Akt1

REGULATION

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 PDK-1 (dPDK-1) mutants have been isolated and the in vivo roles of their kinases have been characterized. Drosophila deficient in the dPDK-1 gene exhibit lethality and an apoptotic phenotype in the embryonic stage. Conversely, overexpression of dPDK-1 increases cell and organ size in a Drosophila PI3K-dependent manner. dPDK-1 not only can activate Drosophila Akt/PKB (Dakt1), but also substitutes the in vivo functions of its mammalian ortholog to activate Akt/PKB. This functional interaction between dPDK-1 and Dakt1 was further confirmed through genetic analyses in Drosophila. However, cAMP-dependent protein kinase, which has been proposed as a possible target of dPDK-1, did not interact with dPDK-1. In conclusion, these findings provide direct evidence that dPDK-1 regulates cell growth and apoptosis during Drosophila development via the PI3K-dependent signaling pathway and demonstrate this Drosophila system to be a powerful tool for elucidating the in vivo functions and targets of PDK-1 (Cho, 2001).

PDK-1 originally was identified as an upstream regulatory kinase of Akt/protein kinase B (PKB). Consequently, the in vivo roles of PDK-1 have been inferred mainly from those of Akt/PKB. Akt/PKB is a growth factor-regulated serine/threonine kinase that contains a pleckstrin homology domain, as does PDK-1. It acts downstream of phosphoinositide 3-kinase (PI3K) to regulate various cellular activities, including glucose metabolism, transcription, and protein translation. Akt/PKB also negatively regulates apoptosis in various ways. To exert its antiapoptotic effects, Akt/PKB either inhibits the activities of proapoptotic proteins, such as BAD and caspase-9, or induces antiapoptotic signals via the NF-kappaB- and forkhead transcription factor-dependent pathways. Recent transgenic studies in Drosophila have revealed an unexpected function of Akt/PKB and the PI3K signaling pathway: the pathway plays an essential role in the control of cell size. When the activities of one or multiple components of the pathway, including PI3K, Drosophila akt1 (Dakt1), and Drosophila p70 S6 kinase, are down-modulated, cell size as well as body size are dramatically reduced in a cell-autonomous manner (Cho, 2001 and references therein).

Recent studies also suggest that PDK-1 is involved in the activation of members of the AGC superfamily of serine/threonine protein kinases, through phosphorylation of their activation loop in response to extracellular stimulations induced by peptide growth factors and hormones. A number of important kinases in this family, including Akt/PKB, p70 S6 kinase, various protein kinase Cs, protein kinase C-related kinases, and cAMP-dependent protein kinase (PKA), have been proposed as either in vivo or in vitro targets of PDK-1. These results implicate that PDK-1 may play the role of a 'master kinase' in regulating these downstream kinases. However, further investigation is required to determine the actual in vivo targets of PDK-1, as it has been revealed that some of the AGC family kinases are not directly phosphorylated by PDK-1 in vivo, despite possessing a putative PDK-1 phosphorylation site at the activation loop and being phosphorylated by PDK-1 in vitro. In addition, although PDK-1 is regarded as a regulator of at least some of these important kinases, the physiological role of the kinase in a multicellular system has not yet been defined at all (Cho, 2001 and references therein).

The PDK-1 Drosophila homolog, dPDK-1 is 54% identical to its human counterpart in the catalytic domain and is also highly homologous in the noncatalytic carboxyl terminus. Flies containing mutations in the dPDK-1 locus were isolated for genetic analyses. Three P-element insertion mutants, EP(3)837, EP(3)3553, and EP(3)3091, have been found containing P-element insertions in either the 5' or intron region of the dPDK-1 gene. In detail, the inserted positions of the P-element in EP(3)837 and EP(3)3553, which have been determined by inverse PCR, are located at 179 bp and 144 bp upstream of the dPDK-1 transcription start site, respectively. The insertion sites and directions of the P-elements are oriented to induce gene expression and imply that these mutants can be used to study the gain of function of dPDK-1. Another EP line, EP(3)3091, has a P-element in the fourth intron of dPDK-1. The insertion site of EP(3)3091 predicts that the transcription of dPDK-1 is disrupted by the insertion of the transposon. Indeed, EP(3)3091 displays a complete lethal phenotype. In addition, another PDK-1-deficient lethal line, DeltadPDK5, has been generated by an imprecise excision of P-element in EP(3)837. This line contains about a 10-kb deletion that includes the first exon of dPDK-1. This mutant fails to complement the lethality of EP(3)3091, suggesting that both lines are alleles of dPDK-1 mutants. Thus, EP(3)3091 and DeltadPDK5 are hereafter referred to as dPDK-1¹ and dPDK-1², respectively (Cho, 2001).

None of the homozygous dPDK-1¹ and dPDK-1² flies emerge as larva, and both display an embryonic lethality. To isolate dPDK-1¹ homozygous individuals, a GFP balancer chromosome was used. The GFP-negative embryos were selected as dPDK-1 homozygotes. All of the hatched larvae from dPDK-1¹ or dPDK-1²/TM3, GFP, Ser females show GFP expression. dPDK-1¹ homozygous embryos produce no ventral cuticles, and they do not develop into the larval stage. These results are similar to those seen in the mutation of Dakt1, whose mammalian homologs are well-known targets of PDK-1. Briefly, absence of maternal and zygotic Dakt1 activity also results in an embryonic lethality, along with defective cuticle formation (Cho, 2001).

Whether dPDK-1 also is involved in the cell survival-signaling pathway was tested. TUNEL assays were performed with dPDK-1¹ homozygous embryos to examine the involvement of the kinase in apoptosis. Apoptotic activity is dramatically induced in the dPDK-1 zygotic loss-of-function mutant. The induced apoptosis in dPDK-1¹ mutant embryos is extensively suppressed by expression of Dakt1 using the hs-GAL4-UAS system. Collectively, these results strongly suggest that PDK-1 plays an important role in Drosophila embryonic development and apoptosis (Cho, 2001).

A series of components in the PI3K pathway including Dakt1 and Drosophila p70 S6 kinase modulate cell size in a cell-autonomous manner. Thus, whether overexpression of dPDK-1 affects cell size was examined using the GAL4-UAS system. dPDK-1 was overexpressed under the control of gmr-GAL4, which directs expression of the gene in the developing eye. This ectopic overexpression of dPDK-1 causes an increase in ommatidia size, ~1.33-fold bigger than controls. In addition, the effect of overexpression of dPDK-1 was examined in a specific compartment of the wing disk. The wing disk is composed of two compartments (dorsal and ventral), which fold and generate the flattened wing blade. When dPDK-1 is ectopically overexpressed in the dorsal compartment with ap-GAL4 driver, the wing of EP(3)837 is convex toward the dorsal side. This is likely the result of an increase in the size of the cells in the dorsal compartment. Indeed, a similar situation is observed in the UAS-Drosophila p70 S6 kinase flies. These results suggest that dPDK-1 regulates cell and organ size (Cho, 2001).

Despite the fact that there is no clear evidence on how the intrinsic kinase activity of PDK-1 is regulated, the kinase has been found to act downstream of PI3K. Thus, whether dPDK-1 and PI3K can genetically interact was examined in fly lines in which dPDK-1 was coexpressed with PI3K or a dominant negative Dp110 (PI3KDN). Overexpression of the PI3K catalytic subunit, Dp110, increases cell size, whereas overexpression of a PI3KDN results in the opposite phenotype. This change in cell size results in the change of organ and body size. Overexpression of PI3KDN under ptc-GAL4 (the driver induces GAL4 expression throughout the anterior compartment with a stripe of maximal intensity along the border of anterior/posterior compartment extending into the posterior compartment) results in reduction of the distance between L3 and L4 veins. However, this phenotype is strongly suppressed by coexpression of dPDK-1 with PI3KDN, suggesting that dPDK-1 acts as a vital downstream effector of PI3K in cell and compartment size control. Conversely, overexpression of the PI3K wild-type causes an increase in the distance between L3 and L4 veins, and coexpression of dPDK-1 and PI3K further increases the distance in a synergistic manner. These results provide strong in vivo evidence that dPDK-1 functions downstream of Drosophila PI3K in the control of cell and compartment size (Cho, 2001).

To determine whether dPDK-1 functions in a manner similar to its mammalian counterpart, myc-tagged dPDK-1, myc-tagged human PDK-1 (hPDK-1), and/or HA-tagged human Akt/PKB were transiently expressed in COS cells. As expected, dPDK-1 strongly induces human Akt/PKB activity, to levels comparable to those induced by hPDK-1. Conversely, coexpression of a dominant negative hPDK-1 or a dominant negative dPDK-1 strongly inhibits the epidermal growth factor-induced activation of human Akt/PKB. These results indicate that the Drosophila ortholog of PDK-1 can properly function and substitute its mammalian counterpart to relay the growth factor-induced activation signals to a mammalian Akt/PKB (Cho, 2001).

Whether dPDK-1 can activate Dakt1 in Drosophila was examined. To test this, dPDK-1 and HA-tagged Dakt1 were coexpressed in the Drosophila eye using the gmr-GAL4 driver, and the phosphotransferase activities of Dakt1 were measured from the head extracts of gmr-GAL4, gmr-GAL4; UAS-HA-Dakt1, or gmr-GAL4; UAS-HA-Dakt1/EP(3)837. Dakt1 activity is strongly increased in the flies coexpressing dPDK-1. Consistent with this increased activity, an electrophoretically retarded Akt/PKB band, corresponding to a highly phosphorylated and activated form, is observed. This biochemical evidence strongly supports that Dakt1 is indeed a physiological target of dPDK-1 (Cho, 2001).

To further confirm the in vivo roles of dPDK-1, genetic interactions between Dakt1 and dPDK-1 were examined in flies. Overexpression of Dakt1 in the Drosophila eye increases eye size and generates a bulging eye with enlarged ommatidia. In addition to this change in size, the ommatidia array becomes irregular, and eye bristles are enlarged with a frequent loss of number. When dPDK-1 is coexpressed with Dakt1 in the eye, it displays a severely crumpled morphology. The eye bristles are enlarged even more severely, and the boundaries of all ommatidia and photoreceptor cells disappear. These dPDK-1/Dakt1 phenotypes are further enhanced by an increased dose of gmr-GAL4 driver. These findings, taken together, clearly demonstrate the functional and genetic interactions between dPDK-1 and Dakt1 (Cho, 2001).

The genetic interactions between dPDK-1 and Drosophila PKA were examined. Although PKA has been proposed to be a putative substrate of PDK-1, the in vivo relevance of this has not been clearly determined. When the catalytic subunit of Drosophila PKA (dPKAc) is overexpressed in the developing eye of Drosophila, the eye is discolored and swells up with wrinkles. Scanning electron microscopic views of the eye show that the boundaries of all ommatidia and photoreceptor cells disappear. However, unlike Akt/PKB, coexpression of dPDK-1 does not affect these phenotypes of dPKAc. Furthermore, the regulatory subunit of Drosophila PKA (dPKAr) also does not interact with dPDK-1. These results support that PKA is not regulated by PDK-1 in Drosophila, which is highly consistent with recent results that PKA is phosphorylated and activated normally in a PDK-1-deficient cell line. These results strongly support that the Drosophila system is a physiologically relevant tool for determining the actual in vivo targets of PDK-1 (Cho, 2001).

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).

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 (Dp110^D954A) 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 Dp110^D954A 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).

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).

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, Dakt^myr, were expressed in late-prepupal salivary glands. Dakt^myr 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 Dakt^myr 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, L^fee. Quantitative RT-PCR and genetic analysis revealed that L^fee 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 L^si heterozygote exhibits slightly reduced eyes that are nicked near the anterior D/V boundary, similar to the major fee phenotype. In the L^si 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 L^rev6-3 had smaller eyes than fee flies. In addition, fee could to be recombined with L^rev6-3, 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 L^fee (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 (L^fee and L^rev/+), but not by its complete loss (L^rev 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 L^fee 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 L^fee 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 Stat92E^ts), 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).

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