTPase Rac1, acting upstream of PI 3-kinase in linking the TCR to Akt/PKB. This is the first report of a membrane receptor employing Rac1 as a downstream transducer for Akt/PKB activation (Genot, 2000).

The protein kinase Akt plays a central role in a number of key biological functions including protein synthesis, glucose homeostasis, and the regulation of cell survival or death. The mechanism by which tyrosine kinase growth factor receptors stimulate Akt has been recently defined. In contrast, the mechanism of activation of Akt by other cell surface receptors is much less well understood. For G protein-coupled receptors (GPCRs), conflicting data suggest that these receptors stimulate Akt in a cell type-specific manner by a yet to be fully elucidated mechanism. Here, advantage was taken of the availability of cells, where Akt activity could not be enhanced by agonists acting on this large family of cell surface receptors, such as NIH 3T3 cells, to investigate the pathway linking GPCRs to Akt. Evidence is presented that expression of phosphatidylinositol 3-kinase (PI3K) beta is necessary and sufficient to transmit signals from G proteins to Akt in these murine fibroblasts and the activation of PI3Kbeta may represent the most likely mechanism whereby GPCRs stimulate Akt, since the vast majority of cells do not express PI3Kgamma, a known G protein-sensitive PI3K isoform. Furthermore, available evidence indicates that GPCRs activate Akt by a pathway distinct from that utilized by growth factor receptors, since it involves the tyrosine phosphorylation-independent activation of PI3Kbeta by G protein betagamma dimers (Murga, 2000).

Regulator of G protein signaling (RGS) proteins are GTPase-activating proteins for heterotrimeric G proteins. One of the best-studied RGS proteins, RGS4, accelerates the rate of GTP hydrolysis by all G(i) and G(q) alpha subunits yet has been shown to exhibit receptor selectivity. Although RGS4 is expressed primarily in brain, its effect on modulating the activity of serotonergic receptors has not yet been reported. In the present study, transfected BE(2)-C human neuroblastoma cells expressing human 5-HT(1B) receptors were used to demonstrate that RGS4 can inhibit the coupling of 5-HT(1B) receptors to cellular signals. Serotonin and sumatriptan were found to stimulate activation of extracellular signal-regulated kinase. This activation is attenuated, but not completely inhibited, by RGS4. Similar inhibition by RGS4 of the protein kinase Akt was also observed. Since RGS4 is expressed at high levels in brain, these results suggest that it may play a role in regulating serotonergic pathways (Lione, 2000).

The protein-tyrosine phosphatase Shp-2 is a positive modulator of the Ras/mitogen-activated protein kinase pathway and a putative substrate of the transforming non-receptor tyrosine kinase v-Src. To characterize the role of Shp-2 in cellular transformation and signaling by v-Src, v-Src was expressed in normal and Shp-2-deficient mouse embryo fibroblasts. Expression of Shp-2 is necessary for morphological transformation by v-Src: Shp-2+/+ cells become rounded or spindly upon v-Src expression, whereas Shp-2-deficient cells remain relatively flat. v-Src-induces reorganization of the actin cytoskeleton and the formation of podosomes are compromised in Shp-2-deficient cells. Shp-2 deficiency also reduces v-Src-induced activation of the anti-apoptotic protein kinase Akt. The reduced activation of Akt in Shp-2-deficient cells correlates with a reduction in the association of the p85 regulatory subunit of PI3-kinase with the adapter protein Cbl. Activation of PI3-kinase by v-Src may be mediated by the association of the adapter protein Cbl with the p85 subunit. Since activation of Akt is dependent on PI3-kinase, this suggests that the effect of Shp-2 on Akt activation may be mediated, at least in part, by its effects on the interaction between PI3-kinase and Cbl. The defect in activation of the Akt survival pathway also correlates with enhanced sensitivity of Shp-2-deficient cells to an apoptosis-inducing agent. These results implicate Shp-2 in v-Src-induced cytoskeletal reorganization and activation of the Akt cell survival pathway (Hakak, 2000).

The opposing effects on proliferation mediated by G-protein-coupled receptor isoforms differing in their COOH termini could be correlated with the abilities of the receptors to differentially activate p38, implicated in apoptotic events, or phosphatidylinositol 3-kinase (PI 3-K), which provides a source of survival signals. These contrasting growth responses of the somatostatin sst(2) receptor isoforms, which couple to identical Galpha subunit pools [Galpha(i3) > Galpha(i2) >> Galpha(0)], were both inhibited following betagamma sequestration. The sst[2(a)] receptor-mediated ATF-2 activation and inhibition of proliferation induced by basic fibroblast growth factor (bFGF) are dependent on prolonged phosphorylation of p38. In contrast, cell proliferation and the associated transient phosphorylation of Akt and p70(rsk) induced by sst[2(b)] receptors are blocked by the PI 3-K inhibitor LY 294002. Stimulation with bFGF alone has no effect on the activity of either p38 or Akt but markedly enhances p38 phosphorylation mediated by sst[2(a)] receptors, suggesting that a complex interplay exists between the transduction cascades activated by these distinct receptor types. In addition, although all receptors mediate a sustained activation of extracellular signal-regulated kinases (ERK1 and ERK2), induction of the tumor suppressor p21(cip1) is detected only following amplification of ERK and p38 phosphorylation by concomitant bFGF and sst[2(a)] receptor activation. Expression of constitutively active Akt in the presence of a p38 inhibitor enables a proliferative response to be detected in sst[2(a)] receptor-expressing cells. These findings demonstrate that the duration of activation and a critical balance between the mitogen-activated protein kinase and PI 3-K pathways are important for controlling cell proliferation and that the COOH termini of the sst(2) receptor isoforms may determine the selection of appropriate betagamma-pairings necessary for interaction with distinct kinase cascades (Sellers, 2000).

The second messenger ceramide (N-alkylsphingosine) has been implicated in a host of cellular processes including growth arrest and apoptosis. Ceramide has been reported to have effects on both protein kinases and phosphatases and may constitute an important component of stress response in various tissues. The relationship between ceramide signaling and the activation of an important signaling pathway, phosphatidylinositol (PI) 3-kinase and its downstream target, protein kinase B (PKB), has been examined. PKB activation is observed following stimulation of cells with the cytokine granulocyte-macrophage colony-stimulating factor. Addition of cell-permeable ceramide analogs, C(2)- or C(6)-ceramide, causes a partial loss (50%-60%) of PKB activation. This reduction is not a result of decreased PI(3,4,5)P(3) or PI(3,4)P(2) generation by PI 3-kinase. Two residues of PKB (threonine 308 and serine 473) require phosphorylation for maximal PKB activation. Serine 473 phosphorylation is consistently reduced by treatment with ceramide, whereas threonine 308 phosphorylation remains unaffected. In further experiments, ceramide appears to accelerate serine 473 dephosphorylation, suggesting the activation of a phosphatase. Consistent with this, the reduction in serine 473 phosphorylation is inhibited by the phosphatase inhibitors okadaic acid and calyculin A. Surprisingly, threonine 308 phosphorylation is abolished in cells treated with these inhibitors, revealing a novel mechanism of regulation of threonine 308 phosphorylation. These results demonstrate that PI 3-kinase-dependent kinase 2-catalyzed phosphorylation of serine 473 is the principal target of a ceramide-activated phosphatase (Schubert, 2000).

Renewal of the gastrointestinal epithelium involves a coordinated process of terminal differentiation and programmed cell death. Integrins have been implicated in the control of apoptotic processes in various cell types. The role of integrins in the regulation of apoptosis in gastrointestinal epithelial cells has been examined with the use of a rat small intestinal epithelial cell line (RIE1) as a model. Overexpression of the integrin alpha5 subunit in RIE1 cells confers protection against several proapoptotic stimuli. In contrast, overexpression of the integrin alpha2 subunit has no effect on cell survival. The antiapoptotic effect of the alpha5 subunit is partially retained by a mutated version that has a truncation of the cytoplasmic domain. The antiapoptotic effects of the full-length or truncated alpha5 subunit are reversed upon treatment with inhibitors of phosphatidylinositol 3-kinase (PI-3-kinase), suggesting that the alpha5beta1 integrin might interact with the PI-3-kinase/Akt survival pathway. When cells overexpressing alpha5 are allowed to adhere to fibronectin, there is a moderate activation of protein kinase B (PKB)/Akt, whereas no such effect is seen in alpha2-overexpressing cells adhering to collagen. Furthermore, in cells overexpressing alpha5 and adhering to fibronectin, there is a dramatic enhancement of the ability of growth factors to stimulate PKB/Akt; again, this is not seen in cells overexpressing alpha2 subunit and adhering to collagen or fibronectin. Expression of a dominant negative version of PKB/Akt in RIE cells blocks to ability of alpha5 to enhance cell survival. Thus, the alpha5beta1 integrin seems to protect intestinal epithelial cells against proapoptotic stimuli by selectively enhancing the activity of the PI-3-kinase/Akt survival pathway (Lee, 2000).

The integrin cytoplasmic domain modulates cell proliferation, adhesion, migration, and intracellular signaling. The beta(1) integrin subunits, beta(1C) and beta(1A), that contain variant cytoplasmic domains differentially affect cell proliferation: beta(1C) inhibits proliferation, whereas beta(1A) promotes it. The ability of beta(1C) and beta(1A) to modulate integrin-mediated signaling events that affect cell proliferation and survival was investigated in Chinese hamster ovary stable cell lines expressing either human beta(1C) or human beta(1A). The different cytodomains of either beta(1C) or beta(1A) do not affect either association with the endogenous alpha(2), alpha(V), and alpha(5) subunits or cell adhesion to fibronectin or TS2/16, a mAb to human beta(1). Upon engagement of endogenous and exogenous integrins by fibronectin, cells expressing beta(1C) show significantly inhibited extracellular signal-regulated kinase (ERK) 2 activation compared with beta(1A) stable cell lines. In contrast, focal adhesion kinase phosphorylation and Protein Kinase B/AKT activity are not affected. Selective engagement of the exogenously expressed beta(1C) by TS2/16 leads to stimulation of Protein Kinase B/AKT phosphorylation but not of ERK2 activation; in contrast, beta(1A) engagement induces activation of both proteins. Ras activation is strongly reduced in beta(1C) stable cell lines in response to fibronectin adhesion and expression of constitutively active Ras [Ras 61 (L)] which rescues beta(1C)-mediated down-regulation of ERK2 activation. Inhibition of cell proliferation in beta(1C) stable cell lines is attributable to an inhibitory effect of beta(1C) on the Ras/MAP kinase pathway because expression of activated MAPK kinase rescues beta(1C) antiproliferative effect. These findings show that the beta(1C) variant, by means of a unique signaling mechanism, selectively inhibits the MAP kinase pathway by preventing Ras activation without affecting either survival signals stimulated by integrins or cellular interactions with the extracellular matrix. These findings highlight a role for beta(1)-specific cytodomain sequences in maintaining an intracellular balance of proliferation and survival signals (Fornaro, 2000).

The alpha(v)beta(3) integrin has been shown to bind several ligands, including osteopontin and vitronectin. Its role in modulating cell migration and downstream signaling pathways in response to specific extracellular matrix ligands has been investigated in this study. Highly invasive prostate cancer PC3 cells that constitutively express alpha(v)beta(3) adhere and migrate on osteopontin and vitronectin in an alpha(v)beta(3)-dependent manner. However, exogenous expression of alpha(v)beta(3) in noninvasive prostate cancer LNCaP [beta(3)-LNCaP] cells mediates adhesion and migration on vitronectin but not on osteopontin. Activation of alpha(v)beta(3) by epidermal growth factor stimulation is required to mediate adhesion to osteopontin but is not sufficient to support migration on this substrate. Alpha(v)beta(3)-mediated cell migration requires activation of the phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (PKB/AKT) pathway since wortmannin, a PI 3-kinase inhibitor, prevents PC3 cell migration on both osteopontin and vitronectin; furthermore, alpha(v)beta(3) engagement by osteopontin and vitronectin activates the PI 3-kinase/AKT pathway. Migration of beta(3)-LNCaP cells on vitronectin also occurs through activation of the PI 3-kinase pathway; however, AKT phosphorylation is not increased upon engagement by osteopontin. Furthermore, phosphorylation of focal adhesion kinase (FAK), known to support cell migration in beta(3)-LNCaP cells, is detected on both substrates. Thus, in PC3 cells, alpha(v)beta(3) mediates cell migration and PI 3-kinase/AKT pathway activation on vitronectin and osteopontin; in beta(3)-LNCaP cells, alpha(v)beta(3) mediates cell migration and PI 3-kinase/AKT pathway activation on vitronectin, whereas adhesion to osteopontin does not support alpha(v)beta(3)-mediated cell migration and PI 3-kinase/AKT pathway activation. It is concluded that alpha(v)beta(3) exists in multiple functional states that can bind either selectively vitronectin or both vitronectin and osteopontin and that alpha(v)beta(3) can differentially activate cell migration and intracellular signaling pathways in a ligand-specific manner (Zheng, 2000).

The serine/threonine kinase Akt (also known as protein kinase B) is activated in response to various stimuli by a mechanism involving phosphoinositide 3-kinase (PI3-K). Akt provides a survival signal that protects cells from apoptosis induced by growth factor withdrawal, but its function in other forms of stress is less clear. The role of PI3-K/Akt during the cellular response to oxidant injury has been examined. H2O2 treatment elevates Akt activity in multiple cell types in a time- and dose-dependent manner. Expression of a dominant negative mutant of p85 (regulatory component of PI3-K) and treatment with inhibitors of PI3-K (wortmannin and LY294002) prevent H2O2-induced Akt activation. Akt activation by H2O2 also depends on epidermal growth factor receptor (EGFR) signaling; H2O2 treatment leads to EGFR phosphorylation, and inhibition of EGFR activation prevents Akt activation by H2O2. Since H2O2 causes apoptosis of HeLa cells, whether or not alterations of PI3-K/Akt signaling would affect this response was investigated. Wortmannin and LY294002 treatment significantly enhances H2O2-induced apoptosis, whereas expression of exogenous myristoylated Akt (an activated form) inhibits cell death. Constitutive expression of v-Akt likewise enhances survival of H2O2-treated NIH3T3 cells. These results suggest that H2O2 activates Akt via an EGFR/PI3-K-dependent pathway and that elevated Akt activity confers protection against oxidative stress-induced apoptosis (Wang, 2000).

NGF is a target-derived growth factor for developing sympathetic neurons. Application of NGF exclusively to distal axons of sympathetic neurons leads to an increase in PI3-K signaling in both distal axons and cell bodies. In addition, there is a more critical dependence on PI3-K for survival of neurons supported by NGF acting exclusively on distal axons as compared to neurons supported by NGF acting directly on cell bodies. Interestingly, PI3-K signaling within both cell bodies and distal axons contributes to survival of neurons. The requirement of PI3-K signaling in distal axons for survival may be explained by the finding that inhibition of PI3-K in the distal axons attenuates retrograde signaling. Therefore, a single TrkA effector, PI3-K, has multiple roles within spatially distinct cellular locales during retrograde NGF signaling (Kuruvilla, 2000).

Dissociated sympathetic neurons obtained from newborn rat superior cervical ganglia and grown in compartmentalized cultures were to assess the subcellular distribution and state of activation of PI3-K and its downstream effector Akt (protein kinase B). Neurons were maintained under conditions in which cell bodies and proximal axons (hereafter referred to as the cell body compartment) were exposed to medium containing a neutralizing antibody directed against NGF (alpha-NGF), while distal axons, which are >1 mm away from cell bodies, were exposed to medium containing NGF. These conditions resemble in vivo conditions in which neurons are maintained by NGF acting exclusively on distal axons (Kuruvilla, 2000).

It was asked whether binding of NGF to receptors exclusively on distal axons regulates the activities of PI3-K and Akt in distal axons and/or cell bodies. For these experiments, NGF was removed from medium bathing distal axons for 24 hr. Then, distal axons were exposed to the same medium (control) or medium containing NGF for various times. The activation states of TrkA and Akt were assessed in extracts prepared from cell body and distal axon compartments by immunoblotting using antibodies that recognize the activated, phosphorylated forms of these proteins. P-Trk (Y490) antibodies recognize TrkA when phosphorylated on Tyr-490, which is the Shc recognition site. P-Akt antibodies recognize Akt when phosphorylated on Ser-473, which is necessary for its catalytic activity. Application of NGF to distal axons results in increased levels of P-TrkA (Y490) and P-Akt within distal axons, which are maximal after 20 min. Increases in both P-TrkA (Y490) and P-Akt are also detected in cell bodies but with slower kinetics. A small but reproducible increase in both P-TrkA (Y490) and P-Akt is detected in extracts of cell bodies within 20 min, and a more robust increase is seen at 8 hr. The appearance of P-TrkA (Y490) and P-Akt in both distal axons and cell bodies is coincident with the appearance of PI3-K activity associated with phosphotyrosine immunoprecipitates. Additionally, withdrawal of NGF from distal axons of neurons, which had been grown with medium containing a high concentration of NGF (100 ng/ml) on distal axons and alpha-NGF on cell bodies, leads to a decrease in the levels of both P-TrkA (Y490) and P-Akt in distal axons and in cell bodies. Thus, NGF acting on TrkA receptors on distal axons regulates the phosphorylation/activation of TrkA, PI3-K, and Akt both locally within distal axons and retrogradely to proximal axons and cell bodies of sympathetic neurons (Kuruvilla, 2000).

These results support the idea that PI3-K signaling within both cell bodies and distal axons is necessary for survival of neurons supported by NGF acting on distal axons. Moreover, the requirement of PI3-K signaling in distal axons is more apparent when a submaximal concentration of NGF is used to support survival. How does PI3-K signaling within distal axons contribute to survival? It was found that PI3-K activity in distal axons controls retrograde NGF transport and retrograde signaling, which may be critical for survival. Complete inhibition of PI3-K in distal axons, as assessed by levels of P-Akt, attenuates retrograde transport of NGF by ~80% in two compartment chambers and 65% in three compartment chambers. Thus, there is a small but significant amount of retrograde transport that occurs in a PI3-K-independent manner. These observations may account for the finding that inhibition of PI3-K in distal axons has more dire consequences for neurons supported by 0.5 ng/ml NGF acting on distal axons than for those supported by 50 ng/ml NGF acting on distal axons. Neurons grown in a low, submaximal concentration of NGF are more vulnerable than neurons supported by a high concentration of NGF to a 65%-80% reduction in retrograde signaling (Kuruvilla, 2000). The precise role of PI3-K signaling in distal axons for ligand-dependent internalization, retrograde transport, and retrograde signaling is not clear. It is possible that products of the PI3-K catalyzed reaction are critical for the ligand-dependent production of clathrin-coated pits, into which NGF and TrkA are initially internalized. In support of this idea, there is an essential role for the pleckstrin homology (PH) domain of the GTPase dynamin for receptor-mediated endocytosis. Further, dynamin is required for retrograde transport of NGF in sympathetic neurons. Since the dynamin PH domain binds to phosphoinositide products of the PI3-K-catalyzed reaction, PI3-K activity associated with TrkA may be critical for recruitment of dynamin to regions of the plasma membrane destined to invaginate to form NGF/TrkA-containing clathrin-coated signaling organelles. Similarly, AP-2, which is involved in clathrin coat formation and vesicle sorting at the plasma membrane, contains an amino-terminal phosphoinositide binding domain that is required for its targeting to the plasma membrane. Thus, it is tempting to speculate that PI3-K signaling in distal axons is needed for survival because this TrkA effector controls membrane recruitment of key regulators of NGF/TrkA endocytosis and retrograde TrkA signaling (Kuruvilla, 2000).

If PI3-K in distal axons is required for retrograde signaling, what is the role of PI3-K in cell bodies in neurons supported by NGF acting exclusively on distal axons? Inhibition of PI3-K in cell bodies leads to near complete apoptosis of neurons within 48 hr, but inhibition of PI3-K exclusively in cell bodies does not affect retrograde transport of NGF. Under these conditions, P-Akt in cell bodies is completely blocked, but levels of P-Akt in distal axons are unaffected. These observations indicate that PI3-K and Akt signaling in distal axons alone cannot support neuronal survival. Since constitutively active PI3-K and Akt can support survival of sympathetic neurons, it is speculated that PI3-K signaling in cell bodies is necessary for survival because it supports Akt signaling and phosphorylation of Akt substrates that mediate the prosurvival effects of PI3-K. Indeed, it seems likely that many of the substrates of Akt function, at least in part, within cell bodies. Substrates of Akt include BAD, caspase-9, IKK, the transcription factor forkhead, and, possibly, CREB. By extension, these data support the idea that phosphorylation of Akt substrates within distal axons cannot support neuronal survival. This may be because critical substrates of Akt are either not present in distal axons or that they are present in distal axons but cannot move in the phosphorylated forms from distal axons to cell bodies to affect the apoptotic machinery. P-Akt itself does not move from distal axons to cell bodies to an appreciable extent so the same is likely to be true for products of Akt-catalyzed phosphorylation reactions (Kuruvilla, 2000).

Growth factor signal transduction mechanisms in neurons are arguably more complex than in most other cell types due to the striking morphological specializations of neurons. Most neurons have long axons that can extend centimeters or even one meter from their cell bodies, and target-derived growth factor signals must be propagated over long distances to influence survival and gene expression within cell bodies. These retrograde signals must be integrated with signals coming from dendrites and those emanating from the membrane of the cell body itself. The present study shows that the same NGF effector pathway, the PI3-K pathway, can have different functions in distinct parts of the same neuron during long-range retrograde signaling. Interestingly, the activity of the PI3-K signaling in distal axons indirectly regulates TrkA signaling pathways in cell bodies, including the PI3-K effector pathway. Thus, there exists interdependence of TrkA effector pathways in distinct cellular locales whereby ligand-dependent TrkA effector signaling in one compartment, the distal axon, controls effector signaling in another, the cell body (Kuruvilla, 2000).

The role of integrins in leukocyte apoptosis is unclear: some studies suggest enhancement, others inhibition. ß2-integrin engagement on neutrophils can either inhibit or enhance apoptosis depending on the activation state of the integrin and the presence of proapoptotic stimuli. Both clustering and activation of alphaMß2 delays spontaneous, or unstimulated, apoptosis, maintains mitochondrial membrane potential, and prevents cytochrome c release. In contrast, in the presence of proapoptotic stimuli, such as Fas ligation, TNFalpha, or UV irradiation, ligation of active alphaMß2 results in enhanced mitochondrial changes and apoptosis. Clustering of inactive integrins does not show this proapoptotic effect and continues to inhibit apoptosis. This discrepancy can be attributed to differential signaling in response to integrin clustering versus activation. Clustered, inactive alphaMß2 is capable of stimulating the kinases ERK and Akt. Activated alphaMß2 stimulates Akt, but not ERK. When proapoptotic stimuli are combined with either alphaMß2 clustering or activation, Akt activity is blocked, allowing integrin activation to enhance apoptosis. Clustered, inactive alphaMß2 continues to inhibit stimulated apoptosis due to maintained ERK activity. Therefore, ß2-integrin engagement can both delay and enhance apoptosis in the same cell, suggesting that integrins can play a dual role in the apoptotic progression of leukocytes (Whitlock, 2000).

Protein kinase B (PKB/Akt) is a regulator of cell survival and apoptosis. To become fully activated, PKB/Akt requires phosphorylation at two sites, threonine 308 and serine 473, in a phosphatidylinositol (PI) 3-kinase-dependent manner. The kinase responsible for phosphorylation of threonine 308 is the PI 3-kinase-dependent kinase-1 (PDK-1), whereas phosphorylation of serine 473 has been suggested to be regulated by PKB/Akt autophosphorylation in a PDK-1-dependent manner. However, the integrin-linked kinase (ILK: see Drosophila Integrin linked kinase) has also been shown to regulate phosphorylation of serine 473 in a PI 3-kinase-dependent manner. Whether ILK phosphorylates this site directly or functions as an adapter molecule has been debated. In-gel kinase assay and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry show that biochemically purified ILK can phosphorylate PKB/Akt directly. Co-immunoprecipitation analysis of cell extracts demonstrates that ILK can complex with PKB/Akt as well as PDK-1 and that ILK can disrupt PDK-1/PKB association. The amino acid residue serine 343 of ILK within the activation loop is required for kinase activity as well as for its interaction with PKB/Akt. Mutational analysis of ILK further shows a crucial role for arginine 211 of ILK within the phosphoinositide phospholipid binding domain in the regulation of PKB-serine 473 phosphorylation. A highly selective small molecule inhibitor of ILK activity also inhibits the ability of ILK to phosphorylate PKB/Akt in vitro and in intact cells. These data demonstrate that ILK is an important upstream kinase for the regulation of PKB/Akt (Persad, 2001).

PDK1 functions as a master kinase, phosphorylating and activating PKB/Akt, S6K and RSK (See Drosophila RSK ). To learn more about the roles of PDK1, mice were generated that either lacked PDK1 or possessed PDK1 hypomorphic alleles, expressing only ~10% of the normal level of PDK1. PDK1-/- embryos die at embryonic day 9.5, displaying multiple abnormalities including lack of somites, forebrain and neural crest derived tissues; however, development of hind- and mid-brain proceed relatively normally. In contrast, hypomorphic PDK1 mice are viable and fertile, and insulin injection induces the normal activation of PKB, S6K and RSK. Nevertheless, these mice are 40%-50% smaller than control animals. The organ volumes from the PDK1 hypomorphic mice are reduced proportionately. The volume of a number of PDK1-deficient cells is reduced by 35%-60%; PDK1 deficiency does not affect cell number, nuclear size or proliferation. Genetic evidence is provided that PDK1 is essential for mouse embryonic development, and regulates cell size independently of cell number or proliferation, as well as insulin's ability to activate PKB, S6K and RSK (Lawlor, 2002).

Growth factors promote cell survival and cell motility, presumably through the activation of Akt and the Rac and Cdc42 GTPases, respectively. Because Akt is dispensable for Rac/Cdc42 regulation of actin reorganization, it has been assumed that Rac and Cdc42 stimulate cell motility independent of Akt in mammalian cells. However, this study demonstrates that Akt is essential for Rac/Cdc42-regulated cell motility in mammalian fibroblasts. A dominant-negative Akt inhibits cell motility stimulated by Rac/Cdc42 or by PDGF treatment, without affecting ruffling membrane-type actin reorganization. Akt is activated by expression of Rac and Cdc42; colocalization of endogenous phosphorylated Akt with Rac and Cdc42 is observed at the leading edge of fibroblasts. Importantly, expression of active Akt but not the closely related kinase SGK is sufficient for increasing cell motility. This effect of Akt is cell autonomous and not mediated by inhibition of GSK3. Dominant-negative Akt but not SGK reverses the increased cell motility phenotype of fibroblasts lacking the PTEN tumor suppressor gene. Taken together, these results suggest that Akt promotes cell motility downstream of Rac/Cdc42 in growth factor-stimulated cells and in invasive PTEN-deficient cells (Higuchi, 2002).

Blood vessel formation is a complex morphological process that is only beginning to be understood at the molecular level. A novel and critical role is demonstrated for the small GTPase, RhoB, in vascular development. RhoB null mice have retarded vascular development in the retina characterized by altered sprout morphology. Moreover, pharmaceutical means to deplete RhoB in neonatal rats is associated with apoptosis in the sprouting endothelial cells of newly forming vessels. Similarly, acute depletion of RhoB by antisense or dominant-negative strategies in primary endothelial cell culture models leads to apoptosis and failures in tube formation. A novel link has been identified between RhoB and the Akt survival signaling pathway to explain these changes. Confocal microscopy reveals that RhoB is highly localized to the nuclear margin with a small percentage found inside the nucleus. Similarly, total Akt is found throughout the cell but has increased accumulation at the nuclear margin, and active phosphorylated Akt is found primarily inside the nucleoplasm, where it partially colocalizes with the RhoB therein. This colocalization is functionally relevant, because when RhoB is depleted, Akt is excluded from the nucleus and total cellular Akt protein is decreased in a proteosome-dependent manner. Because the function of RhoB in vivo appears to be rate limiting only for endothelial cell sprouting, it is proposed that RhoB has a novel stage-specific function to regulate endothelial cell survival during vascular development. RhoB may offer a therapeutic target in diseases such as cancer, diabetic retinopathy, and macular degeneration, where the disruption of sprouting angiogenesis would be desirable (Adini, 2003).

Despite genetic evidence establishing angiopoietin-1 (Ang-1) as an essential regulator of vascular development, the molecular mechanisms underlying Ang-1 function are almost completely uncharacterized. This study demonstrates that Ang-1, via Akt activation, is a potent inhibitor of the forkhead transcription factor FKHR (FOXO1), identifying a nuclear signaling pathway through which Ang-1 modulates gene expression. Microarray analysis was used to show that FKHR, whose function in endothelial cells has not previously been elucidated, regulates many genes associated with vascular destabilization and remodeling (including angiopoietin-2, an Ang-1 antagonist) and endothelial cell apoptosis (e.g., survivin, TRAIL). Ang-1 inhibits FKHR-mediated changes in gene expression and FKHR-induced apoptosis. Analysis of gene expression changes induced by an activated version of Akt confirms that FKHR is a major target through which Akt regulates transcription in endothelial cells. RNA interference was used to demonstrate that FKHR is required for the expression of genes (including Ang-2) that have important vascular functions. These data suggest a novel, tissue-specific role for the Akt/FKHR pathway in the vasculature and suggest a mechanistic basis for the previously described actions of Ang-1 as a regulator of endothelial cell survival and blood vessel stability (Daly, 2004).

Plexins are receptors for the axonal guidance molecules known as semaphorins, and the semaphorin 4D (Sema4D) receptor plexin-B1 induces repulsive responses by functioning as an R-Ras GTPase-activating protein (GAP). This study characterized the downstream signalling of plexin-B1-mediated R-Ras GAP activity, inducing growth cone collapse. Sema4D suppresses R-Ras activity in hippocampal neurons, in parallel with dephosphorylation of Akt and activation of glycogen synthase kinase (GSK)-3beta. Ectopic expression of the constitutively active mutant of Akt or treatment with GSK-3 inhibitors suppressea the Sema4D-induced growth cone collapse. Constitutive activation of phosphatidylinositol-3-OH kinase (PI(3)K), an upstream kinase of Akt and GSK-3beta, also blocka the growth cone collapse. The R-Ras GAP activity is necessary for plexin-B1-induced dephosphorylation of Akt and activation of GSK-3beta and is also required for phosphorylation of a downstream kinase of GSK-3beta, collapsin response mediator protein-2. Plexin-A1 also induces dephosphorylation of Akt and GSK-3beta through its R-Ras GAP activity. It is concluded that plexin-B1 inactivates PI(3)K and dephosphorylates Akt and GSK-3beta through R-Ras GAP activity, inducing growth cone collapse (Ito, 2006).

Oligodendrocytes in the central nervous system (CNS) produce myelin sheaths that insulate axons to ensure fast propagation of action potentials. beta1 integrins regulate the myelination of peripheral nerves, but their function during the myelination of axonal tracts in the CNS is unclear. This study shows that genetically modified mice lacking beta1 integrins in the CNS present a deficit in myelination but no defects in the development of the oligodendroglial lineage. Instead, in vitro data show that beta1 integrins regulate the outgrowth of myelin sheaths. Oligodendrocytes derived from mutant mice are unable to efficiently extend myelin sheets and fail to activate AKT (also known as AKT1), a kinase that is crucial for axonal ensheathment. The inhibition of PTEN, a negative regulator of AKT, or the expression of a constitutively active form of AKT restores myelin outgrowth in cultured beta1-deficient oligodendrocytes. These data suggest that beta1 integrins play an instructive role in CNS myelination by promoting myelin wrapping in a process that depends on AKT (Barros, 2009).

Disrupted-in-schizophrenia 1 (DISC1), a susceptibility gene for major mental illnesses, regulates multiple aspects of embryonic and adult neurogenesis. This study shows that DISC1 suppression in newborn neurons of the adult hippocampus leads to overactivated signaling of AKT, another schizophrenia susceptibility gene. Mechanistically, DISC1 directly interacts with KIAA1212, an AKT binding partner that enhances AKT signaling in the absence of DISC1, and DISC1 binding to KIAA1212 prevents AKT activation in vitro. Functionally, multiple genetic manipulations to enhance AKT signaling in adult-born neurons in vivo exhibit similar defects as DISC1 suppression in neuronal development that can be rescued by pharmacological inhibition of mammalian target of rapamycin (mTOR), an AKT downstream effector. This study identifies the AKT-mTOR signaling pathway as a critical DISC1 target in regulating neuronal development and provides a framework for understanding how multiple susceptibility genes may functionally converge onto a common pathway in contributing to the etiology of certain psychiatric disorders (Kim, 2009).

Little is known about the architecture of cellular microenvironments that support stem and precursor cells during tissue development. Although adult stem cell niches are organized by specialized supporting cells, in the developing cerebral cortex, neural stem/precursor cells reside in a neurogenic niche lacking distinct supporting cells. This study finds that neural precursors themselves comprise the niche and regulate their own development. Precursor-precursor contact regulates beta-catenin signaling and cell fate. In vivo knockdown of N-cadherin reduces beta-catenin signaling, migration from the niche, and neuronal differentiation in vivo. N-cadherin engagement activates beta-catenin signaling via Akt, suggesting a mechanism through which cells in tissues can regulate their development. These results suggest that neural precursor cell interactions can generate a self-supportive niche to regulate their own number (Zhang, 2010).

Whether Akt might link N-cadherin to β-catenin activation in cortical precursors was investigated . It was found that function-blocking antibodies to N-cadherin or shRNA to N-cadherin led to a significant reduction in phosphorylated (active) Akt in primary cortical precursors. To test the link between Akt activation and phosphorylation of β-catenin at Ser552, Akt was inhibited in neural precursors using triciribine (API-2), a small molecule Akt pathway inhibitor. Triciribine treatment of primary cortical precursors reduced the fraction of cells expressing β-catenin Ser552 in a dose-dependent fashion. Finally, expression of a dominant-negative (kinase-dead) Akt also reduced both baseline β-catenin signaling in high-density primary cortical precursor cultures as well as Wnt-stimulated β-catenin signaling. To confirm whether Akt functions downstream of N-cadherin to mediate β-catenin signaling, myristoylated (active) Akt was coexpressed along with shRNA to N-cadherin and β-catenin signaling was measured by TOP-flash reporters. It was found that that myrAkt rescued β-catenin signaling following N-cadherin knockdown. It was also found that myrAkt alone could increase β-catenin signaling, a finding consistent with the idea that this pathway may exist in parallel with the canonical Wnt signaling pathway. Together, these observations suggest that N-cadherin engagement leads to phosphorylation of Akt and subsequent Akt-mediated phosphoryation and activation of β-catenin (Zhang, 2010).

Cell-to-cell variability in populations has been widely observed in mammalian cells. This heterogeneity can result from random stochastic events or can be deliberately maintained through regulatory processes. In the latter case, heterogeneity should confer a selective advantage that benefits the entire population. Using multicolor flow cytometry, this study has uncovered robust heterogeneity in phosphoinositide 3-kinase (PI3K) activity in MCF10A cell populations, which had been previously masked by techniques that only measure population averages. AKT activity is bimodal in response to EGF stimulation and correlates with PI3K protein level, such that only cells with high PI3K protein can activate AKT. It was further shown that heterogeneity in PI3K protein levels is invariably maintained in cell populations through a degradation/resynthesis cycle that can be regulated by cell density. Given that the PI3K pathway is one of the most frequently upregulated pathways in cancer, it is proposed that heterogeneity in PI3K activity is beneficial to normal tissues by restricting PI3K activation to only a subset of cells. This may serve to protect the population as a whole from overactivating the pathway, which can lead to cellular senescence or cancer. Consistent with this, it was show that oncogenic mutations in p110α (H1047R and E545K) partially evade this negative regulation, resulting in increased AKT activity in the population (Yuan, 2011).

DEPTOR is an mTOR inhibitor: DEPTOR expression is necessary to maintain PI3K and Akt activation

The mTORC1 and mTORC2 pathways regulate cell growth, proliferation, and survival. This study identified DEPTOR as an mTOR-interacting protein whose expression is negatively regulated by mTORC1 and mTORC2. The gene for DEPDC6 is found only in vertebrates, and encodes a protein with tandem N-terminal DEP (dishevelled, egl-10, pleckstrin) domains and a C-terminal PDZ (postsynaptic density 95, discs large, zonula occludens-1) domain. Loss of DEPTOR activates S6K1, Akt, and SGK1, promotes cell growth and survival, and activates mTORC1 and mTORC2 kinase activities. DEPTOR overexpression suppresses S6K1 but, by relieving feedback inhibition from mTORC1 to PI3K signaling, activates Akt. Consistent with many human cancers having activated mTORC1 and mTORC2 pathways, DEPTOR expression is low in most cancers. Surprisingly, DEPTOR is highly overexpressed in a subset of multiple myelomas harboring cyclin D1/D3 or c-MAF/MAFB translocations. In these cells, high DEPTOR expression is necessary to maintain PI3K and Akt activation and a reduction in DEPTOR levels leads to apoptosis. Thus, this study identified a novel mTOR-interacting protein whose deregulated overexpression in multiple myeloma cells represents a mechanism for activating PI3K/Akt signaling and promoting cell survival (Peterson, 2009).

Loss-of-function data indicate that DEPTOR inhibits both the mTORC1 and mTORC2 pathways. However, by inhibiting mTORC1, DEPTOR overexpression relieves mTORC1-mediated inhibition of PI3K, causing an activation of PI3K and, paradoxically, of mTORC2-dependent outputs, like Akt (Peterson, 2009).

mTOR interacts with DEPTOR via its PDZ domain, and so far there is no information about the function of the tandem DEP domains the protein also contains. In other proteins, DEP domains mediate protein-protein interactions, but in numerous DEPTOR purifications additional DEPTOR-interacting proteins were not identified, besides the known components of mTORC1 and mTORC2. Therefore, based on current evidence, DEPTOR appears dedicated to mTOR regulation, and it is proposed that in vertebrates it is likely to be involved in regulating other outputs of the mTOR signaling network besides the growth and survival pathways examined in this study. The mTOR complexes and DEPTOR negatively regulate each other, suggesting the existence of a feedforward loop in which the loss of DEPTOR leads to an increase in mTOR activity, which then further reduces DEPTOR expression. This type of regulatory circuit should result in DEPTOR expression being tightly coupled to mTOR activity, and, interestingly, it was noted that DEPTOR mRNA levels strongly anticorrelate with cell size, a readout of mTORC1 activity (Peterson, 2009).

About 28% of human multiple myelomas (MMs) overexpress DEPTOR. These results are consistent with a published survey of 67 MM tumors and 43 MM cell lines, in which 21% were shown to possess copy number gains and associated expression increases of the genes within a 6 Mb region of chromosome 8q24 that contains DEPTOR. Furthermore, it appears that deregulated overexpression of c-MAF and MAFB is an additional, perhaps even more prevalent, mechanism for increasing DEPTOR expression in MMs. The related c-MAF and MAFB transcription factors are expressed (frequently as the result of chromosomal translocations) in a large fraction of MMs, but not in the plasma cells from which they are derived. Consistent with c-MAF playing a key role in promoting DEPTOR expression, a knockdown of c-MAF in a MM cell line having a c-MAF translocation decreases the expression of DEPTOR and mimics the effects of a DEPTOR knockdown on mTOR and PI3K signaling. The levels of the DEPTOR and c-MAF or MAFB mRNAs highly correlate with each other and, importantly, DEPTOR expression correlates with poor survival in patients with multiple myeloma (Peterson, 2009).

In many multiple myeloma cell lines, DEPTOR is massively overexpressed compared to the levels found in other cancer cell lines, such as HeLa cells. In these cells, the great overexpression of DEPTOR inhibits mTORC1 growth signaling and drives outputs dependent on PI3K. Interestingly, a reduction in DEPTOR expression to the lower levels seen in non-multiple myeloma cell lines causes cell death via apoptosis. This suggests that a pharmacologically induced reduction in DEPTOR expression or disruption of the DEPTOR-mTOR interaction could have therapeutic benefits for the treatment of multiple myeloma. There has been progress in developing small-molecule inhibitors of protein-protein interactions mediated by PDZ domains, so it is conceivable that blockers of the DEPTOR-mTOR interaction could be made (Peterson, 2009).

Although a number of other cancer cell lines have high levels of DEPTOR, as a class only multiple myelomas appear to consistently overexpress it. Besides activating PI3K/Akt signaling, DEPTOR overexpression in MM cells may provide these cells with benefits that are not relevant in other cancer types or perhaps even detrimental. For example, the high demand that MM cells place on the protein synthesis machinery to produce large amounts of immunoglobulins, causes a significant ER stress, which renders these cells susceptible to apoptosis induction via agents that induce further ER stress, such as proteasome inhibitors. DEPTOR overexpression, by partial inhibition of protein synthesis through the suppression of mTORC1, may reduce the levels of ER stress below the threshold that triggers apoptosis. In contrast, in other cancer cells in which ER stress is not a significant factor, DEPTOR overexpression may be selected against because reduced rates of protein synthesis may not be tolerated. That mTORC1-stimulated protein synthesis leads to ER stress is already appreciated as TSC1 or TSC2 null cells have increased sensitivity to ER stress-induced death (Peterson, 2009).

It is curious that DEPTOR is overexpressed mostly in MMs characterized by chromosomal translocations instead of those that are hyperdiploid because of aneuploidy. Elevated DEPTOR expression might be tolerated better in the nonhyperdiploid MMs because aneuploidy itself increases sensitivity to conditions, like mTORC1 inhibition, that interfere with protein synthesis. Moreover, the state of high mTORC2 and low mTORC1 signaling that this work indicates that some MM cells prefer cannot be achieved by mutations that activate PI3K signaling, perhaps explaining why multiple myelomas exhibit low rates of PTEN-inactivating or PI3K-activating mutations (Peterson, 2009).

Effects of Akt mutation

To elucidate the functions of the serine/threonine kinase Akt/PKB in vivo, mice were generated lacking both akt1 and akt2 genes. Akt1/Akt2 double-knockout (DKO) mice exhibit severe growth deficiency and die shortly after birth. These mice display impaired skin development because of a proliferation defect, severe skeletal muscle atrophy because of a marked decrease in individual muscle cell size, and impaired bone development. These defects are strikingly similar to the phenotypes of IGF-1 receptor-deficient mice and suggest that Akt may serve as the most critical downstream effector of the IGF-1 receptor during development. In addition, Akt1/Akt2 DKO mice display impeded adipogenesis. Specifically, Akt1 and Akt2 are required for the induced expression of PPARgamma, the master regulator of adipogenesis, establishing a new essential role for Akt in adipocyte differentiation. Overall, the combined deletion of Akt1 and Akt2 establishes in vivo roles for Akt in cell proliferation, growth, and differentiation. These functions of Akt were uncovered despite the observed lower level of Akt activity mediated by Akt3 in Akt1/Akt2 DKO cells, suggesting that a critical threshold level of Akt activity is required to maintain normal cell proliferation, growth, and differentiation (Peng, 2003).

AKT structure and regulation by phosphorylation

Protein kinase B/Akt plays crucial roles in promoting cell survival and mediating insulin responses. The enzyme is stimulated by phosphorylation at two regulatory sites: Thr 309 of the activation segment and Ser 474 of the hydrophobic motif, a conserved feature of many AGC kinases. Analysis of the crystal structures of the unphosphorylated and Thr 309 phosphorylated states of the PKB kinase domain provides a molecular explanation for regulation by Ser 474 phosphorylation. Activation by Ser 474 phosphorylation occurs via a disorder to order transition of the alphaC helix with concomitant restructuring of the activation segment and reconfiguration of the kinase bilobal structure. These conformational changes are mediated by a phosphorylation-promoted interaction of the hydrophobic motif with a channel on the N-terminal lobe induced by the ordered alphaC helix and are mimicked by peptides corresponding to the hydrophobic motif of PKB and potently by the hydrophobic motif of PRK2 (Yang, 2002).

Tumor suppressor genes evolved as negative effectors of mitogen and nutrient signaling pathways, such that mutations in these genes can lead to pathological states of growth. Tuberous sclerosis (TSC) is a potentially devastating disease associated with mutations in two tumor suppressor genes, TSC1 and 2, that function as a complex to suppress signaling in the mTOR/S6K/4E-BP pathway. However, the inhibitory target of TSC1/2 and the mechanism by which it acts are unknown. Evidence is provided that TSC1/2 is a GAP for the small GTPase Rheb and that insulin-mediated Rheb activation is PI3K dependent. Moreover, Rheb overexpression induces S6K1 phosphorylation and inhibits PKB phosphorylation, as do loss-of-function mutations in TSC1/2, but contrary to earlier reports Rheb has no effect on MAPK phosphorylation. Finally, coexpression of a human TSC2 cDNA harboring a disease-associated point mutation in the GAP domain, failed to stimulate Rheb GTPase activity or block Rheb activation of S6K1 (Garami, 2003).

Activation of AKT by membrane translocation

The role of subcellular localization in the regulation of protein kinase B (PKB) activation has been investigated. The myristoylation/palmitylation motif from the Lck tyrosine kinase was attached to the N terminus of protein kinase B to alter its subcellular location. Myristoylated/palmitylated (m/p)-PKBalpha is associated with the plasma membrane of transfected cells, whereas the wild-type kinase is mostly cytosolic. The activity of m/p-PKBalpha is 60-fold higher compared with the unstimulated wild-type enzyme, and can not be stimulated further by growth factors or phosphatase inhibitors. In vivo 32P labeling and mutagenesis has demonstrated that m/p-PKBalpha activity is due to phosphorylation on Thr308 and Ser473, two amino acids that are normally modified on PKB following stimulation of the cells with insulin or insulin-like growth factor-1 (IGF-1). A dominant negative form of phosphoinositide 3-kinase (PI3-K) does not affect m/p-PKBalpha activity. The pleckstrin homology (PH) domain of m/p-PKBalpha is not required for its activation or phosphorylation on Thr308 and Ser473, suggesting that this domain may serve as a membrane-targeting module. Consistent with this view, PKBalpha is translocated to the plasma membrane within minutes after stimulation with IGF-1. This translocation requires the PH domain and is sensitive to wortmannin. These results indicate that PI3-K activity is required for translocation of PKB to the plasma membrane, where its activation occurs through phosphorylation of the same sites that are induced by insulin or IGF-1. Following activation the kinase detaches from the membrane and translocates to the nucleus (Andjelkovic, 1997).

Protein kinase B (PKB) is a proto-oncogene that is activated in signaling pathways initiated by phosphoinositide 3-kinase. Chromatographic separation of brain cytosol reveals a kinase activity that phosphorylates and activates PKB only in the presence of phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3]. Phosphorylation occurs exclusively on threonine-308, a residue implicated in activation of PKB in vivo. PtdIns(3,4,5)P3 was determined to have a dual role: its binding to the pleckstrin homology domain of PKB is required to allow phosphorylation by the upstream kinase and it directly activates the upstream kinase (Stokoe, 1997).

Protein kinase B (PKB) is activated in response to phosphoinositide 3-kinases and their lipid products phosphatidylinositol 3,4, 5-trisphosphate [PtdIns(3,4,5)P3] and PtdIns(3,4)P2 in the signaling pathways used by a wide variety of growth factors, antigens, and inflammatory stimuli. PKB is a direct target of these lipids, but this regulation is complex. The lipids can bind to the pleckstrin homologous domain of PKB, causing its translocation to the membrane, and also enable upstream, Thr308-directed kinases to phosphorylate and activate PKB. Four isoforms of these PKB kinases were purified from sheep brain. They bind PtdIns(3,4,5)P3 and associate with lipid vesicles containing it. These kinases contain an NH2-terminal catalytic domain and a COOH-terminal pleckstrin homologous domain, and their heterologous expression augments receptor activation of PKB, which suggests they are the primary signal transducers that enable PtdIns(3,4,5)P3 or PtdIns- (3,4)P2 to activate PKB and hence to control signaling pathways regulating cell survival, glucose uptake, and glycogen metabolism (Stephens, 1998).

Protein kinase B (PKB) is involved in the regulation of apoptosis, protein synthesis and glycogen metabolism in mammalian cells. Phosphoinositide-dependent protein kinase (PDK-1) activates PKB in a manner dependent on phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3], which is also needed for the translocation of PKB to the plasma membrane. It has been proposed that the amount of PKB activated is determined exclusively as a result of its translocation, and that a constitutively active pool of membrane-associated PDK-1 simply phosphorylates all the PKB made available. The effects of membrane localization of PDK-1 on PKB activation has been investigated. Ectopically expressed PDK-1 translocated to the plasma membrane in response to platelet-derived growth factor (PDGF) and translocation is sensitive to wortmannin, an inhibitor of phosphoinositide 3-kinase. Translocation of PDK-1 also occurs upon its co-expression with constitutively active phosphoinositide 3-kinase, but not with an inactive form. Overexpression of PDK-1 enhances the ability of PDGF to activate PKB. PDK-1 disrupted in the pleckstrin homology (PH) domain, which does not translocate to the membrane, does not increase PKB activity in response to PDGF, whereas membrane-targeted PDK-1 activates PKB to the extent that it can not be activated further by PDGF. It is concluded that in response to PDGF, binding of Ptdlns (3,4,5)P3 and/or Ptdlns(3,4)P2 to the PH domain of PDK-1 causes its translocation to the plasma membrane where it co-localizes with PKB, significantly contributing to the scale of PKB activation (Anderson, 1998).

Two products of PI 3-kinase activation, PtdIns(3,4,5)P3 and its immediate breakdown product PtdIns(3,4)P2, trigger physiological processes by interacting with proteins possessing pleckstrin homology (PH) domains. One of the best characterized PtdIns(3,4,5)P3/PtdIns(3,4)P2 effector proteins is protein kinase B (PKB), also known as Akt. PKB possesses a PH domain located at its N terminus, and this domain binds specifically to PtdIns(3,4,5)P3 and PtdIns(3,4)P2 with similar affinity. Following activation of PI 3-kinase, PKB is recruited to the plasma membrane by virtue of its interaction with PtdIns(3,4,5)P3/PtdIns(3,4)P2. PKB is then activated by the 3-phosphoinositide-dependent protein kinase-1 (PDK1), which like PKB, possesses a PtdIns(3,4,5)P3/PtdIns(3,4)P2 binding PH domain. This study describes the high-resolution crystal structure of the isolated PH domain of PKBalpha in complex with the head group of PtdIns(3,4,5)P3. The head group has a significantly different orientation and location compared to other Ins(1,3,4,5)P4 binding PH domains. Mutagenesis of the basic residues that form ionic interactions with the D3 and D4 phosphate groups reduces or abolishes the ability of PKB to interact with PtdIns(3,4,5)P3 and PtdIns(3,4)P2. The D5 phosphate faces the solvent and forms no significant interactions with any residue on the PH domain, and this explains why PKB interacts with similar affinity with both PtdIns(3,4,5)P3 and PtdIns(3,4)P2 (Thomas, 2002).

Full activation of PKB requires phosphorylation on residues Thr308 and Ser473. While the Thr308 kinase, named 3-phosphoinositide-dependent kinase-1 (PDK1), has been extensively characterized, the identity of the Ser473 kinase remains unclear. This study focused on the plasma membrane (PM) fraction because membrane localization is sufficient to activate PKB, and this suggests that PKB upstream kinases are constitutively active at the membrane. A constitutively active PKB Ser473 kinase activity has been identified that is enriched in buoyant, detergent-insoluble plasma membrane rafts that are distinct from the cytosolic distribution of PKB and PDK1. This Ser473 kinase activity is released from the membrane by high salt, and gel filtration analysis shows that the kinase responsible is present in a large complex of >500 kDa. Two major phosphoproteins and integrin-linked kinase (ILK) were detected in partially purified PKB Ser473 kinase preparations. In contrast to previous observations, however, ILK immunoprecipitates do not retain Ser473 kinase activity. Thus, a novel raft-associated PKB Ser473 kinase has been identifed, implicating a role for lipid rafts in PKB signaling (Hill, 2002).

Mutations of NPHS1 or NPHS2, the genes encoding nephrin and podocin, as well as the targeted disruption of CD2-associated protein (CD2AP), lead to heavy proteinuria, suggesting that all three proteins are essential for the integrity of glomerular podocytes, the visceral glomerular epithelial cells of the kidney. It has been speculated that these proteins participate in common signaling pathways; however, it has remained unclear which signaling proteins are actually recruited by the slit diaphragm protein complex in vivo. This study demonstrates that both nephrin and CD2AP interact with the p85 regulatory subunit of phosphoinositide 3-OH kinase (PI3K) in vivo, recruit PI3K to the plasma membrane, and, together with podocin, stimulate PI3K-dependent AKT signaling in podocytes. Using two-dimensional gel analysis in combination with a phosphoserine-specific antiserum, this study demonstrates that the nephrin-induced AKT mediates phosphorylation of several target proteins in podocytes. One such target is Bad; its phosphorylation and inactivation by 14-3-3 protects podocytes against detachment-induced cell death, suggesting that the nephrin-CD2AP-mediated AKT activity can regulate complex biological programs. These findings reveal a novel role for the slit diaphragm proteins nephrin, CD2AP, and podocin and demonstrate that these three proteins, in addition to their structural functions, initiate PI3K/AKT-dependent signal transduction in glomerular podocytes (Huber, 2003).

The endosomal protein Appl1 mediates Akt substrate specificity and cell survival in vertebrate development

During development of multicellular organisms, cells respond to extracellular cues through nonlinear signal transduction cascades whose principal components have been identified. Nevertheless, the molecular mechanisms underlying specificity of cellular responses remain poorly understood. Spatial distribution of signaling proteins may contribute to signaling specificity. This hypothesis by investigating the role of the Rab5 effector Appl1 (adaptor protein containing pH domain, PTB domain, and Leucine zipper motif, also termed DIP13α and β), an endosomal protein that interacts with transmembrane receptors and Akt. In zebrafish, Appl1 regulates Akt activity and substrate specificity, controlling GSK-3β but not TSC2. Consistent with this pattern, Appl1 is selectively required for cell survival, most critically in highly expressing tissues. Remarkably, Appl1 function requires its endosomal localization. Indeed, Akt and GSK-3β, but not TSC2, dynamically associate with Appl1 endosomes upon growth factor stimulation. It is proposed that partitioning of Akt and selected effectors onto endosomal compartments represents a key mechanism contributing to the specificity of signal transduction in vertebrate development (Schenck, 2008).

Based on these data, the current model of Akt regulation and specificity needs to be refined to take into account the contribution of APPL endosomes to the signaling mechanisms. Previously, Akt phosphorylation at Thr308 has been reported to uncouple signaling to FoxO1/3 transcription factors from other Akt effectors. With APPL1, a crucial missing link has been provided between the dependence on Rab5 for the activity of Akt (Hunker, 2006; Su, 2006) and its functional specification. It is proposed that receptors internalized into APPL endosomes are exposed to a molecular membrane environment enriched in selected signaling factors, thus 'channeling' signaling flow downstream of Akt to evoke cell survival uncoupled from growth and proliferation. A platform for selective recruitment and activation of signaling components is envisioned. The protein and lipid composition of such platform needs to be thoroughly established, but it is likely to depend on a combinatorial use of protein-protein and protein-lipid interactions. For example, APPL proteins utilize coincidence detection of membrane curvature and presence of Rab5 to achieve proper organelle targeting. Rab5 alone is not sufficient since APPL localization also crucially depends on its BAR domain. A similar combinatorial use of binding sites is likely to ensure the recruitment of downstream signaling components such as Akt and GSK-3β. However, such recruitment appears to be transient, suggesting that dynamic interactions rather than stable signaling complexes on the Appl endosomes account for signal propagation via Akt and GSK-3β. The precise kinetics and biochemical features of these interactions need to be evaluated using ad hoc developed quantitative live cell imaging techniques (Schenck, 2008).

Regulation of Akt transcription

Peroxisome proliferator-activated receptors (PPARs: distantly related to Drosophila Eip75B) are ligand-activated transcription factors that belong to the nuclear hormone receptor family. Three isotypes that have distinct tissue distributions and functions have been found in vertebrates. Important roles of PPARalpha and PPARgamma in lipid homeostasis and in inflammation have been unveiled. Little is known about the exact function of PPARß, although it has been implicated in colon tumorigenesis, and embryonic development. In adult epidermis, PPARß is undetectable in interfollicular keratinocytes. However, the expression of PPARß is reactivated upon proliferative stimuli such as cutaneous injury, suggesting a role of PPARß in regulating keratinocyte proliferation/differentiation processes. Apoptosis, differentiation, and proliferation are cellular responses that play a pivotal role in wound healing. During this process PPARß translates inflammatory signals into prompt keratinocyte responses. PPARß modulates Akt1 activation via transcriptional upregulation of ILK and PDK1, revealing a mechanism for the control of Akt1 signaling. The resulting higher Akt1 activity leads to increased keratinocyte survival following growth factor deprivation or anoikis. PPARß also potentiates NF-kappaB activity and MMP-9 production, which can regulate keratinocyte migration. Together, these results provide a molecular mechanism by which PPARß protects keratinocytes against apoptosis and may contribute to the process of skin wound closure (Di-Poï, 2002).

Degradation of Akt

The serine threonine kinase Akt is a core survival factor that underlies a variety of human diseases. Although regulatory phosphorylation and dephosphorylation have been well documented, the other posttranslational mechanisms that modulate Akt activity remain unclear. This study shows that tetratricopeptide repeat domain 3 (TTC3) is an E3 ligase that interacts with Akt. TTC3 contains a canonical RING finger motif, a pair of tetratricopeptide motifs, a putative Akt phosphorylation site, and nuclear localization signals, and is encoded by a gene within the Down syndrome (DS) critical region on chromosome 21. TTC3 is an Akt-specific E3 ligase that binds to phosphorylated Akt and facilitates its ubiquitination and degradation within the nucleus. Moreover, DS cells exhibit elevated TTC3 expression, reduced phosphorylated Akt, and accumulation in the G(2)M phase, which can be reversed by TTC3 siRNA or Myr-Akt. Thus, interaction between TTC3 and Akt may contribute to the clinical symptoms of DS (Suizu, 2009).

Akt targets Raf

The signaling pathway comprising Raf, MEK (mitogen-activated protein kinase, or ERK kinase), and ERK (extracellular signal-regulated kinase) lies downstream of the small guanine nucleotide binding protein Ras and mediates several apparently conflicting cellular responses, such as proliferation, apoptosis, growth arrest, differentiation, and senescence, depending on the duration and strength of the external stimulus and on cell type. Another pathway that lies downstream of Ras includes phosphatidylinositol (PI) 3-kinase and Akt (or protein kinase B) and also regulates these cellular responses, acting either synergistically with or in opposition to the Raf pathway. Coordination of the two pathways in a single cellular response may depend on cell type or the stage of differentiation. Akt interacts with Raf and phosphorylates this protein at a highly conserved serine residue in its regulatory domain in vivo. This phosphorylation of Raf by Akt inhibits activation of the Raf-MEK-ERK signaling pathway and shifts the cellular response in a human breast cancer cell line from cell cycle arrest to proliferation. These observations provide a molecular basis for cross talk between two signaling pathways at the level of Raf and Akt. These results demonstrate that Akt antagonizes Raf activity by direct phosphorylation of Ser259. This modification creates a binding site for 14-3-3 protein, a negative regulator of Raf. Similarly, phosphorylation of BAD or the forkhead transcription factor FKHRL1 by Akt also promotes binding of 14-3-3 protein. In all three instances, phosphorylation by Akt inactivates the function of its substrate. Cross talk between the Raf-MEK-ERK and the PI 3-kinase-Akt pathways, mediated by direct interaction of Akt with and its phosphorylation of Raf, may switch the biological response from growth arrest to proliferation, as shown for MCF-7 cells, and may also modulate senescence or differentiation as shown for myoblast differentiation, depending on the cellular system (Zimmermann, 1999).

AKT targets GSK-3

GSK3 is inhibited by serine phosphorylation in response to insulin or growth factors and in vitro by either MAP kinase-activated protein (MAPKAP) kinase-1 (also known as p90rsk) or p70 ribosomal S6 kinase (p70S6k). However, agents that prevent the activation of both MAPKAP kinase-1 and p70S6k by insulin in vivo do not block the phosphorylation and inhibition of GSK3. Another insulin-stimulated protein kinase inactivates GSK3 under these conditions, and it is the product of the proto-oncogene protein kinase B (PKB, also known as Akt/RAC). Like the inhibition of GSK3, the activation of PKB is prevented by inhibitors of phosphatidylinositol (PI) 3-kinase (Cross, 1995).

Activation of phosphatidylinositide 3'-OH kinase (PI 3-kinase) is implicated in mediating a variety of growth factor-induced responses, among which are the inactivation of glycogen synthase kinase-3 (GSK-3) and the activation of the serine/threonine protein kinase B (PKB). GSK-3 inactivation occurs through phosphorylation of Ser-9, and several kinases, such as protein kinase C, mitogen-activated protein kinase-activated protein kinase-1 [p90(Rsk)], p70(S6kinase), and also PKB have all been shown to phosphorylate this site in vitro. In the light of the many candidates to mediate insulin-induced GSK-3 inactivation, the role of PKB has been investigated by constructing a PKB mutant that exhibits dominant-negative function (inhibition of growth factor-induced activation of PKB at expression levels similar to wild-type PKB), because currently no such mutant has been reported. The PKB mutant (PKB-CAAX) acts as an efficient inhibitor of PKB activation and also of insulin-induced GSK-3 regulation. Furthermore, it has been shown that PKB and GSK-3 co-immunoprecipitate, indicating a direct interaction between GSK-3 and PKB. An additional functional consequence of this interaction is implicated by the observation that the oncogenic form of PKB, gagPKB, induces a cellular relocalization of GSK-3 from the cytosolic to the membrane fraction. These results demonstrate that PKB activation is both necessary and sufficient for insulin-induced GSK-3 inactivation and establish a linear pathway from insulin receptor to GSK-3. Regulation of GSK-3 by PKB is likely through direct interaction, since both proteins co-immunoprecipitate. This interaction also results in a translocation of GSK-3 to the membrane in cells expressing transforming gagPKB (van Weeren 1998).

Activation of the Ras/Raf/ERK pathway extends the half-life of the Myc protein and thus enhances the accumulation of Myc activity. Investigated were two N-terminal phosphorylation sites in Myc, Thr 58 and Ser 62, known to be regulated by mitogen stimulation. Phosphorylation of these two residues is critical for determining the stability of Myc. Phosphorylation of Ser 62 is required for Ras-induced stabilization of Myc, likely mediated through the action of ERK. Conversely, phosphorylation of Thr 58, likely mediated by GSK-3 but dependent on the prior phosphorylation of Ser 62, is associated with degradation of Myc. Further analysis demonstrates that the Ras-dependent PI-3K pathway is also critical for controlling Myc protein accumulation, likely through the control of GSK-3 activity. These observations thus define a synergistic role for multiple Ras-mediated phosphorylation pathways in the control of Myc protein accumulation during the initial stage of cell proliferation (Sears, 2000).

The amino acid sequence surrounding Ser 62 represents a consensus ERK recognition sequence, and evidence has been presented that ERK can mediate the phosphorylation of Myc at Ser 62. Mutation of Ser 62 prevents mitogen- and Ras-induced stabilization of Myc. Moreover, phosphorylation at Ser 62 is enhanced under conditions where Myc is stabilized. The importance of Ser 62 in the control of Myc stability is seen in the strict requirement for the stabilization of Myc by Ras, but seen from work that has demonstrated an impaired transforming function when Ser 62 is altered. In contrast, phosphorylation at Thr 58 coincides with a decreased stability of Myc and mutations that prevent Thr 58 phosphorylation lead to stable Myc protein. Once again, this coincides with work that has shown that alteration of Thr 58 enhances the transforming activity of Myc and that mutations at this site are common in Myc proteins derived from tumors. Various lines of work suggest that the GSK-3 protein kinase is most likely responsible for the phosphorylation of Myc at Thr 58. Thr 58 lies within an established consensus, and GSK-3 has been shown to phosphorylate Thr 58 in Myc in vitro. However, unlike ERK, which is tightly regulated by cell growth, the level of GSK-3 protein is constant and does not fluctuate with cell growth. Nevertheless, despite the continual presence of GSK-3 protein, the activity of the kinase is regulated during the initial phase of cell proliferation. In particular, GSK-3 activity is inhibited through the action of PI-3K/AKT. Thus, as Ras initiates the PI-3K/AKT pathway, GSK-3 activity is held in check, preventing the phosphorylation of Thr 58. Only when AKT activity declines would GSK-3 then have the capacity to phosphorylate Thr 58 to induce the degradation of Myc. Thus, Ras activation elicits two responses within the cell that can cooperate to enhance Myc stability: a direct effect of ERK and an indirect effect of AKT (Sears, 2000 and references therein).

The inhibition of GSK3 is required for the stimulation of glycogen and protein synthesis by insulin and the specification of cell fate during development. The insulin-induced inhibition of GSK3 and its unique substrate specificity are explained by the existence of a phosphate binding site in which Arg-96 is critical. Thus, mutation of Arg-96 abolishes the phosphorylation of 'primed' glycogen synthase as well as inhibition by PKB-mediated phosphorylation of Ser-9. Hence, the phosphorylated N terminus acts as a pseudosubstrate, occupying the same phosphate binding site used by primed substrates. Significantly, this mutation does not affect phosphorylation of 'nonprimed' substrates in the Wnt-signaling pathway (Axin and ß-catenin), suggesting new approaches to design more selective GSK3 inhibitors for the treatment of diabetes (Frame, 2001).

GSK3 is phylogenetically most closely related to the cyclin-dependent protein kinases (CDKs), such as CDK1 (also called cdc2) and CDK2. However, the specificity of GSK3 is unique in that it requires a priming phosphate located at n + 4 (where n is the site of phosphorylation) in order to phosphorylate many of its substrates, such as glycogen synthase. In contrast, the phosphorylation of Axin and ß-catenin in the Wnt signaling pathway is not known to require a priming phosphate and may rely on high-affinity interactions in a multiprotein complex with GSK3. Thus, Axin binds to both GSK3 and ß-catenin, bringing these proteins into close proximity to facilitate their phosphorylation by GSK3. This study presents evidence for a specific site of interaction between the phosphate of the primed substrate and Arg-96 of GSK3. This same phosphate binding site is also occupied by Ser-9 once it becomes phosphorylated by PKB. The existence of this site helps to explain several features of GSK3, such as its unusual substrate specificity requirements and the mechanism by which it becomes inhibited in response to insulin and growth factors. These findings have important implications for drug development in this area (Frame, 2001).

Axon-dendrite polarity is a cardinal feature of neuronal morphology essential for information flow. A differential distribution of GSK-3ß activity is found in the axon versus the dendrites. A constitutively active GSK-3ß mutant inhibits axon formation, whereas multiple axons formed from a single neuron when GSK-3ß activity is reduced by pharmacological inhibitors, a peptide inhibitor, or siRNAs. An active mechanism for maintaining neuronal polarity was revealed by the conversion of preexisting dendrites into axons upon GSK-3 inhibition. Biochemical and functional data show that the Akt kinase and the PTEN phosphatase are upstream of GSK-3ß in determining neuronal polarity. These results demonstrate that there are active mechanisms for maintaining as well as establishing neuronal polarity, indicate that GSK-3ß relays signaling from Akt and PTEN to play critical roles in neuronal polarity, and suggest that application of GSK-3ß inhibitors can be a novel approach to promote generation of new axons after neural injuries (Jiang, 2005).

Serine (Ser) 9 in GSK-3ß can be regulated by multiple kinases, among which Akt is differentially localized in axons. To test for GSK-3ß regulation by the candidate upstream molecules, the same inhibitors for PI3K as those used previously for studies of neuronal polarity were used. LY294002 significantly inhibits Akt phosphorylation at Ser 473 and GSK-3ß phosphorylation at Ser 9. LY294002 does not affect GSK-3ß phosphorylation at Tyr216. In addition to the biochemical evidences, the distribution of GSK-3ß and pGSK-3ß was examined. LY294002 reduces the ratio of pGSK-3ß over GSK-3ß. aPKC is another candidate GSK-3ß regulator, but GSK-3ß phosphorylation is not affected by the aPKC inhibitor bisindolylmaleimide I (Bis), or by the MAPK inhibitor U0126 or the PKA inhibitor KT5720 (Jiang, 2005).

To test for the functional significance of Akt, attempts were made to examine the effect of inhibiting Akt by using short hairpin RNAs (shRNAs), but they caused neuronal death. The effect of increasing Akt activity on neuronal polarity was examined by using Myr-Akt, a constitutively active Akt derived by fusion with the myristoylation signal of Src. The wild-type Akt does not affect neuronal polarity, whereas Myr-Akt causes the formation of multiple axons (Jiang, 2005).

Because GSK-3ß S9A should not be phosphorylated by Akt after the substation of Ser 9, it was possible to test for the relationship between Akt and GSK-3ß by cotransfection of GSK-3ß S9A and Myr-Akt. GSK-3ß S9A could partially, but not completely, reverse the effect of Myr-Akt on the formation of multiple axons. These results are consistent with the idea of Akt being upstream of GSK-3ß. The partial reversal could result from either incomplete overlap in GSK-3ß S9A and Myr-Akt transfection or the possibility that GSK-3ß constitutes a part, but not all, of the output for Akt (Jiang, 2005).

Detailed examination indicates that Akt plays multiple roles in axon and dendrite development. Myr-Akt increases the number of neurites per neuron both in neurons forming multiple axons, and in neurons with single axons. Myr-Akt increases both the numbers of axons and dendrites from the normal total of five neurites to eight. When the number of axons is increased in Myr-Akt neurons, axon length is shorter than normal, perhaps due to limited materials for an increased number of axons. When the number of axons is not increased in Myr-Akt neurons, the single axon is longer than normal, indicating that Akt promotes axon outgrowth. The length of dendrites is not affected by Myr-Akt (Jiang, 2005).

Only the effect of increasing axon number is shared between Akt activation and GSK-3ß inactivation. GSK-3ß is therefore only one downstream component of Akt, specifically mediating the function of Akt in axon-dendrite polarity, but not in neurite number or axon length (Jiang, 2005).

PTEN is a lipid and protein phosphatase that functions in opposition to PI3K by dephosphorylating the lipid product of PI3K, phosphatidylinositol 3,4,5-trisphosphate (PIP3) . The role of PTEN was examined further and the functional relationship of PTEN and GSK-3ß was characterized. Neuronal polarity is lost upon PTEN overexpression. Detailed analysis indicates that PTEN transfection inhibits axon formation without affecting dendrite formation. The inactive PTEN control (PTEN G129R) does not affect neuronal polarity (Jiang, 2005).

To investigate the role of endogenous PTEN, PTEN siRNA, a small inhibitor RNA (siRNA) construct designed specifically for PTEN, was used, and PTEN SsiRNA, a scrambled control. PTEN siRNA, but not PTEN SsiRNA, reduces PTEN protein levels in hippocampal neurons. PTEN siRNA increases the number of axons at the expense of dendrites. When the level of PTEN protein was examined, the level of PTEN was reduced by more than 50% in PTEN siRNA neurons with multiple axons, whereas PTEN was reduced less than 20% in PTEN siRNA neurons with single axons. Significant reduction of PTEN expression therefore correlates with the formation of multiple axons (Jiang, 2005).

To test for a functional relationship between PTEN and GSK-3ß, two kinds of experiments were performed. The first was to transfect neurons with PTEN and also treat them with SB415286. SB415286 almost completely reverses the effect of PTEN overexpression on axon-dendrite polarity, resulting in multiple axons at the expense of dendrites. The second kind of experiment was to cotransfect PTEN siRNA and GSK-3ß S9A. The effect of PTEN siRNA on multiple axon formation could be inhibited by an S0A mutant of GSK-3ß in which Ser 9 was replaced with alanine. These results indicate that GSK-3ß is downstream of PTEN in axon-dendrite polarity formation because GSK-3ß manipulations dominate over PTEN manipulations (Jiang, 2005).

One role of PTEN does not seem to be downstream of GSK-3ß. When neurons are cotransfected with PTEN siRNA and GSK-3ß S9A, GSK-3ß S9A could not eliminate axons, suggesting that either PTEN siRNA can antagonize the effect of GSK-3ß S9A in axon formation or that GSK-3ß S9A has to act in a PTEN-dependent pathway to inhibit axon formation. In the latter scenario, GSK-3ß activation could be upstream of PTEN in inhibiting axon formation (Jiang, 2005).

Akt targets TSC-2

The S/T-protein kinases activated by phosphoinositide 3-kinase (PI3K) regulate a myriad of cellular processes. An approach using a combination of biochemistry and bioinformatics can identify substrates of these kinases. This approach identifies the tuberous sclerosis complex-2 gene product, tuberin (Drosophila homolog Gigas), as a potential target of Akt/PKB. Upon activation of PI3K, tuberin is phosphorylated on consensus recognition sites for PI3K-dependent S/T kinases. Moreover, Akt/PKB can phosphorylate tuberin in vitro and in vivo. S939 and T1462 of tuberin are PI3K-regulated phosphorylation sites and T1462 is constitutively phosphorylated in PTEN-/- tumor-derived cell lines. Finally, a tuberin mutant lacking the major PI3K-dependent phosphorylation sites can block the activation of S6K1, suggesting a means by which the PI3K-Akt pathway regulates S6K1 activity (Manning, 2002).

Class I phosphoinositide 3-kinases (PI3Ks) are activated by many extracellular growth and survival stimuli. These lipid kinases catalyze the production of the second messengers phosphatidylinositol-3,4-bisphosphate (PtdIns-3,4P2) and phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5P3). Downstream targets containing specialized domains, such as pleckstrin-homology (PH) domains, that specifically bind to these lipid products of PI3K are then activated. These activated proteins control a wide array of cellular processes, including survival, proliferation, protein synthesis, growth, metabolism, cytoskeletal rearrangements, and differentiation. However, there is still much that is not known about the signaling events leading from activation of PI3K effectors to downstream changes in cell physiology (Manning, 2002).

Serine/threonine (S/T) protein kinases can account for much of the functional diversity of PI3K signaling. Akt/protein kinase B and the 70 kDa-S6 kinase 1 (S6K1) are the best characterized of the PI3K-regulated S/T kinases. The mitogen-stimulated activation of both of these kinases is blocked by PI3K-specific inhibitors. Akt contains a PH domain that is specific to PtdIns-3,4P2 and PtdIns-3,4,5P3. Akt is thereby recruited to these PI3K-generated second messengers and to the PDK1 protein kinase, which also specifically binds to these lipids. PDK1 then phosphorylates and activates Akt (Manning, 2002).

The regulation of S6K1 is much more complex, with both PI3K-dependent and -independent signaling pathways involved in its activation. Several PI3K-regulated effectors are known to participate in the activation of S6K1, including PDK1, PKCzeta/lambda, Cdc42, Rac1, and Akt. However, the molecular mechanism of how these contribute to S6K1 activation remains unclear. In addition to mitogen-regulated signaling to S6K1, the metabolic state of the cell and the availability of nutrients control S6K1 activation through the mammalian target of rapamycin (mTOR, also known as FRAP, RAFT, and RAPT). Recent studies suggest that mTOR is also regulated by mitogenic signals. Interestingly, it has been suggested that the point of convergence of the mitogenic and nutrient-sensing signals in the regulation of S6K1 may be at the level of Akt directly phosphorylating mTOR. However, this phosphorylation does not appear to affect mTOR activity or S6K1 activation. Thus, of the PI3K-regulated effectors thought to participate in S6K1 activation, the molecular basis of how Akt regulates S6K1 remains the least well understood (Manning, 2002).

Akt itself has been implicated in many of the PI3K-regulated cellular events, and several substrates have been shown to be phosphorylated in vitro and/or in vivo by Akt. Therefore, the total cellular effect of PI3K activation and subsequent activation of Akt is mediated through a variety of different targets. However, it seems unlikely that the large array of processes controlled by the PI3K-Akt pathway can be accounted for by the current knowledge of downstream targets (Manning, 2002).

An approach has been developed to screen for substrates of PI3K-dependent S/T kinases, such as Akt. This approach uses phospho-specific antibodies generated against a phosphorylated protein kinase consensus recognition motif in combination with a protein database motif scanning program called Scansite ( Scansite is a web-based program that searches protein databases for optimal substrates of specific protein kinases and for optimal binding motifs for specific protein domains with data generated by peptide library screens. The phospho-motif antibody is used to recognize proteins phosphorylated specifically under conditions in which the kinase of interest is active. Scansite is then used to identify candidate substrates of this protein kinase that have the predicted molecular mass of the proteins recognized by the phospho-motif antibody. This approach successfully identifies known substrates of Akt. The tuberous sclerosis complex-2 (TSC2) tumor suppressor gene product, tuberin, is also identified and characterized as an Akt substrate. Furthermore, it is found that overexpression of a tuberin mutant lacking the major Akt phosphorylation sites can inhibit growth factor-induced activation of S6K1. These results provide a biochemical link between the PI3K-Akt pathway and regulation of S6K1 and also indicate a biochemical basis for the disease tuberous sclerosis complex (TSC) (Manning, 2002).

TSC is a common disease affecting an estimated 1 in 6000 individuals and is characterized by the occurrence of widespread benign tumors called hamartomas frequently affecting the brain, skin, kidneys, lungs, eyes, and heart. In approximately 85% of TSC patients, the disease is caused by loss-of-function mutations in one of two tumor suppressor genes, TSC1 and TSC2, which encode hamartin and tuberin, respectively. These two proteins form a complex. Tuberin, which has a region of homology to Rap1 GTPase-activating proteins (GAPs), has been shown to possess in vitro GAP activity toward Rap1. However, the true molecular and cellular functions of the hamartin-tuberin complex have yet to be clearly defined. Furthermore, very little is known about how these tumor suppressor gene products are regulated (Manning, 2002).

Based on genetic studies and the fact that PI3K and Akt are oncogenes while the TSC genes are tumor suppressors, one would predict that the PI3K-Akt-mediated phosphorylation of tuberin would inhibit the function of the tuberin-hamartin complex. In Drosophila, hamartin and tuberin appear to function together to antagonize signaling of the insulin-PI3K-Akt pathway and, thereby, restrict cell growth and proliferation. Most strikingly, loss of just one copy of TSC1 or TSC2 partially rescues the lethality of insulin receptor loss-of-function mutants. This result implies that one of the primary functions of the insulin pathway, at least in Drosophila, is to inhibit the hamartin-tuberin complex. Furthermore, both mouse and Drosophila genetic studies have suggested that the tuberin-hamartin complex functions to inhibit S6K1 (Manning, 2002).

Expression in human cells of the tuberinS939A/T1462A mutant, which lacks the major PI3K-dependent phosphorylation sites, at levels comparable to endogenous tuberin leads to a decrease in growth factor-induced S6K1 phosphorylation and activity. This phosphorylation and subsequent activation of S6K1 has been previously demonstrated to be dependent on PI3K. These results, along with those from genetic studies in other systems, are consistent with a model in which growth factors activate PI3K leading to the phosphorylation of tuberin by Akt. This phosphorylation inhibits the tuberin-hamartin complex, thereby relieving its inhibition of S6K1. In this model, expression of the tuberinS939A/T1462A mutant, which would not be phosphorylated and inhibited, would have a dominant effect over endogenous tuberin and block growth factor-induced S6K1 activation. It will be of great interest to determine the molecular nature of S6K1 inhibition by the tuberin-hamartin complex in the absence of mitogenic stimuli. It is possible that the complex does so upstream of mTOR, because the constitutive activation of S6K1 in TSC1-/- MEFs is sensitive to rapamycin. Alternatively, mTOR might regulate S6K1 in a nutrient-sensitive pathway parallel to the mitogen-sensitive PI3K-Akt-tuberin pathway (Manning, 2002).

Recent studies have suggested that S6K1 activation can occur independent of PI3K and Akt. These studies demonstrate the existence of multiple pathways regulating S6K1 and that the tuberin-hamartin complex might integrate signals from many different inputs. The identification of tuberin as a direct downstream target of the PI3K-Akt pathway provides the missing link between this signaling cascade and control of S6K1 activity (Manning, 2002).

The identification of this biochemical relationship between the mammalian TSC tumor suppressor gene products and the oncogenic PI3K-Akt pathway could have important implications in human diseases. For instance, in approximately 10%-15% of patients diagnosed with TSC, mutations in TSC1 or TSC2 have not been detected. Based on the findings of this study, it is possible that mutations leading to aberrant activation of the PI3K-Akt pathway, such as PTEN mutations, could inhibit the function of the tuberin-hamartin complex by causing constitutive phosphorylation of tuberin. It will be interesting to examine the phosphorylation state of tuberin within hamartomas from such TSC patients. Indeed, in PTEN-/- cell lines derived from both glioblastoma and prostate tumors, growth factor-independent phosphorylation of tuberin on the PI3K-dependent T1462 site is detected (Manning, 2002).

Germline mutations in either PTEN or the TSC genes cause autosomal dominant diseases that are characterized by the occurrence of widespread hamartomas due to loss of heterozygosity at these loci. However, the tissue distribution of these benign tumors varies between patients with loss of PTEN and those with TSC. These differences might be explained by a model in which the tuberin-hamartin complex is the primary growth-inhibiting target of the PI3K-Akt pathway in tissues affected in TSC patients. In other tissues, such as those affected in patients with PTEN mutations, this complex might be one of many targets of the PI3K-Akt pathway. Interestingly, though, recent studies have suggested that mTOR activity is essential for oncogenic transformation of cells by activated PI3K or Akt and for growth of PTEN-/- tumors. Therefore, aberrant phosphorylation and inhibition of the tuberin-hamartin complex, and subsequent increased activity of mTOR and/or S6K1, would likely contribute to tumorigenesis caused by mutations that activate the PI3K-Akt pathway. Future studies using crosses between PTEN and TSC knockout mice should help determine the contribution of the tuberin-hamartin complex in prevention of the variety of tumors caused by uncontrolled signaling through the PI3K-Akt pathway. Finally, the elucidation of a PI3K-Akt-tuberin pathway controlling S6K1 activity will have important implications in the understanding and treatment of the prevalent TSC disease (Manning, 2002).

Normal cellular functions of hamartin and tuberin, encoded by the TSC1 and TSC2 tumor suppressor genes, are closely related to their direct interactions. However, the regulation of the hamartin-tuberin complex in the context of the physiologic role as tumor suppressor genes has not been documented. Insulin or insulin growth factor (IGF) 1 stimulates phosphorylation of tuberin, which is inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 but not by the mitogen-activated protein kinase inhibitor PD98059. Expression of constitutively active PI3K or active Akt, including Akt1 and Akt2, induces tuberin phosphorylation. Akt/PKB associates with hamartin-tuberin complexes, promoting phosphorylation of tuberin and increased degradation of hamartin-tuberin complexes. The ability to form complexes, however, is not blocked. Akt also inhibits tuberin-mediated degradation of p27(kip1), thereby promoting CDK2 activity and cellular proliferation. These results indicate that tuberin is a direct physiological substrate of Akt and that phosphorylation of tuberin by PI3K/Akt is a major mechanism controlling hamartin-tuberin function (Dan, 2002).

TSC1-TSC2 inhibits the p70 ribosomal protein S6 kinase 1 (an activator of translation) and activates the eukaryotic initiation factor 4E binding protein 1 (4E-BP1, an inhibitor of translational initiation). These functions of TSC1-TSC2 are mediated by inhibition of the mammalian target of rapamycin (mTOR). Furthermore, TSC2 is directly phosphorylated by Akt, which is involved in stimulating cell growth and is activated by growth stimulating signals, such as insulin. TSC2 is inactivated by Akt-dependent phosphorylation, which destabilizes TSC2 and disrupts its interaction with TSC1. These data indicate a molecular mechanism for TSC2 in insulin signalling, tumor suppressor functions and in the inhibition of cell growth (Inoki, 2002).

Akt1-Inhibitor of DNA binding2 is essential for growth cone formation and axon growth and promotes central nervous system axon regeneration

Mechanistic studies of axon growth (see Drosophila axonogenesis) during development are beneficial to the search for neuron-intrinsic regulators of axon regeneration. This study discovered that in the developing neuron from rat, Akt signaling (see Drosophila Akt1 and insulin signaling) regulates axon growth and growth cone formation through phosphorylation of serine 14 (S14) on Inhibitor of DNA binding 2 (Id2) (see Drosophila emc). This enhances Id2 protein stability by means of escape from proteasomal degradation, and steers its localization to the growth cone, where Id2 interacts with radixin (see Drosophila Moe) that is critical for growth cone formation. Knockdown of Id2, or abrogation of Id2 phosphorylation at S14, greatly impairs axon growth and the architecture of growth cone. Intriguingly, reinstatement of Akt/Id2 signaling after injury in mouse hippocampal slices redeemed growth promoting ability, leading to obvious axon regeneration. These results suggest that Akt/Id2 signaling is a key module for growth cone formation and axon growth, and its augmentation plays a potential role in CNS axonal regeneration (Ko, 2016).

Akt and NF-kappaB

Activation of the nuclear transcription factor NF-kappaB by inflammatory cytokines requires the successive action of NF-kappaB-inducing kinase (NIK) and an IKB-kinase (IKK) complex composed of IKKalpha and IKKbeta. The Akt serine-threonine kinase is involved in the activation of NF-kappaB by tumor necrosis factor (TNF). TNF activates phosphatidylinositol-3-OH kinase [PI(3)K] and its downstream target Akt (protein kinase B). Wortmannin [a PI(3)K inhibitor], dominant-negative PI(3)K or kinase-dead Akt inhibits TNF-mediated NF-kappaB activation. Constitutively active Akt induces NF-kappaB activity and this effect is blocked by dominant-negative NIK. Conversely, NIK activates NF-kappaB and this is blocked by kinase-dead Akt. Thus, both Akt and NIK are necessary for TNF activation of NF-kappaB. Akt mediates IKKalpha phosphorylation at threonine 23. Mutation of this amino acid blocks phosphorylation by Akt or TNF and activation of NF-kappaB. These findings indicate that Akt is part of a signaling pathway that is necessary for inducing key immune and inflammatory responses (Ozes, 1999).

The mechanisms of cell proliferation and transformation are intrinsically linked to the process of apoptosis: the default of proliferating cells is to die unless specific survival signals are provided. Platelet-derived growth factor (PDGF) is a principal survival factor that inhibits apoptosis and promotes proliferation, but the mechanisms mediating its anti-apoptotic properties are not completely understood. The transcription factor NF-kappaB is important in PDGF signaling. NF-kappaB transmits two signals: one is required for the induction of proto-oncogene c-myc and proliferation, and the second, an anti-apoptotic signal, counterbalances c-Myc cytotoxicity. A putative pathway has been traced whereby PDGF activates NF-kappaB through Ras and phospatidylinositol-3-kinase [PI(3)K] to the PKB/Akt protein kinase and the IkappaB kinase (IKK); NF-kappaB thus appears to be a target of the anti-apoptotic Ras/PI(3)K/Akt pathway. Upon PDGF stimulation, Akt transiently associates in vivo with IKK and induces IKK activation. These findings establish a role for NF-kappaB in growth factor signaling and define an anti-apoptotic Ras/PI(3)K/Akt/IKK/NF-kappaB pathway, thus linking anti-apoptotic signaling with transcription machinery (Romashkova, 1999).

Recent work has suggested a role for the serine/threonine kinase Akt and IkappaB kinases (IKKs) in nuclear factor (NF)-kappaB activation. In this study, the involvement of these components in NF-kappaB activation through a G protein-coupled pathway was examined using transfected HeLa cells that express the B2-type bradykinin (BK) receptor. The function of IKK2, and to a lesser extent, IKK1, is suggested by BK-induced activation of their kinase activities and by the ability of their dominant negative mutants to inhibit BK-induced NF-kappaB activation. BK-induced NF-kappaB activation and IKK2 activity are markedly inhibited by RGS3T, a regulator of G protein signaling that inhibits Galpha(q), and by two Gbetagamma scavengers. Co-expression of Galpha(q) potentiates BK-induced NF-kappaB activation, whereas co-expression of either an activated Galpha(q)(Q209L) or Gbeta(1)gamma(2) induces IKK2 activity and NF-kappaB activation without BK stimulation. BK-induced NF-kappaB activation is partially blocked by LY294002 and by a dominant negative mutant of phosphoinositide 3-kinase (PI3K), suggesting that PI3K is a downstream effector of Galpha(q) and Gbeta(1)gamma(2) for NF-kappaB activation. Furthermore, BK can activate the PI3K downstream kinase Akt, whereas a catalytically inactive mutant of Akt inhibits BK-induced NF-kappaB activation. Taken together, these findings suggest that BK utilizes a signaling pathway that involves Galpha(q), Gbeta(1)gamma(2), PI3K, Akt, and IKK for NF-kappaB activation (Xie, 2000).

The serine/threonine kinase Akt (also known as protein kinase B, PKB) is activated by numerous growth-factor and immune receptors through lipid products of phosphatidylinositol (PI) 3-kinase. Akt can couple to pathways that regulate glucose metabolism or cell survival. Akt can also regulate several transcription factors, including E2F, CREB, and the Forkhead family member Daf-16. Akt regulates signaling pathways that lead to induction of the NF-kappaB family of transcription factors in the Jurkat T-cell line. This induction occurs, at least in part, at the level of degradation of the NF-kappaB inhibitor IkappaB, and is specific for NF-kappaB, since other inducible transcription factors are not affected by Akt overexpression. Furthermore, the effect requires the kinase activity and pleckstrin homology (PH) domain of Akt. Also, Akt does not act alone to induce cytokine promoters and NF-kappaB reporters, because signals from other pathways are required to observe the effect. These studies uncover a previously unappreciated connection between Akt and NF-kappaB induction that could have implications for the control of T-cell growth and survival (Kane, 1999).

While NF-kappaB is considered to play key roles in the development and progression of many cancers, the mechanisms whereby this transcription factor is activated in cancer are poorly understood. A key oncoprotein in a variety of cancers is the serine- threonine kinase Akt, which can be activated by mutations in PI3K, by loss of expression/activity of PTEN, or through signaling induced by growth factors and their receptors. A key effector of Akt-induced signaling is the regulatory protein mTOR (mammalian target of rapamycin). This study shows that mTOR downstream from Akt controls NF-kappaB activity in PTEN-null/inactive prostate cancer cells via interaction with and stimulation of IKK. The mTOR-associated protein Raptor is required for the ability of Akt to induce NF-kappaB activity. Correspondingly, the mTOR inhibitor rapamycin is shown to suppress IKK activity in PTEN-deficient prostate cancer cells through a mechanism that may involve dissociation of Raptor from mTOR. The results provide insight into the effects of Akt/mTOR-dependent signaling on gene expression and into the therapeutic action of rapamycin (Dan, 2008).

Akt targets Foxo family transcription factors

Survival factors can suppress apoptosis in a transcription-independent manner by activating the serine/threonine kinase Akt, which then phosphorylates and inactivates components of the apoptotic machinery, including BAD and Caspase 9. Akt also regulates the activity of FKHRL1 (Drosophila homolog Foxo), a member of the Forkhead family of transcription factors. In the presence of survival factors, Akt phosphorylates FKHRL1, leading to FKHRL1's association with 14-3-3 proteins and FKHRL1's retention in the cytoplasm. Survival factor withdrawal leads to FKHRL1 dephosphorylation, nuclear translocation, and target gene activation. Within the nucleus, FKHRL1 triggers apoptosis most likely by inducing the expression of genes that are critical for cell death, such as the Fas ligand gene (Brunet, 1999).

The regulation of intracellular localization of AFX, a human Forkhead transcription factor, was studied. AFX was recovered as a phosphoprotein from transfected COS-7 cells growing in the presence of FBS, and the phosphorylation was eliminated by wortmannin, a potent inhibitor of phosphatidylinositol (PI) 3-kinase. AFX is phosphorylated in vitro by protein kinase B (PKB), a downstream target of PI 3-kinase, but a mutant protein in which three putative phosphorylation sites of PKB have been replaced by Ala is not recognized by PKB. In Chinese hamster ovary cells (CHO-K1) cultured with serum, the AFX protein fused with green fluorescence protein (AFX-GFP) is localized mainly in the cytoplasm, and wortmannin induces transient nuclear translocation of the fusion protein. The AFX-GFP mutant in which all three phosphorylation sites have been replaced by Ala is detected exclusively in the cell nucleus. AFX-GFP is in the nucleus when the cells are infected with an adenovirus vector encoding a dominant-negative form of either PI 3-kinase or PKB, whereas the fusion protein stays in the cytoplasm when the cells express constitutively active PKB. In CHO-K1 cells expressing AFX-GFP, DNA fragmentation is induced by the stable PI 3-kinase inhibitor LY294002, and the expression of the active form of PKB suppresses this DNA fragmentation. The phosphorylation site mutant of AFX-GFP enhances DNA fragmentation irrespective of the presence and absence of PI 3-kinase inhibitor. These results indicate that the nuclear translocation of AFX is negatively regulated through its phosphorylation by PKB (Takaishi, 1999).

Although genetic analysis has demonstrated that members of the winged helix, or forkhead, family of transcription factors play pivotal roles in the regulation of cellular differentiation and proliferation, both during development and in the adult, little is known of the mechanisms underlying their regulation. The activation of phosphatidylinositol 3 (PI3) kinase by extracellular growth factors induces phosphorylation, nuclear export, and transcriptional inactivation of FKHR1, a member of the FKHR subclass of the forkhead family of transcription factors. Protein kinase B (PKB)/Akt, a key mediator of PI3 kinase signal transduction, phosphorylates recombinant FKHR1 in vitro at threonine-24 and serine-253. Mutants FKHR1(T24A), FKHR1(S253A), and FKHR1(T24A/S253A) are resistant to both PKB/Akt-mediated phosphorylation and PI3 kinase-stimulated nuclear export. These results indicate that phosphorylation by PKB/Akt negatively regulates FKHR1 by promoting export from the nucleus (Biggs, 1999).

The androgen receptor (AR) controls several biological functions including prostate cell growth and apoptosis. However, the mechanism by which AR maintains its stability to function properly remains largely unknown. Akt and Mdm2 have been shown to form a complex with AR and promote phosphorylation-dependent AR ubiquitylation, resulting in AR degradation by the proteasome. The effect of Akt on AR ubiquitylation and degradation is markedly impaired in a Mdm2-null cell line compared with the wild-type cell line, suggesting that Mdm2 is involved in Akt-mediated AR ubiquitylation and degradation. Furthermore, the E3 ligase activity of Mdm2 and phosphorylation of Mdm2 by Akt are essential for Mdm2 to affect AR ubiquitylation and degradation. These results suggest that phosphorylation-dependent AR ubiquitylation and degradation by Akt require the involvement of Mdm2 E3 ligase activity, a novel mechanism that provides insight into how AR is targeted for degradation (Lin, 2002).

IGF-1 can promote AR degradation via activation of the PI3K-Akt pathway. In addition, IL-6 can also down-regulate AR protein levels via the PI3K-Akt pathway. Thus, PI3K-Akt, but not MAPK, may represent the major pathway for growth factor-induced AR protein degradation. While the mechanism responsible for this distinct effect on AR protein degradation is currently unclear, it is likely that phosphorylation of AR at distinct sites by these two pathways may result in different AR conformations, which may then contribute differentially to protein stability. In support of the role of PI3K-Akt in AR degradation, an AR mutant, which is defective in Akt-mediated AR phosphorylation, is remarkably stable compared with the wtAR. Thus, the PI3K-Akt pathway is involved in protein degradation. PI3K-Akt has been associated with p27Kip1 and insulin receptor substrate-1 (IRS-1) stability, and blockage of the PI3K-Akt pathway causes the accumulation of p27Kip1 protein and IRS-1. Therefore, the PI3K-Akt pathway may represent a central pathway for the degradation of several proteins, including AR (Lin, 2002).

The phosphatidylinositol-3-OH-kinase (PI(3)K) effector protein kinase B regulates certain insulin-responsive genes, but the transcription factors regulated by protein kinase B have yet to be identified. Genetic analysis in Caenorhabditis elegans has shown that the Forkhead transcription factor daf-16 is regulated by a pathway consisting of insulin-receptor-like daf-2 and PI(3)K-like age-1. Protein kinase B phosphorylates AFX, a human ortholog of daf-16, both in vitro and in vivo. Inhibition of endogenous PI(3)K and protein kinase B activity prevents protein kinase B-dependent phosphorylation of AFX and reveals residual protein kinase B-independent phosphorylation that requires Ras signalling towards the Ral GTPase. In addition, phosphorylation of AFX by protein kinase B inhibits its transcriptional activity. Together, these results delineate a pathway for PI(3)K-dependent signalling to the nucleus (Kops, 1999).

Forkhead transcription factor FKHR (Foxo1: Drosophila homolog Foxo) is a key regulator of glucose homeostasis, cell-cycle progression, and apoptosis. FKHR is phosphorylated via insulin or growth factor signaling cascades, resulting in its cytoplasmic retention and the repression of target gene expression. The fate has been investigated of FKHR after cells are stimulated by insulin. Insulin treatment is shown to decrease endogenous FKHR proteins in HepG2 cells; this decrease is inhibited by proteasome inhibitors. FKHR is ubiquitinated in vivo and in vitro, and insulin enhances the ubiquitination in the cells. In addition, the signal to FKHR degradation from insulin is mediated by the phosphatidylinositol 3-kinase pathway, and mutation of FKHR at the serine or threonine residues phosphorylated by protein kinase B, a downstream target of phosphatidylinositol 3-kinase, inhibits the ubiquitination in vivo and in vitro. Finally, efficient ubiquitination of FKHR requires both phosphorylation and cytoplasmic retention in the cells. These results demonstrate that the insulin-induced phosphorylation of FKHR leads to the multistep negative regulation, not only by the nuclear exclusion but also the ubiquitination-mediated degradation (Matsuzaki, 2003).

Growth factor receptors promote cell growth and survival by stimulating the activities of phosphatidylinositol 3-kinase and Akt/PKB. Akt activation causes proteasomal degradation of substrates that control cell growth and survival. Expression of activated Akt triggers proteasome-dependent declines in the protein levels of the Akt substrates tuberin, FOXO1, and FOXO3a. The addition of proteasome inhibitors stabilizes the phosphorylated forms of multiple Akt substrates, including tuberin and FOXO proteins. Activation of Akt triggers the ubiquitination of several proteins containing phosphorylated Akt substrate motifs. Together the data indicate that activated Akt stimulates proteasomal degradation of its substrates and suggest that Akt-dependent cell growth and survival are induced through the degradation of negative regulators of these processes (Plas, 2003).

Myc synergizes with Ras and PI3-kinase in cell transformation, yet the molecular basis for this behavior is poorly understood. Myc is shown to recruit TFIIH, P-TEFb and Mediator to the cyclin D2 and other target promoters, while the PI3-kinase pathway controls formation of the preinitiation complex and loading of RNA polymerase II. The PI3-kinase pathway involves Akt-mediated phosphorylation of FoxO transcription factors. In a nonphosphorylated state, FoxO factors inhibit induction of multiple Myc target genes, Myc-induced cell proliferation and transformation by Myc and Ras. Abrogation of FoxO function enables Myc to activate target genes in the absence of PI3-kinase activity and to induce foci formation in primary cells in the absence of oncogenic Ras. It is suggested that the cooperativity between Myc and Ras is at least in part due to the fact that Myc and FoxO proteins control distinct steps in the activation of an overlapping set of critical target genes (Bouchard, 2004).

FoxOs inhibit mTORC1 and activate Akt by inducing the expression of Sestrin3 and Rictor

FoxO transcription factors and TORC1 are conserved downstream effectors of Akt. This study unraveled regulatory circuits underlying the interplay between Akt, FoxO, and mTOR. Activated FoxO1 inhibits mTORC1 by TSC2-dependent and TSC2-independent mechanisms. First, FoxO1 induces Sestrin3 (Sesn3) gene expression. Sesn3, in turn, inhibits mTORC1 activity in Tsc2-proficient cells. Second, FoxO1 elevates the expression of Rictor, leading to increased mTORC2 activity that consequently activates Akt. In Tsc2-deficient cells, the elevation of Rictor by FoxO increases mTORC2 assembly and activity at the expense of mTORC1, thereby activating Akt while inhibiting mTORC1. FoxO may act as a rheostat that maintains homeostatic balance between Akt and mTOR complexes' activities. In response to physiological stresses, FoxO maintains high Akt activity and low mTORC1 activity. Thus, under stress conditions, FoxO inhibits the anabolic activity of mTORC1, a major consumer of cellular energy, while activating Akt, which increases cellular energy metabolism, thereby maintaining cellular energy homeostasis (Chen, 2010; see graphical abstract).

Ablation in mice of the mTORC components raptor, rictor, or mLST8 reveals that mTORC2 is required for signaling to Akt-FOXO and PKCa, but not S6K1

The mTOR kinase controls cell growth, proliferation, and survival through two distinct multiprotein complexes, mTORC1 and mTORC2. mTOR and mLST8 are in both complexes, while raptor and rictor are part of only mTORC1 and mTORC2, respectively. To investigate mTORC1 and mTORC2 function in vivo, mice deficient for raptor, rictor, or mLST8 were generated. Like mice null for mTOR, those lacking raptor die early in development. However, mLST8 null embryos survive until e10.5 and resemble embryos missing rictor. mLST8 is necessary to maintain the rictor-mTOR, but not the raptor-mTOR, interaction, and both mLST8 and rictor are required for the hydrophobic motif phosphorylation of Akt/PKB and PKCα, but not S6K1. Furthermore, insulin signaling to FOXO3, but not to TSC2 or GSK3β, requires mLST8 and rictor. Thus, mTORC1 function is essential in early development, mLST8 is required only for mTORC2 signaling, and mTORC2 is a necessary component of the Akt-FOXO and PKCα pathways (Guertin, 2007).

The mammalian target of rapamycin (mTOR) kinase is the catalytic subunit of at least two distinct signaling complexes, referred to as mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Studies with cultured cell lines indicate that the complexes participate in different pathways and recognize distinct substrates, the specificity of which is determined by unique mTOR-interacting proteins. mTORC1 controls cell growth in part by phosphorylating S6 Kinase 1 (S6K1) and the eIF-4E-binding protein 1 (4E-BP1), known regulators of protein synthesis. It has been proposed that mTORC2 phosphorylates and activates Akt/PKB, which regulates cell proliferation, growth, survival, and metabolism. Full activation of Akt/PKB requires phosphorylation of S473 of the hydrophobic motif, the site proposed to depend on mTORC2, as well as phosphorylation of T308 of the activation loop by PDK1. The clinically valuable drug rapamycin specifically inhibits mTORC1 activity, although recent studies indicate that prolonged rapamycin treatment can also inhibit mTORC2 assembly and function in some cell types. Both complexes participate in signaling pathways associated with human diseases, including tuberous sclerosis complex (TSC), lymphangioleiomyomatosis (LAM), Cowden disease, Peutz-Jeghers syndrome (PJS), neurofibromatosis, familial cardiac hypertrophy, and cancers characterized by hyperactivation of PI3K/Akt (Guertin, 2007).

The roles of mTORC1 and mTORC2 during mammalian development are not well understood. In addition to mTOR, mTORC1 contains raptor (regulatory-associated protein of mTOR) and mLST8 (also called GβL). mTORC2 also contains mTOR and mLST8, but instead of raptor, this complex contains rictor (rapamycin-insensitive companion of mTOR). Germline disruption of mTOR in mice causes embryonic lethality at or around implantation. However, when these mice were engineered, it was not known that mTOR is part of two distinct complexes and pathways. With this new information, it becomes difficult to interpret the phenotypes of the mTOR null mice because these animals lack both mTORC1 and mTORC2 function. Thus, to determine the in vivo role of each branch of the mTOR signaling network, mice lacking the expression of raptor, rictor, and mLST8 were generated and characterized. It is conclude that during mammalian development, mTORC1 and mTORC2 have different, but essential, roles; that mLST8 is required only for mTORC2 function; and that mTORC2 is a crucial component of the Akt/PKB-FOXO and PKCα signaling networks (Guertin, 2007).

Akt/PKB regulates actin organization and cell motility via Girdin/APE

The serine/threonine kinase Akt is well known as an important regulator of cell survival and growth and has also been shown to be required for cell migration in different organisms. However, the mechanism by which Akt functions to promote cell migration is not understood. This study identifies an Akt substrate, designated Girdin/APE (Akt-phosphorylation enhancer), which is an actin binding protein. Database searches reveal homologs in mouse, rat, and Drosophila, but no apparent matches in Caenorhabditis elegans and Dictyostelium. Girdin expresses ubiquitously and plays a crucial role in the formation of stress fibers and lamellipodia. Akt phosphorylates serine at position 1416 in Girdin, and phosphorylated Girdin accumulates at the leading edge of migrating cells. Cells expressing mutant Girdin, in which serine 1416 is replaced with alanine, form abnormal elongated shapes and exhibit limited migration and lamellipodia formation. These findings suggest that Girdin is essential for the integrity of the actin cytoskeleton and cell migration and provide a direct link between Akt and cell motility (Enomoto, 2005).

The structure of Girdin predicted by the COILS algorithm showed a tendency to assume an alpha-helical coiled-coil conformation in its middle domain, between Ala-253 and Lys-1375, with a high coiled-coil probability of 1.0. The predicted coiled-coil domain contains 135 continuous heptad repeats ([abcdefg]135) that are typical of alpha-helical coiled-coils. The 9.5 kb Girdin transcript was found to be expressed ubiquitously in various human tissues by high-stringency Northern blot analysis (Enomoto, 2005).

Four different regions can be distinguished in the Girdin molecule based on the sequences of its subunits, subcellular localization, and functions: an N-terminal region that seems to facilitate the formation of a dimer (NT), an extremely long coiled-coil region, a region that binds to the plasma membrane through the interaction with phosphoinositides (CT1), and a C-terminal region that encompasses an actin binding site (CT2). The amino acid sequence of the CT2 domain shows no homology with the calponin homology (CH) domain, a common actin binding domain that is present in most actin binding proteins such as alpha-actinin, filamins, fimbrin, spectrins, cortexillins, and dystrophin, suggesting that Girdin represents a novel class of actin binding proteins (Enomoto, 2005).

Analysis of the sequence of Girdin reveals that it includes 135 heptad repeats, (abcdefg)135, between Leu-253 and Lys-1375 that correspond to a central rod domain. Within the repeats, positions a and d are preferentially occupied by hydrophobic residues like Leu, Ile, Met, or Val; this is consistent with the signature of canonical coiled-coil structures that wind around each other in a superhelix. The oligomerization properties of coiled-coil sequences are determined by the distribution of alpha-branched residues in the a or d positions. Val and Ile in position a favor dimerization, they favor tetramerization in position d, and their presence in both a and d positions facilitate the formation of trimers. In the coiled-coil sequence of Girdin, 22 repeats have Val or Ile in the a position, whereas they are present in the d position of only 9 repeats, suggesting that the coiled-coil domain of Girdin tends to form a dimer. This is consistent with the findings suggested by gel filtration that the NT domain of Girdin forms a dimer (Enomoto, 2005).

The possession of two actin binding sites enables crosslinking or bundling proteins to link filaments and to stabilize higher-order assemblies of actin filaments. Possessing two actin binding CT2 domains in juxtaposition, the dimeric Girdin molecules seem to be designed to gather actin filaments together into bundles or a meshwork. Consistent with this possibility are the findings of immunofluorescent staining and electron microscopy that the depletion of Girdin interfers with actin networks, leading to the disruption of stress fibers, cortical actin filaments, and actin meshwork at the leading edge. During migration, the Girdin knockdown cells produce multiple protrusions, resulting in limited directional migration. These observations indicate that Girdin fulfils an essential function in determining the stability and integrity of actin bundles and meshwork. These mediate a variety of important biological processes. Eukaryotic cells have a fail-safe mechanism in the multiple actin crosslinking proteins that share overlapping functions. The phenotypic consequences of the depletion of Girdin indicate that the presence of other proteins cannot completely compensate for its loss. Because the speculated primary structure, molecular size, and putative function of Girdin are reminiscent of those of filamin, it is important to clarify the functional difference and synergism between the two (Enomoto, 2005).

The CT1 domain of Girdin associates with the plasma membrane through the cluster of basic amino acid residues Arg-1389 to Lys-1407. This positively charged sequence is related to a consensus sequence for PI(4,5)P2 binding, which has been found in gelsolin, villin, profilin, vinculin, and other various cytoskeletal proteins. Unexpectedly, the basic amino acid cluster in Girdin does not bind to PI(4,5)P2, but binds to PI(4)P and binds weakly to PI(3)P. Considering that PI(4)P, but not PI(3)P, is abundant in mammalian cells, it is plausible to conclude that Girdin binds to PI(4)P, which resides in the membranes of mammalian cells in an amount equal to that of PI(4,5)P2. It is speculated that it stabilizes the cortical actin filaments by anchoring them at the plasma membrane (Enomoto, 2005).

Akt phosphorylates Girdin in vitro and in intact cells. The phosphorylation of Girdin is induced by EGF and during cell migration, suggesting a significance for phosphorylation in physiological cellular events. In migrating Vero fibroblasts, the phosphorylated Girdin preferentially localizes to lamellipodia at the leading edge, which is in line with observations that activated Akt is also localized at the leading edge during migration in mammalian cells. It is plausible that Akt, activated downstream of PI3K, translocates from the cytosol to the leading edge through its PH domain, and subsequently phosphorylates Girdin on the actin filaments at the front of the cells (Enomoto, 2005). How does Akt regulate the function of Girdin by phosphorylation? Insight into this issue comes from the observation that the phosphorylation of the CT domain of Girdin affects in vitro binding to PI(4)P. Because the phosphorylation site is present in the neighborhood of the phosphoinositide binding site, it is speculated that phosphorylation induces a conformational change around these sites, and this change in turn alters affinity for the phosphoinositide. It was further found that the phosphorylated CT domain retains the property of actin binding, and its affinity for F-actin is comparable to that of the nonphosphorylated form. Based on these observations, it is speculated that phosphorylation by Akt releases Girdin from PI(4)P and allows it to localize at the leading edge in order to crosslink the newly generated actin filaments in the lamellipodium network (Enomoto, 2005).

Spatial coordination of actin polymerization and ILK-Akt2 activity during endothelial cell migration

Eukaryotic cell migration proceeds by cycles of protrusion, adhesion, and contraction, regulated by actin polymerization, focal adhesion assembly, and matrix degradation. However, mechanisms coordinating these processes remain largely unknown. This study shows that local regulation of thymosin-beta4 (Tbeta4) binding to actin monomer (G-actin) coordinates actin polymerization with metalloproteinase synthesis to promote endothelial cell motility. In particular and quite unexpectedly, FRET analysis reveals diminished interaction between Tbeta4 and G-actin at the cell leading edge despite their colocalization there. Profilin-dependent dissociation of G-actin-Tbeta4 complexes simultaneously liberates actin for filament assembly and facilitates Tbeta4 binding to integrin-linked kinase (ILK) in the lamellipodia. Tbeta4-ILK complexes then recruit and activate Akt2, resulting in matrix metalloproteinase-2 production. Thus, the actin-Tbeta4 complex constitutes a latent coordinating center for cell migratory behavior, allowing profilin to initiate a cascade of events at the leading edge that couples actin polymerization to matrix degradation (Fan, 2009).

ILK phosphorylates PKB/Akt and binds PINCH, paxillin, and parvin (see Drosophila Parvin), all important components regulating cell migration. ILK is critical for EC migration and angiogenesis. The mechanism by which Tβ4 stimulates MMP expression is unclear. Because Tβ4 coimmunoprecipitates with ILK-PINCH, activation of Akt in the ILK-Akt complex could be responsible for MMP expression and increased cell migration. These studies show an interaction of Tβ4 with the kinase domain of ILK, and that formation of the ternary Tβ4–ILK-Akt2 complex increases Akt2 phosphorylation with a consequent increase in MMP-2 expression and cell migration. These Tβ4-mediated processes are regulated by G-actin binding to Tβ4: dissociation of actin–Tβ4 complex promotes Tβ4-ILK interaction and MMP-2 expression. Thus, these results provide a molecular mechanism for Tβ4-promoted protease synthesis and cell migration (Fan, 2009).

The Akt family consists of three isoforms: Akt1, Akt2, and Akt3. The role of Akt2 in cell migration is controversial: Akt2 knockout elevates Rac and Pak1 activities to increase fibroblast cell motility, but knockdown studies indicate that Tβ4-inducible Akt2 activation induces protease synthesis to facilitate EC migration. Consistent with these findings, transient knockdown or overexpression studies show a stimulatory role of Akt2 in cell migration and invasion in multiple cell types. Thus, Akt2 may have dual roles in induction of MMP and inhibition of migratory signaling, including Rac. Akt1 is critical for actin polymerization and cell motility, particularly for EC migration and in vivo angiogenesis. The data show that Tβ4 stimulates stronger ILK-mediated activation of Akt2 than that of Akt1. It is speculated that weaker Akt1 induction results in a tighter localization of active Akt1 to the leading edge, enhancing local actin polymerization (Fan, 2009).

Opposing roles for Akt1 and Akt2 in Rac/Pak signaling and cell migration

The Akt/PKB isoforms have different roles in animals, with Akt2 primarily regulating metabolic signaling and Akt1 regulating growth and survival. This study shows distinct roles for Akt1 and Akt2 in mouse embryo fibroblast cell migration and regulation of the cytoskeleton. Akt1-deficient cells responded poorly to platelet-derived growth factor while Akt2-deficient cells had a dramatically enhanced response, resulting in a substantial increase in dorsal ruffling. Swapping domains between Akt1 and Akt2 demonstrated that the N-terminal region containing the pleckstrin homology domain and a linker region distinguishes the two isoforms, while the catalytic domains are interchangeable. Akt2 knock-out cells also migrated faster than wild-type cells, especially through extracellular matrix (ECM), while Akt1 knock-out cells migrated more slowly than wild-type cells. Consistently, Akt2 knock-out cells had elevated Pak1 and Rac activities, suggesting that Akt2 inhibits Rac and Pak1. Both Akt2 and Akt1 associated in complexes with Pak1, but only Akt2 inhibited Pak1 in kinase assays, suggesting an underlying molecular basis for the different cellular phenotypes. Together these data provide evidence for an unexpected functional link between Akt2 and Pak1 that opposes the actions of Akt1 on cell migration (Zhou, 2006).

PDGF controls contact inhibition of locomotion by regulating N-cadherin during neural crest migration

A fundamental property of neural crest (NC) migration is Contact inhibition of locomotion (CIL), a process by which cells change their direction of migration upon cell contact. CIL has been proven to be essential for NC migration in amphibian and zebrafish by controlling cell polarity in a cell contact dependent manner. Cell contact during CIL requires the participation of the cell adhesion molecule N-cadherin (see Drosophila CadN), which starts to be expressed by NC cells as a consequence of the switch between E- and N-cadherins during epithelial to mesenchymal transition (EMT). However, the mechanism that controls the upregulation of N-cadherin remains unknown. This study shows that PDGFRα (see Drosophila Pvr) and its ligand PDGF-A (see Drosophila Pvf1) are co-expressed in migrating cranial NC. Inhibition of PDGF-A/PDGFRα blocks NC migration by inhibiting N-cadherin and, consequently impairing CIL. Moreover, PI3K/AKT (see Drosophila Akt) was found to be a downstream effector of the PDGFRα cellular response during CIL. These results lead to a proposal that PDGF-A/PDGFRα signalling is a tissue-autonomous regulator of CIL by controlling N-cadherin upregulation during EMT. Finally, it was shown that once NC have undergone EMT, the same PDGF-A/PDGFRα works as NC chemoattractant guiding their directional migration (Bahm, 2017).

Growth factor-dependent trafficking of cerebellar NMDA receptors via protein kinase B/Akt phosphorylation of NR2C

NMDA receptor subunit composition varies throughout the brain, providing molecular diversity in NMDA receptor function. The NR2 subunits (NR2A-D) in large part dictate the distinct functional properties of NMDA receptors and differentially regulate receptor trafficking. Although the NR2C subunit is highly enriched in cerebellar granule cells and plays a unique role in cerebellar function, little is known about NR2C-specific regulation of NMDA receptors. This study demonstrates that PKB/Akt directly phosphorylates NR2C on serine 1096 (S1096). In addition, 14-3-3epsilon was identified as an NR2C interactor, whose binding is dependent on S1096 phosphorylation. Both growth factor stimulation and NMDA receptor activity lead to a robust increase in both phosphorylation of NR2C on S1096 and surface expression of cerebellar NMDA receptors. Finally, NR2C expression, unlike NR2A and NR2B, supports neuronal survival. Thus, these data provide a direct mechanistic link between growth factor stimulation and regulation of cerebellar NMDA receptors (Chen, 2009).

Growth factors play a critical role in regulating neuronal survival during development. Cerebellar granule cells, in particular, are known to undergo a period of extensive apoptosis during development, which is precisely regulated by growth factors. IGF-1 has been demonstrated to promote cerebellar granule cell survival both in vitro and in vivo by blocking apoptosis. IGF-1 increases PI3 kinase activity leading to increased PKB activity and stimulation of downstream kinase/signaling cascades. PKB phosphorylates a variety of substrates, many of which regulate mitochondrial function and apoptosis. Mice lacking various isoforms of PKB have an overall reduction in cell number in many tissues, and specifically in the brain when the PKBγ isoform is absent. The growth factor/PKB signaling pathway has been specifically implicated in regulating cerebellar granule cell survival. Furthermore, there is evidence that PKB can potentiate NMDA receptor responses in cerebellar granule cells, suggesting a link between growth factor signaling and receptor activation in these neurons. However, until now, there was no mechanism described linking growth factor signaling, PKB activity, and NMDA receptor expression (Chen, 2009).

This study found that growth factor signaling and PKB phosphorylation increase surface expression of NR2C-containing NMDA receptors. Importantly, overexpression of NR2C was shown to protect neurons from NMDA-induced toxicity. Consistent with a role for phosphorylation of S1096 in receptor trafficking, NR2C S1096A was less effective at neuronal protection than NR2C WT, supporting a role for the PI3K/PKB pathway in neuronal survival. Overexpression of NR2C S1096A also has a protective effect compared to NR2A or NR2B, which is probably due to the increased level of NR2C surface expression due to exogenous overexpression. For example, the S1096A mutation does not completely abolish the surface expression of NR2C when overexpressed in cerebellar granule cells. These results suggest that surface-expressed NR2C S1096A, even if decreased compared to that of NR2C WT, is sufficient to support neuronal survival. How does NR2C contribute to neuronal survival? The answer probably lies in the fact that NR2C-containing NMDA receptors have unique channel properties and that NR2C may mediate specific signaling pathways to activate downstream survival molecules. In conclusion, these findings that PKB directly phosphorylates cerebellar NMDA receptors and increases their surface expression reveals a novel molecular mechanism by which growth factor signaling can directly affect NMDA receptor activity (Chen, 2009).

Other AKT targets

BAD is a distant member of the Bcl-2 family that promotes cell death. Phosphorylation of BAD prevents this. BAD phosphorylation induced by interleukin-3 (IL-3) is inhibited by specific inhibitors of phosphoinositide 3-kinase (PI 3-kinase). Akt, a survival-promoting serine-threonine protein kinase, is activated by IL-3 in a PI 3-kinase-dependent manner. Active, but not inactive, forms of Akt are found to phosphorylate BAD in vivo and in vitro at the same residues that are phosphorylated in response to IL-3. Thus, the proapoptotic function of BAD is regulated by the PI 3-kinase-Akt pathway (del Paso, 1997).

Growth factors can promote cell survival by activating the phosphatidylinositide-3'-OH kinase and its downstream target, the serine-threonine kinase Akt. However, the mechanism by which Akt functions to promote survival is not understood. Growth factor activation of the PI3'K/Akt signaling pathway culminates in the phosphorylation of the BCL-2 family member BAD, thereby suppressing apoptosis and promoting cell survival. Akt phosphorylates BAD in vitro and in vivo, and blocks the BAD-induced death of primary neurons in a site-specific manner. These findings define a mechanism by which growth factors directly inactivate a critical component of the cell-intrinsic death machinery (Datta, 1997).

In eukaryotes, entry into M-phase of the cell cycle is induced by activation of cyclin B-Cdc2 kinase. At G2-phase, the activity of its inactivator, a member of the Wee1 family of protein kinases (see Drosophila Wee1), exceeds that of its activator, Cdc25C phosphatase. However, at M-phase entry the situation is reversed, such that the activity of Cdc25C exceeds that of the Wee1 family. The mechanism of this reversal is unclear. In oocytes from the starfish Asterina pectinifera, the kinase Akt (or protein kinase B) phosphorylates and downregulates Myt1, a member of the Wee1 family. This switches the balance of regulator activities and causes the initial activation of cyclin B-Cdc2 at the meiotic G2/M-phase transition. These findings identify Myt1 as a new target of Akt, and demonstrate that Akt functions as an M-phase initiator (Okumura, 2002).

Growth factors and hormones activate protein translation by phosphorylation and inactivation of the translational repressors, the eIF4E-binding proteins (4E-BPs), through a wortmannin- and rapamycin-sensitive signaling pathway. The mechanism by which signals emanating from extracellular signals lead to phosphorylation of 4E-BPs is not well understood. The activity of the serine/threonine kinase Akt/PKB is shown to be required in a signaling cascade that leads to phosphorylation and inactivation of 4E-BP1. PI 3-kinase elicits the phosphorylation of 4E-BP1 in a wortmannin- and rapamycin-sensitive manner, whereas activated Akt-mediated phosphorylation of 4E-BP1 is wortmannin resistant but rapamycin sensitive. A dominant negative mutant of Akt blocks insulin-mediated phosphorylation of 4E-BP1, indicating that Akt is required for the in vivo phosphorylation of 4E-BP1. Importantly, an activated Akt induces phosphorylation of 4E-BP1 on the same sites that are phosphorylated upon serum stimulation. Similar to what has been observed with serum and growth factors, phosphorylation of 4E-BP1 by Akt inhibits the interaction between 4E-BP1 and eIF-4E. Furthermore, phosphorylation of 4E-BP1 by Akt requires the activity of FRAP/mTOR. FRAP/mTOR may lie downstream of Akt in this signaling cascade. These results demonstrate that the PI 3-kinase-Akt signaling pathway, in concert with FRAP/mTOR, induces the phosphorylation of 4E-BP1 (Gingras, 1998).

Investigated were the roles of Akt (protein kinase B) and the atypical lambda isoform of protein kinase C (PKClambda), both of which act downstream of phosphoinositide 3-kinase, in the activation of glycogen synthase and phosphorylation of 4E-BP1 (PHAS-1) in response to insulin. A mutant Akt (Akt-AA) in which the phosphorylation sites targeted by growth factors are replaced by alanine was shown to inhibit insulin-induced activation of both Akt and glycogen synthase in L6 myotubes. Expression of a mutant Akt in which Lys179 in the kinase domain was replaced by aspartate also inhibits insulin-induced activation of glycogen synthase but has no effect on insulin activation of endogenous Akt. A kinase-defective mutant of PKClambda (lambdaDeltaNKD), which prevents insulin-induced activation of PKClambda, does not affect the activation of glycogen synthase by insulin. Insulin-induced phosphorylation of 4E-BP1 is inhibited by Akt-AA in Chinese hamster ovary cells. However, lambdaDeltaNKD has no effect on 4E-BP1 phosphorylation induced by insulin. These data suggest that Akt, but not PKClambda, is required for insulin activation of glycogen synthase and for insulin-induced phosphorylation of 4E-BP1 (Takata, 1999).

Recent studies indicate that phosphatidylinositide-3OH kinase (PI3K)-induced S6 kinase (S6K1: see Drosophila RPS6-p70-protein kinase) activation is mediated by protein kinase B (PKB). Support for this hypothesis has largely relied on results obtained with highly active, constitutively membrane-localized alleles of wild-type PKB, whose activity is independent of PI3K. The importance of PKB signaling in S6K1 activation was examined. In parallel, both the inactivation of glycogen synthase kinase 3beta (GSK-3beta) and the phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) were monitored as respective markers of the rapamycin-insensitive and -sensitive branches of the PI3K signaling pathway. The results demonstrate that two activated PKBalpha mutants, whose basal activity is equivalent to that of insulin-induced wild-type PKB, inhibit GSK-3beta to the same extent as a highly active, constitutively membrane-targeted wild-type PKB allele. However, of these two mutants, only the constitutively membrane-targeted allele of PKB induces S6K1 activation. Furthermore, an interfering mutant of PKB, which blocks insulin-induced PKB activation and GSK-3beta inactivation, has no effect on S6K1 activation. Surprisingly, all the activated PKB mutants, regardless of constitutive membrane localization, induce 4E-BP1 phosphorylation and the interfering PKB mutant blocks insulin-induced 4E-BP1 phosphorylation. The results demonstrate that PKB mediates S6K1 activation only as a function of constitutive membrane localization, whereas the activation of PKB appears both necessary and sufficient to induce 4E-BP1 phosphorylation independent of its intracellular location (Dufner, 1999).

The pleckstrin homology (PH) domain of the protooncogenic serine/threonine protein kinase PKB/Akt can bind phosphoinositides. A yeast-based two-hybrid system was employed that identified inosine-5' monophosphate dehydrogenase (IMPDH) type II as specifically interacting with PKB/Akts PH domain. IMPDH catalyzes the rate-limiting step of de novo guanosine-triphosphate (GTP) biosynthesis. Using purified fusion proteins, PKB/Akts PH domain and IMPDH associate in vitro and this association moderately activates IMPDH. Purified PKB/Akt also associates with IMPDH in vitro. PKB/Akt or IMPDH can be pulled-down from mammalian cell lysates using glutathione-S-transferase (GST)-IMPDH or GST-PH domain fusion proteins, respectively. Additionally, PKB/Akt and IMPDH can be co-immunoprecipitated from COS cell lysates and active PKB/Akt can phosphorylate IMPDH in vitro. These results implicate PKB/Akt in the regulation of GTP biosynthesis through its interaction with IMPDH, which is involved in providing the GTP pool used by signal transducing G-proteins (Ingleyab, 2000).

The role of the protein kinase Akt in cell migration is incompletely understood. Sphingosine-1-phosphate (S1P)-induced endothelial cell migration requires the Akt-mediated phosphorylation of the G protein-coupled receptor (GPCR) EDG-1. Activated Akt binds to EDG-1 and phosphorylates the third intracellular loop at the T236 residue. Transactivation of EDG-1 by Akt is not required for Gi-dependent signaling but is indispensable for Rac activation, cortical actin assembly, and chemotaxis. Indeed, T236AEDG-1 mutant sequesters Akt and acts as a dominant-negative GPCR to inhibit S1P-induced Rac activation, chemotaxis, and angiogenesis. Transactivation of GPCRs by Akt may constitute a specificity switch to integrate rapid G protein-dependent signals into long-term cellular phenomena such as cell migration. How GPCRs regulate Rac is poorly understood. EDG-1 activates Rac activity in endothelial cells and transfected CHO cells. Akt activity is required for EDG-1 to activate Rac. Akt and Rac are involved in a complex regulatory network to modulate actin dynamics and cell migration. Since EDG-1 phosphorylation by Akt is needed for Rac activation, it is likely that phosphorylated EDG-1 interacts with upstream mediators of Rac -- for example, the exchange factors such as Tiam. Indeed, S1P treatment of endothelial cells results in translocation of Tiam I and Rac to cell membrane (Lee, 2001).

Basement membranes (BM) are important for epithelial differentiation, cell survival, and normal and metastatic cell migration. Much is known about their breakdown and remodeling, yet their positive regulation is poorly understood. Analysis of a fibroblast growth factor (FGF) receptor mutation has raised the possibility that protein kinase B (Akt/PKB) activated by FGF is connected to the expression of certain laminin and type IV collagen isotypes. This hypothesis was tested; constitutively active Akt/PKB, an important downstream element of phosphoinositide 3'-kinase signaling, was shown to induce the synthesis of laminin-1 and collagen IV isotypes and cause their translocation to the BM. By using promoter-reporter constructs, constitutively active phosphoinositide 3'-kinase-p110 or Akt/PKB was shown to activate, whereas dominant negative Akt/PKB was shown to inhibit, transcription of laminin beta1 and collagen IV alpha1 in differentiating C2 myoblast- and insulin-induced Chinese hamster ovary-T cell cultures. These results suggest that Akt/PKB activated by receptor tyrosine kinases is involved in the positive regulation of BM formation. Thus, Akt/PKB activates laminin and collagen IV at the level of transcription. Akt/PKB controls numerous transcription factors. Whether the forkhead family, the NFkappa B system, or other mechanisms connect Akt/PKB activation with the transcription of laminin and collagen IV chains remains to be determined. It is tempting, nevertheless, to speculate that this regulation represents the positive side of BM remodeling, whereas metalloproteinases represent its negative side. Such positive regulation of BM formation could result in the local amplification of cell signaling mediated by the various signaling molecules associated with the BM (Li, 2001).

Heregulin (HRG)-induced tyrosine phosphorylation of the Gab2 docking protein is enhanced by pretreatment with wortmannin, indicating negative regulation via a PI3-kinase-dependent pathway. This represents phosphorylation by the serine/threonine kinase protein kinase B (PKB), since PKB constitutively associates with Gab2 (Drosophila homolog: Daughter of sevenless), phosphorylates Gab2 on a consensus phosphorylation site (Ser159) in vitro and inhibits Gab2 tyrosine phosphorylation. However, expression of Gab2 mutated at this site (S159A Gab2) not only enhances HRG-induced Gab2 tyrosine phosphorylation and association with Shc and ErbB2, but also markedly increased tyrosine phosphorylation of ErbB2 and other cellular proteins and amplifies activation of the ERK and PKB pathways. The impact of this negative regulation is further emphasized by a potent transforming activity for S159A Gab2, but not wild-type Gab2, in fibroblasts. These studies establish Gab2 as a proto-oncogene, and a model is presented in which receptor recruitment of Gab2 is tightly regulated via an intimate association with PKB. Release of this negative constraint enhances growth factor receptor signaling, possibly since Gab2 binding limits dephosphorylation and disassembly of receptor-associated signaling complexes (Lynch, 2002).

Overactivation of ionotropic glutamate receptors can induce neuronal death, a phenomenon known as excitotoxicity. Cell survival during this response is determined by a balance among signaling cascades, including those that recruit the Akt and JNK pathways. A novel interaction is described between Akt1 and JNK interacting protein 1 (JIP1), a JNK pathway scaffold. Direct association between Akt1 and JIP1 is observed in primary neurons. Neuronal exposure to an excitotoxic stimulus decreases the Akt1-JIP1 interaction and concomitantly increases association between JIP1 and JNK. Akt1 interaction with JIP1 inhibits JIP1-mediated potentiation of JNK activity by decreasing JIP1 binding to specific JNK pathway kinases. Consistent with this view, neurons from Akt1-deficient mice exhibited higher susceptibility to kainate excitotoxicity than wild-type littermates. Overexpression of Akt1 mutants that bind JIP1 reduced excitotoxic apoptosis. These results suggest that Akt1 binding to JIP1 acts as a regulatory gate preventing JNK activation, which is released under conditions of excitotoxic injury (Yano, 2002).

In several systems, the JNK pathway plays a positive role in apoptosis. Glutamate or kainate exposure activates the JNK pathway in primary neurons; this activation is responsible for subsequent apoptotic death. How is an excitotoxicity-specific JNK response generated? The JIP family of JNK scaffolds (also islet-brain [IB] or JNK/stress-activated kinase-associated protein [JSAP]) has been suggested to play a critical role in assembling specific JNK signaling pathway components. Each member of this scaffold family can bind JNK, MKK7, and a mixed-lineage kinase (MLK, a MAPKKK family) on different regions of JIP. Experiments using transiently transfected cell lines have suggested that JIP1 can amplify MLK-induced JNK activation. Recent evidence from mice genetically deficient in JIP1 has demonstrated that this scaffold is a stimulus-specific, positive regulator of JNK activity in vivo. Significantly, JIP1 gene deletion confers higher resistance to kainate-induced neuronal death in mice and neuronal culture, indicating that JIP1 plays an positive role in AMPA/kainate receptor-mediated apoptotic signaling (Yano, 2002).

JNK activity can be antagonized by Akt kinase activity in numerous cell systems, and this crosstalk may underlie many of the prosurvival effects of Akt. The Akt family of Ser/Thr-directed protein kinases (Akt1-3 or protein kinase Balpha-gamma) are important mediators of cell survival in response to growth factors and stimuli that elicit calcium influx. Akt kinases have been suggested to phosphorylate a number of proapoptotic proteins directly, thereby leading to suppression of death signals. This study describes a novel mechanism of Akt-JNK crosstalk in neurons undergoing excitotoxic apoptosis. Evidence that Akt1 binding to JIP1 decreases JIP1's ability to enhance JNK activity by interfering with JIP1-mediated assembly of an active JNK signaling complex. Excitotoxic kainate exposure decreases the neuronal interaction between Akt1 and JIP1 and increases formation of JNK-JIP1 complexes, suggesting that Akt1 interaction with JIP1 acts as a negative switch for JNK activity. Consistent with this model, Akt1 gene deletion renders neurons more susceptible to kainate-induced neuronal death, and ectopic expression of Akt1 binding mutants decreases kainate toxicity (Yano, 2002).

In the search for neuroprotective factors in Huntington's disease, it was found that insulin growth factor 1 via activation of the serine/threonine kinase Akt/PKB is able to inhibit neuronal death specifically induced by mutant huntingtin containing an expanded polyglutamine stretch. The IGF-1/Akt pathway has a dual effect on huntingtin-induced toxicity, since activation of this pathway also results in a decrease in the formation of intranuclear inclusions of mutant huntingtin. huntingtin is a substrate of Akt and phosphorylation of huntingtin by Akt is crucial to mediate the neuroprotective effects of IGF-1. Akt is altered in Huntington's disease patients. Taken together, these results support a potential role of the Akt pathway in Huntington's disease (Humbert, 2002).

Although the complete neuroprotective effect mediated by IGF-1 requires phosphorylation of mutant huntingtin at S421, in the absence of phosphorylation of huntingtin, IGF-1 is still able to induce some neuroprotection. This suggests that in the context of mutant huntingtin-induced cell death, IGF-1 mediates its neuroprotective effect not only via a direct action of Akt on huntingtin protein but also via the phosphorylation of other substrates that increase neuronal survival. Several substrates of Akt such as Bad, FOXOs, and caspase-9 that promote neuronal survival when phosphorylated by Akt have been described. Those substrates, as well as others that remain to be identified, could also participate in mediating the neuroprotective effect elicited by IGF-1 and Akt on mutant huntingtin-induced cell death (Humbert, 2002).

The role of the PI 3-kinase cascade in regulation of cell growth is well established. PKB (protein kinase B) is a key downstream effector of the PI 3-kinase pathway and is best known for its antiapoptotic effects and the role it plays in initiation of S phase. PKB activity is high in the G2/M phase of the cell cycle in epithelial cells. Inhibition of the PI 3-kinase pathway in MDCK cells induces apoptosis at the G2/M transition, prevents activation of cyclin B-associated kinase, and prohibits entry of the surviving cells into mitosis. All of these consequences of the inhibition of PI 3-kinase are relieved by expression of a constitutively active form of PKB (caPKB), indicating that PKB plays a role in regulation of the G2/M phase. Inhibition of PI 3-kinase results in activation of Chk1, whereas constitutively active PKB inhibits the ability of Chk1 to become activated in response to treatment with hydroxyurea. Preliminary data show that PKB phosphorylates the Chk1 polypeptide in vitro on serine 280. These results not only implicate PKB activity in transition through the G2/M stage of the cell cycle, but they also suggest the existence of crosstalk between the PI 3-kinase pathway and the key regulators of the DNA damage checkpoint machinery (Shtivelman, 2002).

An affinity purification method has been used to identify substrates of protein kinase B/Akt. One protein that associates with 14-3-3 in an Akt-dependent manner is shown to be the Yes-associated protein (YAP: Drosophila homolog Yorkie), which is phosphorylated by Akt at serine 127, leading to binding to 14-3-3. Akt promotes YAP localization to the cytoplasm, resulting in loss from the nucleus where it functions as a coactivator of transcription factors including p73. p73-mediated induction of Bax expression following DNA damage requires YAP function and is attenuated by Akt phosphorylation of YAP. YAP overexpression increases, while YAP depletion decreases, p73-mediated apoptosis following DNA damage, in an Akt inhibitable manner. Akt phosphorylation of YAP may thus suppress the induction of the proapoptotic gene expression response following cellular damage (Basu, 2003).

YAP is a 65 kDa protein (sometimes termed YAP65 or YAP1) that was originally identified due to its interaction with the Src family tyrosine kinase Yes. YAP contains either one or two WW domains depending on alternative splicing and also a PDZ interaction motif, an SH3 binding motif, and a coiled-coil domain. YAP has been reported to interact with p53 binding protein-2, an important regulator of the apoptotic activity of p53. Through its carboxyl terminus, YAP binds to the PDZ-containing protein EBP50, a submembranous scaffolding protein. YAP is a transcriptional coactivator that binds and activates Runx transcription factors and the four TEAD/TEF transcription factors. YAP is homologous to TAZ (45% identity), a transcriptional coactivator that is regulated by interaction with 14-3-3 and PDZ domain-containing proteins. YAP also interacts with the p53 family member p73, resulting in an enhancement of p73's transcriptional activity. YAP phosphorylation by Akt suppresses its ability to promote p73-mediated transcription of proapoptotic genes in response to DNA damaging agents and the resulting cell death. This extends the range of mechanisms whereby Akt can promote cellular survival in the face of apoptotic stimuli (Basu, 2003).

The effects of insulin on the mammalian target of rapamycin, mTOR, were investigated in 3T3-L1 adipocytes. mTOR protein kinase activity was measured in immune complex assays with recombinant PHAS-I as substrate. Insulin-stimulated kinase activity is clearly observed when immunoprecipitations are conducted with the mTOR antibody, mTAb2. Insulin also increases by severalfold the 32P content of mTOR, determined after purifying the protein from 32P-labeled adipocytes with rapamycin.FKBP12 agarose beads. Insulin affects neither the amount of mTOR immunoprecipitated nor the amount of mTOR detected by immunoblotting with mTAb2. However, the hormone markedly decreases the reactivity of mTOR with mTAb1, an antibody that activates the mTOR protein kinase. The effects of insulin on increasing mTOR protein kinase activity and on decreasing mTAb1 reactivity are abolished by incubating mTOR with protein phosphatase 1. Interestingly, the epitope for mTAb1 is located near the COOH terminus of mTOR in a 20-amino acid region that includes consensus sites for phosphorylation by protein kinase B (PKB). Experiments were performed in MER-Akt cells to investigate the role of PKB in controlling mTOR. These cells express a PKB-mutant estrogen receptor fusion protein that is activated when the cells are exposed to 4-hydroxytamoxifen. Activating PKB with 4-hydroxytamoxifen mimics insulin by decreasing mTOR reactivity with mTAb1 and by increasing the PHAS-I kinase activity of mTOR. These findings support the conclusion that insulin activates mTOR by promoting phosphorylation of the protein via a signaling pathway that contains PKB (Scott, 1999).

Components of intracellular signaling that mediate the stimulation-dependent recycling of integrins are being identified, but key transport effectors that are the ultimate downstream targets remain unknown. ACAP1, a GAP for ARF6, has been shown to function as a transport effector in the cargo sorting of transferrin receptor (TfR) that undergoes constitutive recycling. This study shows that ACAP1 also participates in the regulated recycling of integrin β1 to control cell migration. However, in contrast to TfR recycling, the role of ACAP1 in β1 recycling requires its phosphorylation by Akt, which is, in turn, regulated by a canonical signaling pathway. Disrupting the activities of either ACAP1 or Akt, or their assembly with endosomal β1, inhibits β1 recycling and cell migration. These findings advance an understanding of how integrin recycling is achieved during cell migration, and also address a basic issue of how intracellular signaling can interface with transport to achieve regulated recycling (Li, 2005).

The ARF family of small GTPases initiates intracellular transport by regulating the recruitment of coat proteins and other cargo-sorting adaptors from the cytosol to membrane. The GAPs for these small GTPases in the better-characterized transport pathways have been shown to function not only as negative upstream regulators of ARFs, but also as their effectors, by being components of coat complexes. An important implication of the cumulative findings on ACAP1 as a cargo-sorting device is that this role will be relevant for a broad range of cellular activities that are known to involve endocytic recycling. Besides cell migration, which itself underlies a wide range of physiologic and pathologic events, other important examples that require endocytic recycling include insulin-stimulated recycling of glucose transporters, cell polarity, cytokinesis, and phagocytosis. Thus, the future investigation of a potential role for ACAP1 in these examples will likely contribute to a better mechanistic understanding of how these events are achieved (Li, 2005),

In fully grown mouse oocytes, a decrease in cAMP concentration precedes and is linked to CDK1 (cyclin-dependent kinase 1) activation. The molecular mechanism for this coupling, however, is not defined. PKB (protein kinase B, also called AKT) is implicated in CDK1 activation in lower species. During resumption of meiosis in starfish oocytes, MYT1, a negative regulator of CDK1, is phosphorylated by PKB in an inhibitory manner. It can imply that PKB is also involved in CDK1 activation in mammalian oocytes. Activation of PKB and CDK1 was monitored during maturation of mouse oocytes. PKB phosphorylation and activation preceded GVBD (germinal vesicle breakdown) in oocytes maturing either in vitro or in vivo. Activation was transient and PKB activity was markedly reduced when virtually all of the oocytes had undergone GVBD. PKB activation was independent of CDK1 activity, because although butyrolactone I prevented CDK1 activation and GVBD, PKB was nevertheless transiently phosphorylated and activated. LY-294002, an inhibitor of phosphoinositide 3-kinase-PKB signalling, suppressed activation of PKB and CDK1 as well as resumption of meiosis. OA (okadaic acid)-sensitive phosphatases are involved in PKB-activity regulation, because OA induced PKB hyperphosphorylation. During resumption of meiosis, PKB phosphorylated on Ser(473) is associated with nuclear membrane and centrosome, whereas PKB phosphorylated on Thr(308) is localized on centrosome only. The results of the present paper indicate that PKB is involved in CDK1 activation and resumption of meiosis in mouse oocytes. The presence of phosphorylated PKB on centrosome at the time of GVBD suggests its important role for an initial CDK1 activation (Kalous, 2005).

Acquisition of epidermal barrier function, that serves to prevent water loss, occurs late in mouse gestation. Several days before birth a wave of barrier acquisition sweeps across murine fetal skin, converging on dorsal and ventral midlines. The molecular pathways active during epidermal barrier formation were investigated. Akt signaling increased as the barrier wave crossed epidermis and Jun was transiently dephosphorylated. Inhibitor experiments on embryonic explants showed that the dephosphorylation of Jun was dependent on both Akt and protein phosphatase 2A (Pp2a). Inhibition of Pp2a and Akt signaling also caused defects in epidermal barrier formation. These data are compatible with a model for developmental barrier acquisition mediated by Pp2a regulation of Jun dephosphorylation, downstream of Akt signaling. Support for this model was provided by siRNA-mediated knockdown of Ppp2r2a (Pr55alpha or B55alpha), a regulatory subunit of Pp2a expressed in an Akt-dependent manner in epidermis during barrier formation. Ppp2r2a reduction caused significant increase in Jun phosphorylation and interfered with the acquisition of barrier function, with barrier acquisition being restored by inhibition of Jun phosphorylation. These data provide strong evidence that Ppp2r2a is a regulatory subunit of Pp2a that targets this phosphatase to Jun, and that Pp2a action is necessary for barrier formation. This study therefore describes a novel Akt-dependent Pp2a activity that acts at least partly through Jun to affect initial barrier formation during late embryonic epidermal development (O'Shaughnessy, 2009).

AKT, cell survival and development

Dictyostelium Akt/PKB is homologous to mammalian Akt/PKB and is required for cell polarity and proper chemotaxis during early development. The kinase activity of Akt/PKB kinase is activated in response to chemoattractants in neutrophils and in Dictyostelium by the chemoattractant cAMP functioning via a pathway involving a heterotrimeric G protein and PI3-kinase. Dictyostelium contains several kinases structurally related to Akt/PKB, one of which, PKBR-1, is investigated here for its role in cell polarity, movement and cellular morphogenesis during development. PKBR-1 has a kinase and a carboxy-terminal domain related to those of Akt/PKB, but no PH domain. Instead, it has an amino-terminal myristoylation site, which is required for its constitutive membrane localization. Like Akt/PKB, PKBR-1 is activated by cAMP through a G-protein-dependent pathway, but does not require PI3-kinase, probably because of the constitutive membrane localization of PKBR-1. This is supported by experiments demonstrating the requirement for membrane association for activation and in vivo function of PKBR-1. PKBR-1 protein is found in all cells throughout early development but is then restricted to the apical cells in developing aggregates, which are thought to control morphogenesis. PKBR-1 null cells arrest development at the mound stage and are defective in morphogenesis and multicellular development. These phenotypes are complemented by Akt/PKB, suggesting functional overlap between PKBR-1 and Akt/PKB. Akt/PKB PKBR-1 double knockout cells exhibit growth defects and show stronger chemotaxis and cell-polarity defects than Akt/PKB null cells. These results expand the previously known functions of Akt/PKB family members in cell movement and morphogenesis during Dictyostelium multicellular development. The results suggest that Akt/PKB and PKBR-1 have overlapping effectors and biological function: Akt/PKB functions predominantly during aggregation to control cell polarity and chemotaxis, whereas PKBR-1 is required for morphogenesis during multicellular development (Meili, 2000).

Studies in Dictyostelium have shown that the p110-related phosphatidylinositol-3-kinases PI3K1 and PI3K2 are required for proper development, pinocytosis chemotaxis, and chemoattractant-mediated activation of PKB. Insights into the mechanism by which PI3K regulates chemotaxis derive from studies on PKB in mammalian leukocytes and Dictyostelium cells. PKB activation requires its translocation to the plasma membrane by binding of its PH domain to PtdIns(3,4,5)P3 and PtdIns(3,4)P2 produced upon activation of PI3K, leading to PKB activation. In leukocytes and Dictyostelium cells, chemoattractants mediate PKB activation through a G-protein-coupled pathway that requires the activity of the respective PI3Ks. Chemoattractant stimulation of neutrophils and Dictyostelium cells results in a transient localization of a GFP fusion of the PH domains from the Dictyostelium and mammalian PKBs to the plasma membrane. When these cells are placed in a chemoattractant gradient, membrane localization of the PKB-PH-GFP fusion is restricted to the leading edge, as is the case for other PH-domain-containing proteins in Dictyostelium. In Dictyostelium, translocation of the PKB-PH domain GFP fusion is PI3K-dependent. PI3 kinase and protein kinase B (PKB or Akt) control cell polarity and chemotaxis, in part, through the regulation of PAKa, a structural homolog of mammalian PAKs (p21-activated kinase) that is required for myosin II assembly. PI3K and PKB mediate PAKa's subcellular localization, PAKa's activation in response to chemoattractant stimulation, and chemoattractant-mediated myosin II assembly. Mutation of the PKB phosphorylation site in PAKa to Ala blocks PAKa's activation and inhibits PAKa redistribution in response to chemoattractant stimulation, whereas an Asp substitution leads to an activated protein. Addition of the PI3K inhibitor LY294002 results in a rapid loss of cell polarity and the axial distribution of actin, myosin, and PAKa. These results provide a mechanism by which PI3K regulates chemotaxis (Chung, 2001).

The serine/threonine kinase Akt has been implicated in the control of cell survival and metabolism. Disruption is reported of the most ubiquitously expressed member of the akt family of genes, akt1, in the mouse. Akt-/- mice are viable but smaller when compared to wild-type littermates. In addition, the life span of Akt-/- mice, upon exposure to genotoxic stress, is shorter. However, Akt-/- mice do not display a diabetic phenotype. Increased spontaneous apoptosis in testes, and attenuation of spermatogenesis is observed in Akt-/- male mice. Increased spontaneous apoptosis is also observed in the thymi of Akt-/- mice, and Akt-/- thymocytes are more sensitive to apoptosis induced by gamma-irradiation and dexamethasone. Finally, Akt-/- mouse embryo fibroblasts (MEFs) are more susceptible to apoptosis induced by TNF, anti-Fas, UV irradiation, and serum withdrawal (Chen, 2001).

The relatively subtle phenotype of Akt1-/- mice suggests that Akt2 and Akt3 may substitute to some extent for Akt1, as was shown for the Akt1 and Akt2 in Caenorhabditis elegans. Since Akt2 and Akt3 are expressed in both testes and thymus, it is not clear why only these particular organs are affected by the ablation of Akt1. One possibility is that germ cells and thymus cells are exclusively dependent on Akt for their survival and therefore even a reduced threshold level of Akt activity is sufficient to affect their survival. Alternatively, despite a similar level of expression of the other Akt isoforms in these organs, Akt1 is more profoundly activated in the cells of these organs and/or may have exclusive protein substrates in these cells. Further studies including deletions of akt2 and akt3 genes are required to verify these possibilities (Chen, 2001).

In Drosophila, the PTEN/PI 3-kinase/Akt signaling pathway is associated with cell survival, organism size and metabolism. Disruption of the Akt gene in Drosophila impairs normal cell survival during embryogenesis, and results in a decreased cell size. The disruption of the akt-1 gene in the mouse, which by itself does not impair embryogenesis, has been shown here to affect cell survival and organism size, as well as cause growth retardation in adult mice. It remains to be seen if the combined disruption of the three akt genes in the mouse will result in embryonic lethality as a result of impaired cell survival during embryogenesis (Chen, 2001).

Surprisingly, despite multiple downstream effectors of Akt and the ubiquitous expression of Akt1, ablation of Akt1 by itself does not have a gross phenotypic impact. This observation implies that reduced threshold level of Akt activity can be tolerated and therefore suggests that small molecules aimed at reducing Akt activity could be excellent therapeutic regimens for the treatment of cancers in which the PI 3-kinase/Akt pathway is constitutively activated (Chen, 2001).

A neurosecretory pathway regulates a reversible developmental arrest and metabolic shift at the Caenorhabditis elegans dauer larval stage. Defects in an insulin-like signaling pathway cause arrest at the dauer stage. Two C. elegans Akt/PKB homologs, akt-1 and akt-2, transduce insulin receptor-like signals that inhibit dauer arrest and AKT-1 and AKT-2 signaling are indispensable for insulin receptor-like signaling in C. elegans. A loss-of-function mutation in the Fork head transcription factor DAF-16 relieves the requirement for Akt/PKB signaling, which indicates that AKT-1 and AKT-2 function primarily to antagonize DAF-16. This is the first evidence that the major target of Akt/PKB signaling is a transcription factor. An activating mutation in akt-1, revealed by a genetic screen, as well as increased dosage of wild-type akt-1 relieves the requirement for signaling from AGE-1 PI3K, which acts downstream of the DAF-2 insulin/IGF-1 receptor homolog. This demonstrates that Akt/PKB activity is not necessarily dependent on AGE-1 PI3K activity. akt-1 and akt-2 are expressed in overlapping patterns in the nervous system and in tissues that are remodeled during dauer formation (Paradis, 1998).

Extracellular signals often result in simultaneous activation of both the Raf-MEK-ERK and PI3K-Akt pathways (where ERK is extracellular-regulated kinase, MEK is mitogen-activated protein kinase or ERK kinase, and PI3K is phosphatidylinositol 3-kinase). However, these two signaling pathways exert opposing effects on muscle cell hypertrophy. Manipulation of these pathways during muscle differentiation indicates that inhibition of the Ras-Raf-MEK-ERK pathway promotes differentiation, whereas inhibition of PI3K blocks differentiation. However, the roles of these two pathways in the process of skeletal muscle hypertrophy has not previously been evaluated. C2C12 myoblasts normally proliferate and are mononucleated. When deprived of serum at confluence, they fuse and differentiate into postmitotic, elongated, and multinucleated myotubes. The hypertrophic action of insulin-like growth factor-1 (IGF-1) on muscle cells in vivo is mimicked by the addition of IGF-1 during the differentiation of C2C12 myotubes in vitro, resulting in the generation of thicker myotubes. In addition to inducing hypertrophy of myotubes in vivo, IGF-1 has been shown to activate both the Raf-MEK-ERK pathway and the PI3K-Akt pathway. The roles of these two pathways in the differentiation and hypertrophy of C2C12 myotubes were examined by genetic manipulation. Expression of a constitutively active form of Raf (c.a.-Raf) results in the generation of smaller and thinner myotubes, whereas expression of a dominant negative form of Raf (d.n.-Raf) results in markedly thicker myotubes. Thus, inhibition of the Raf-MEK-ERK pathway induces a hypertrophic phenotype similar to that elicited by IGF-1 treatment. In contrast, activation of the Akt pathway by expression of a constitutively active form of Akt (c.a.-Akt) results in a hypertrophic phenotype more pronounced than that observed with d.n.-Raf and characterized by multinucleated myotubes that are both thickened and shortened. Thus, genetic manipulation of the Raf-MEK-ERK and PI3K-Akt pathways reveals opposing phenotypic effects of these pathways during muscle differentiation, with the Raf-MEK-ERK pathway inhibiting development of the hypertrophic phenotype and the PI3K-Akt pathway promoting it. The PI3K-Akt pathway inhibits the Raf-MEK-ERK pathway; this cross-regulation depends on the differentiation state of the cell: Akt activation inhibits the Raf-MEK-ERK pathway in differentiated myotubes, but not in their myoblast precursors. The stage-specific inhibitory action of Akt correlates with its stage-specific ability to form a complex with Raf, suggesting the existence of differentially expressed mediators of an inhibitory Akt-Raf complex (Rommel, 1999).

The Akt/protein kinase B serine/threonine kinase is a downstream effector of phosphoinositide 3-kinase (PI3K). Akt is an important component of mitogenic and antiapoptotic signaling pathways and is implicated in neoplastic transformation. Thyroid cells in culture retain a differentiated phenotype consisting of epithelial cell morphology and the expression of several tissue-specific genes. The survival and proliferation of these cells depend on thyrotropin and a mixture of five additional hormones that includes insulin. The regulation of proliferation and the expression of the thyroid differentiation program are intimately connected processes. As a result, oncogenes that induce hormone-independent proliferation invariably impair the expression of the thyroid-specific differentiation markers. Given that thyrotropin and insulin stimulate Akt activation in thyroid cells, the effects of Akt on thyroid cell proliferation, survival, and differentiation were determined. To this end, constitutively active myristylated Akt (myrAkt) was expressed in PC Cl 3 thyroid cells. The myrAkt-expressing cells continue to proliferate, even in the absence of hormones, and they are resistant to programmed cell death induced by starvation. These effects are paralleled by the induction of the G1 cyclins D3 and E and by the inhibition of induction of the proapoptotic Fas, Fas ligand, and BAD genes in starved cells. However, in marked contrast with several other oncogenes, myrAkt does not interfere with the expression of thyroid differentiation functions. These results unveil the existence of an Akt-triggered thyroid cell pathway that modulates proliferation and survival without affecting the expression of the thyroid cell differentiated phenotype (De Vita, 2000).

The serine/threonine kinase protein kinase B (PKB)/Akt mediates cell survival in a variety of systems. Transgenic mice expressing a constitutively active form of PKB (gag-PKB) have been generated to examine the effects of PKB activity on T lymphocyte survival. Thymocytes and mature T cells overexpressing gag-PKB display increased active PKB, enhanced viability in culture, and resistance to a variety of apoptotic stimuli. PKB activity prolongs the survival of CD4(+)CD8(+) double positive (DP) thymocytes in fetal thymic organ culture, but is unable to prevent antigen-induced clonal deletion of thymocytes expressing the major histocompatibility complex class I-restricted P14 T cell receptor (TCR). In mature T lymphocytes, PKB can be activated in response to TCR stimulation, and peptide-antigen-specific proliferation is enhanced in T cells expressing the gag-PKB transgene. Both thymocytes and T cells overexpressing gag-PKB display elevated levels of the antiapoptotic molecule Bcl-X(L). In addition, the activation of peripheral T cells leads to enhanced NF-kappaB activation via accelerated degradation of the NF-kappaB inhibitory protein IkappaBalpha. These data highlight a physiological role for PKB in promoting survival of DP thymocytes and mature T cells, and provide evidence for the direct association of three major survival molecules [PKB, Bcl-X(L), and NF-kappaB] in vivo in T lymphocytes (Jones, 2000).

Phosphoinositide 3-kinase (PI3K) has been shown to regulate cell and organ size in Drosophila, but the role of PI3K in vertebrates in vivo is not well understood. To examine the role of PI3K in intact mammalian tissue, transgenic mice expressing constitutively active or dominant-negative mutants of PI3K in the heart have been created and characterized. Cardiac-specific expression of constitutively active PI3K results in mice with larger hearts, while dominant-negative PI3K results in mice with smaller hearts. The increase or decrease in heart size is associated with comparable increase or decrease in myocyte size. Cardiomyopathic changes, such as myocyte necrosis, apoptosis, interstitial fibrosis or contractile dysfunction, are not observed in either of the transgenic mice. Thus, the PI3K pathway is necessary and sufficient to promote organ growth in mammals (Shioi, 2000).

What are the downstream targets of PI3K that are involved in the regulation of organ size? The available information does not provide a definitive answer to this question. However, Akt, a well characterized downstream target of PI3K, is likely to be one of the major mediators of this process. Akt is necessary and sufficient for phosphorylation and subsequent inactivation of 4E-BP1, a repressor of mRNA translation. Akt can also activate p70S6K in some contexts, although activation of p70S6K might not be solely dependent on Akt. p70S6K is upregulated in caPI3K hearts and downregulated in dnPI3K hearts. The amount of phosphorylated S6 protein correlates with the activation of p70S6K. The inhibition of the p70S6K pathway by rapamycin at nanomolar concentrations selectively suppresses an increase in protein synthesis of cultured neonatal myocytes in response to growth factors. Interestingly, rapamycin does not inhibit other phenotypic changes associated with myocyte hypertrophy, such as re-activation of fetal genes and sarcomere organization. This raises the possibility that p70S6K may selectively regulate cell size via controlling the rate of protein synthesis. Gene disruption of p70S6K is known to result in smaller body size in mice. In Drosophila, deficiency of the S6K gene is associated with a reduction in body size associated with smaller cells (Shioi, 2000 and references therein).

Nerve growth factor (NGF) induces dramatic axon growth from responsive embryonic peripheral neurons. However, the roles of the various NGF-triggered signaling cascades in determining specific axon morphological features remain unknown. Activated and inhibitory mutants of Trk effectors were transfected into sensory neurons lacking the proapoptotic protein Bax. This allowed axon growth to be studied in the absence of NGF, enabling the contributions of individual signaling mediators to be observed. While Ras is both necessary and sufficient for NGF-stimulated axon growth, the Ras effectors Raf and Akt induce distinct morphologies. Activated Raf-1 causes axon lengthening comparable to NGF, while active Akt increases axon caliber and branching. These results suggest that the different Trk effector pathways mediate distinct morphological aspects of developing neurons (Markus, 2002).

The signaling pathway of insulin/insulin-like growth factor-1/phosphatidylinositol-3 kinase/Akt is known to regulate longevity as well as resistance to oxidative stress in the nematode Caenorhabditis elegans. This regulatory process involves the activity of DAF-16, a forkhead transcription factor. Although reduction-of-function mutations in components of this pathway have been shown to extend the lifespan in organisms ranging from yeast to mice, activation of Akt has been reported to promote proliferation and survival of mammalian cells. Akt activity has been shown to increase along with cellular senescence; inhibition of Akt extends the lifespan of primary cultured human endothelial cells. Constitutive activation of Akt promotes senescence-like arrest of cell growth via a p53/p21-dependent pathway, and inhibition of forkhead transcription factor FOXO3a by Akt is essential for this growth arrest to occur. FOXO3a influences p53 activity by regulating the level of reactive oxygen species. These findings reveal a novel role of Akt in regulating the cellular lifespan and suggest that the mechanism of longevity is conserved in primary cultured human cells and that Akt-induced senescence may be involved in vascular pathophysiology (Miyauchi, 2004).

Spermatogonial stem cells have unique properties to self-renew and support spermatogenesis throughout their lifespan. Although glial cell line-derived neurotrophic factor (GDNF) has recently been identified as a self-renewal factor for spermatogonial stem cells, the molecular mechanism of spermatogonial stem cell self-renewal remains unclear. The present study assessed the role of the phosphoinositide-3 kinase (PI3K)-Akt pathway using a germline stem (GS) cell culture system that allows in vitro expansion of spermatogonial stem cells. Akt was rapidly phosphorylated when GDNF was added to the GS cell culture, and the addition of a chemical inhibitor of PI3K prevented GS cell self-renewal. Furthermore, conditional activation of the myristoylated form of Akt-Mer (myr-Akt-Mer) by 4-hydroxy-tamoxifen induced logarithmic proliferation of GS cells in the absence of GDNF for at least 5 months. The myr-Akt-Mer GS cells expressed spermatogonial markers and retained androgenetic imprinting patterns. In addition, they supported spermatogenesis and generated offspring following spermatogonial transplantation into the testes of infertile recipient mice, indicating that they are functionally normal. These results demonstrate that activation of the PI3K-Akt pathway plays a central role in the self-renewal division of spermatogonial stem cells (Lee, 2007).

Akt and apoptosis: Akt targets caspase and BCL2 family members

Caspases are intracellular proteases that function as initiators and effectors of apoptosis. The kinase Akt and p21-Ras, an Akt activator, induce phosphorylation of pro-caspase-9 (pro-Casp9) in cells. Cytochrome c-induced proteolytic processing of pro-Casp9 is defective in cytosolic extracts from cells expressing either active Ras or Akt. Akt phosphorylates recombinant Casp9 in vitro on serine-196 and inhibits its protease activity. Mutant pro-Casp9(Ser196Ala) is resistant to Akt-mediated phosphorylation and inhibition in vitro and in cells, resulting in Akt-resistant induction of apoptosis. Thus, caspases can be directly regulated by protein phosphorylation (Cardone, 1998).

Caspase-9 is one caspase upstream of caspase-3 and its activation is stimulated by Apaf-1/cytochrome c and inhibited by Akt signals. BAD phosphorylation by Akt is an essential step for growth factor-mediated inhibition of caspase activation. Recently, it has been shown that human caspase-9 is phosphorylated by Akt and that its protease activity is reduced. To clarify the molecular mechanism of regulation of caspase-9 activation in neuronal apoptosis, two alternative splicing products of mouse caspase-9, caspase-9L and caspase-9S, were isolated from a P19 embryonal carcinoma cell cDNA library. Curiously, the Akt phosphorylation sites and motifs found in human caspase-9 are absent in both mouse caspase-9L and -9S. Mouse caspase-9 is not phosphorylated by activated Akt in vitro. Reverse transcription polymerase chain reaction analysis shows that the absent Akt motif is not limited to caspase-9 expressed in P19 embryonal carcinoma cells but also occurs in caspase-9 expressed in mouse, rat, and monkey. These results suggest that inhibition of caspase-9 activation by Akt-dependent phosphorylation is not generalized across species (Fujita, 1999).

Phosphoinositide 3 kinase/Akt pathway plays an essential role in neuronal survival. However, the cellular mechanisms by which Akt suppresses cell death and protects neurons from apoptosis remain unclear. Transient expression of constitutively active Akt inhibits ceramide-induced death of hybrid motor neuron 1 cells. Stable expression of either constitutively active Akt or Bcl-2 inhibits apoptosis, but only Bcl-2 prevents the release of cytochrome c from mitochondria, suggesting that Akt regulates apoptosis at a postmitochondrial level. Consistent with this, overexpressing active Akt rescues cells from apoptosis without altering expression levels of endogenous Bcl-2, Bcl-x, or Bax. Akt inhibits apoptosis induced by microinjection of cytochrome c and lysates from cells expressing active Akt inhibit cytochrome c-induced caspase activation in a cell-free assay, while lysates from Bcl-2-expressing cells have no effect. Addition of cytochrome c and dATP to lysates from cells expressing active Akt do not activate caspase-9 or -3 and immunoprecipitated Akt added to control lysates blocks cytochrome c-induced activation of the caspase cascade. Taken together, these data suggest that Akt inhibits activation of caspase-9 and -3 by posttranslational modification of a cytosolic factor downstream of cytochrome c and before activation of caspase-9 (Zhou, 2000).

A growing number of downstream targets of Akt have been identified, including glycogen synthase kinase-3, BAD, human caspase-9, and transcription factors such as CREB, Forkhead, and NFkappaB. While each of these has been implicated as an important target for Akt to inhibit apoptosis, in the present case, caspase-9 appears to be the most likely candidate since Akt phosphorylates human caspase-9 and inhibits its activity, which would provide a potential mechanism for Akt to inhibit caspase activation at a postmitochondrial level. However, [32P]orthophosphate labeling of cells expressing active Akt indicates that mouse caspase-9 is not phosphorylated by Akt. This may be explained by recent reports that the consensus Akt phosphorylation sites on human caspase-9 are not conserved in caspase-9 from other species such as mouse caspase-9 in HMN1 cells. However, the data also suggest that there exists an additional, more general mechanism by which Akt can suppress activation of caspases by cytochrome c at a postmitochondrial stage. When added into vector-cell extracts, immunoisolated active Akt is sufficient to inhibit caspase activation induced by cytochrome c, suggesting that active Akt has a direct effect on inhibiting cytochrome c-induced caspase activation. One such direct target for Akt may be Apaf-1 (Drosophila homolog: Apaf-1-related-killer), which forms a holoenzyme with caspase-9 and regulates caspase-9 activity. Akt phosphorylates Apaf-1 in vitro; however, further studies are needed to determine whether cellular Apaf-1 is a direct target of Akt, and how the phosphorylation status of Apaf-1 may regulate caspase-9 activation (Zhou, 2000).

The serine/threonine kinase Akt/PKB is a major downstream effector of growth factor-mediated cell survival. Activated Akt, like Bcl-2 and Bcl-xL, prevents closure of a mitochondrial permeability transition pore (PT pore) component, the voltage-dependent anion channel (VDAC); intracellular acidification; mitochondrial hyperpolarization; and the decline in oxidative phosphorylation that precedes cytochrome c release. However, unlike Bcl-2 and Bcl-xL, the ability of activated Akt to preserve mitochondrial integrity, and thereby inhibit apoptosis, requires glucose availability and is coupled to glucose metabolism. Hexokinases are known to bind to VDAC and directly couple intramitochondrial ATP synthesis to glucose metabolism. Evidence is provided that such coupling serves as a downstream effector function for Akt: (1) Akt increases mitochondria-associated hexokinase activity; (2) the antiapoptotic activity of Akt requires only the first committed step of glucose metabolism catalyzed by hexokinase and (3) ectopic hexokinase expression mimics the ability of Akt to inhibit cytochrome c release and apoptosis. It is therefore proposed that Akt increases coupling of glucose metabolism to oxidative phosphorylation and regulates PT pore opening via the promotion of hexokinase-VDAC interaction at the outer mitochondrial membrane (Gottlob, 2001).

USP14 regulates autophagy by suppressing K63 ubiquitination of Beclin 1

The ubiquitin-proteasome system (UPS) and autophagy are two major intracellular degradative mechanisms that mediate the turnover of complementary repertoires of intracellular proteomes. Simultaneously activating both UPS and autophagy might provide a powerful strategy for the clearance of misfolded proteins. However, it is not clear whether UPS and autophagy can be controlled by a common regulatory mechanism. K48 deubiquitination by USP14 (see Drosophila Usp14) is known to inhibit UPS. This study shows that USP14 regulates autophagy by negatively controlling K63 ubiquitination of Beclin 1 (see Drosophila Atg6). Furthermore, activation of USP14 by Akt-mediated phosphorylation provides a mechanism for Akt to negatively regulate autophagy by promoting K63 deubiquitination. Data suggest that Akt-regulated USP14 activity modulates both proteasomal degradation and autophagy through controlling K48 and K63 ubiquitination, respectively. Therefore, regulation of USP14 provides a mechanism for Akt to control both proteasomal and autophagic degradation. The study proposes that inhibition of USP14 may provide a strategy to promote both UPS and autophagy for developing novel therapeutics targeting neurodegenerative diseases (Xu, 2016). 

Akt mediates self-renewal division of mouse spermatogonial stem cells

Spermatogonial stem cells have unique properties to self-renew and support spermatogenesis throughout their lifespan. Although glial cell line-derived neurotrophic factor (GDNF) has recently been identified as a self-renewal factor for spermatogonial stem cells, the molecular mechanism of spermatogonial stem cell self-renewal remains unclear. In the present study, the role of the phosphoinositide-3 kinase (PI3K)-Akt pathway was assessed using a germline stem (GS) cell culture system that allows in vitro expansion of spermatogonial stem cells. Akt was rapidly phosphorylated when GDNF was added to the GS cell culture, and the addition of a chemical inhibitor of PI3K prevented GS cell self-renewal. Furthermore, conditional activation of the myristoylated form of Akt-Mer (myr-Akt-Mer) by 4-hydroxy-tamoxifen induced logarithmic proliferation of GS cells in the absence of GDNF for at least 5 months. The myr-Akt-Mer GS cells expressed spermatogonial markers and retained androgenetic imprinting patterns. In addition, they supported spermatogenesis and generated offspring following spermatogonial transplantation into the testes of infertile recipient mice, indicating that they are functionally normal. These results demonstrate that activation of the PI3K-Akt pathway plays a central role in the self-renewal division of spermatogonial stem cells (Lee, 2007).

The majority of early primordial germ cells acquire pluripotency by AKT activation

Primordial germ cells (PGCs) are undifferentiated germ cells in embryos, the fate of which is to become gametes; however, mouse PGCs can easily be reprogrammed into pluripotent embryonic germ cells (EGCs) in culture in the presence of particular extracellular factors, such as combinations of Steel factor (KITL), LIF and bFGF (FGF2). Early PGCs form EGCs more readily than do later PGCs, and PGCs lose the ability to form EGCs by embryonic day (E) 15.5. This study examined the effects of activation of the serine/threonine kinase AKT in PGCs during EGC formation; notably, AKT activation, in combination with LIF and bFGF, enhanced EGC formation and caused approximately 60% of E10.5 PGCs to become EGCs. The results indicate that the majority of PGCs at E10.5 could acquire pluripotency with an activated AKT signaling pathway. Importantly, AKT activation did not fully substitute for bFGF and LIF, and AKT activation without both LIF and bFGF did not result in EGC formation. These findings indicate that AKT signal enhances and/or collaborates with signaling pathways of bFGF and of LIF in PGCs for the acquisition of pluripotency (Matsui, 2014).

Neurofibromatosis-1, acting through Akt, regulates neuroglial progenitor proliferation and glial differentiation in a brain region-specific manner

Recent studies have shown that neuroglial progenitor/stem cells (NSCs) from different brain regions exhibit varying capacities for self-renewal and differentiation. This study used neurofibromatosis-1 (NF1) as a model system to elucidate a novel molecular mechanism underlying brain region-specific NSC functional heterogeneity. Nf1 loss leads to increased NSC proliferation and gliogenesis in the brainstem, but not in the cortex. Using Nf1 genetically engineered mice and derivative NSC neurosphere cultures, it was shown that this brain region-specific increase in NSC proliferation and gliogenesis results from selective Akt hyperactivation. The molecular basis for the increased brainstem-specific Akt activation in brainstem NSCs is the consequence of differential rictor expression, leading to region-specific mammalian target of rapamycin (mTOR)/rictor-mediated Akt phosphorylation and Akt-regulated p27 phosphorylation. Collectively, these findings establish mTOR/rictor-mediated Akt activation as a key driver of NSC proliferation and gliogenesis, and identify a unique mechanism for conferring brain region-specific responses to cancer-causing genetic changes (Lee, 2010).

Akt1 controls the timing and amplitude of vascular circadian gene expression

The AKT signaling pathway is important for circadian rhythms in mammals and flies (Drosophila). However, AKT signaling in mammals is more complicated since there are 3 isoforms of AKT, each performing slightly different functions. This study dealt with the most ubiquitous AKT isoform, Akt1, and its role at the organismal level in the central and vascular peripheral clocks. Akt1-/- mice exhibit relatively normal behavioral rhythms with only minor differences in circadian gene expression in the liver and heart. However, circadian gene expression in the Akt1-/- aorta, compared with control aorta, follows a distinct pattern. In the Akt1-/- aorta, positive regulators of circadian transcription have lower amplitude rhythms and peak earlier in the day, and negative circadian regulators are expressed at higher amplitudes and peak later in the day. In endothelial cells, negative circadian regulators exhibit an increased amplitude of expression, while the positive circadian regulators are arrhythmic with a decreased amplitude of expression. This indicates that Akt1 conditions the normal circadian rhythm in the vasculature more so than in other peripheral tissues where other AKT isoforms or kinases might be important for daily rhythms (Luciano, 2017).

AKT and malignant transformation

Three members have been identified in the protein kinase B (PKB) family: Akt/PKB alpha, AKT2/PKB beta, and AKT3/PKB gamma. Previous studies have demonstrated that only AKT2 is predominantly involved in human malignancies and has oncogenic activity. However, the mechanism of transforming activity of AKT2 is still not well understood. The activation of AKT2 has been demonstrated in human ovarian epithelial cancer cells with several growth factors, including epidermal growth factor, insulin-like growth factor I, insulin-like growth factor II, basic fibroblast growth factor, platelet-derived growth factor, and insulin. The kinase activity and the phosphorylation of AKT2 are induced by the growth factors and blocked by the phosphatidylinositol (PI) 3-kinase inhibitor, wortmannin, and dominant-negative Ras (N17Ras). Moreover, the activated Ras and v-Src, two proteins that transduce growth factor-generated signals, also activate AKT2, and this activation is not significantly enhanced by growth factor stimulation but is abrogated by wortmannin. These results indicate that AKT2 is a downstream target of PI 3-kinase and that Ras and Src function upstream of PI 3-kinase and mediate the activation of AKT2 by growth factors. The findings also provide further evidence that AKT2, in cooperation with Ras and Src, is important in the development of some human malignancies (Liu, 1998).

The Akt/PKB protein kinase is implicated in the control of cell cycle progression and the suppression of apoptosis in cancer cells. A conditionally active form of Akt/PKB (M+ Akt:ER*) was used to study the ability of this protein to influence biological processes that are central to the process of oncogenic transformation of mammalian cells. Activation of M+ Akt:ER* in Rat1 cells elicits alterations in cell morphology and promotes anchorage-independent growth in agarose with high efficiency. Consistent with these observations, activation of M+ Akt:ER* suppresses the apoptosis of Rat1 cells that occurs after the detachment of these cells from extracellular matrix. Furthermore, activation of M+ Akt:ER* is sufficient to promote the progression of quiescent Rat1 cells into the S and G2-M phases of the cell cycle. In accord with this is the observation that activation of M+ Akt:ER* leads to decreased expression of the cyclin-dependent kinase inhibitor p27Kip1 with a concomitant increase in cyclin-dependent kinase-2 activity. Perhaps surprisingly, activation of M+ Akt:ER* or expression of a constitutively active form of Akt leads to rapid activation of MAP/ERK kinase (MEK) and the extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinases in Rat1 cells. However, pharmacological inhibition of MEK by PD098059 does not inhibit the morphological alterations of Rat1 cells that occur after M+ Akt:ER* activation. These data suggest that M+ Akt:ER* can activate a number of pathways in Rat1 cells, leading to significant alterations in a number of biological processes. The conditional transformation system described here will allow further elucidation of the ability of Akt to contribute to both the normal response of cells to mitogenic stimulation and the aberrant proliferation observed in cancer cells (Mirza, 2000).

v-Crk induces cellular tyrosine phosphorylation and transformation of chicken embryo fibroblasts (CEF). The molecular mechanism of the v-Crk-induced transformation was studied. Experiments with Src homology (SH)2 and SH3 domain mutants revealed that the induction of tyrosine phosphorylation of cellular proteins requires only the SH2 domain, but both the SH2 and SH3 domains are required for complete transformation. Analysis of three well defined signaling pathways, the mitogen-activated protein kinase (MAPK) pathway, the Jun N-terminal kinase (JNK) pathway, and the phosphoinositide 3-kinase (PI3K)/AKT pathway, demonstrate that only the PI3K/AKT pathway is constitutively activated in v-Crk-transformed CEF. Both the SH2 and SH3 domains are required for this activation of the PI3K/AKT pathway in CEF. The colony formation of CEF is strongly induced by a constitutively active PI3K mutant, and a PI3K inhibitor, LY294002, suppresses the v-Crk-induced transformation. These results strongly suggest that constitutive activation of the PI3K/AKT pathway plays an essential role in v-Crk-induced transformation of CEF (Akagi, 2000).

Gliomas are the most common primary malignant brain tumors and are classified into four clinical grades, with the most aggressive tumors being grade 4 astrocytomas (also known as glioblastoma multiforme; GBM). Frequent genetic alterations in GBMs result in stimulation of common signal transduction pathways involving Ras, Akt and other proteins. It is not known which of these pathways, if any, are sufficient to induce GBM formation. In a tissue-specific manner, genes encoding activated forms of Ras and Akt have been transferred to astrocytes and neural progenitors in mice. Although neither activated Ras nor Akt alone is sufficient to induce GBM formation, the combination of activated Ras and Akt induces high-grade gliomas with the histological features of human GBMs. These tumors appear to arise after gene transfer to neural progenitors, but not after transfer to differentiated astrocytes. Increased activity of RAS is found in many human GBMs, and Akt activity is increased in most of these tumors, implying that combined activation of these two pathways accurately models the biology of this disease (Holland, 2000).

PTEN is a tumor suppressor gene located on chromosome 10q23 that encodes a protein and phospholipid phosphatase. Somatic mutations of PTEN are found in a number of human malignancies, and loss of expression, or mutational inactivation of PTEN, leads to the constitutive activation of protein kinase B (PKB)/Akt via enhanced phosphorylation of Thr-308 and Ser-473. The integrin-linked kinase (ILK) can phosphorylate PKB/Akt on Ser-473 in a phosphoinositide phospholipid-dependent manner. The activity of ILK is constitutively elevated in a serum- and anchorage-independent manner in PTEN-mutant cells, and transfection of wild-type (WT) PTEN into these cells inhibits ILK activity. Transfection of a kinase-deficient, dominant-negative form of ILK or exposure to a small molecule ILK inhibitor suppresses the constitutive phosphorylation of PKB/Akt on Ser-473, but not on Thr-308, in the PTEN-mutant prostate carcinoma cell lines PC-3 and LNCaP. Transfection of dominant-negative ILK and WT PTEN into these cells also results in the inhibition of PKB/Akt kinase activity. Furthermore, dominant-negative ILK or WT PTEN induces G(1) phase cycle arrest and enhanced apoptosis. Together, these data demonstrate a critical role for ILK in PTEN-dependent cell cycle regulation and survival and indicate that inhibition of ILK may be of significant value in PTEN-mutant tumor therapy (Persad, 2000).


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Biological Overview

date revised: 23 August 2014

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