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).
The initiation factor 4E for eukaryotic translation (eIF4E) binds the messenger RNA 5'-cap structure and is important in the regulation of protein
synthesis. Mammalian eIF4E activity is inhibited when the initiation factor binds to the translational repressors, the 4E-binding proteins (4E-BPS). The Drosophila 4E-BP (d4E-BP) is a downstream target of the phosphatidylinositol-3-OH kinase [PI(3)K] signal-transduction cascade, which affects the interaction of d4E-BP with eIF4E. Ectopic expression of a highly active d4E-BP mutant in wing-imaginal discs causes a reduction of wing size, brought about by a decrease in cell size and number. A marked reduction in cell size is also observed in post-mitotic cells. Expression of d4E-BP in the eye and wing together with PI(3)K or dAkt1, the serine/threonine kinase downstream of PI(3)K, results in suppression of the growth phenotype elicited by these kinases. These results support a role for d4E-BP as an effector of cell growth (Miron, 2001).
Drosophila 4E-BP (d4E-BP) was isolated by interaction cloning from a complementary DNA expression library using 32P-labelled deIF4EI. d4E-BP is identical to Drosophila Thor (Bernal, 2000) and homologous to 4E-BPs from other species. Phosphorylation sites in mammalian 4E-BP1 are conserved in d4E-BP, but the predicted eIF4E-binding motif in d4E-BP (YERAFMK) diverges from the canonical consensus sequence (Miron, 2001).
To examine the binding of d4E-BP to deIF4E, residues within the consensus eIF4E-binding site were mutated. Recombinant proteins were expressed in Escherichia coli, and far Western blotting was performed using 32P-labelled deIF4EI. Mutation of Tyr 54 to Ala (Y54A) or Phe (Y54F), and Met 59 to Ala (M59A) abrogates the interaction of d4E-BP with deIF4E. Mutation of Lys 60 to Ala (K60A) decreases deIF4E binding by 87%, indicating that Lys 60 contributes to deIF4E binding. However, when either Met 59 or Lys 60 are mutated to the consensus Leu, the interaction of d4E-BP with deIF4EI is 2.5-fold higher than with the wild type, and when both Met 59 and Lys 60 are so changed, deIF4E binding increases by 3.4-fold. These results indicate that d4E-BP interacts with deIF4E, albeit more weakly than previously characterized 4E-BPs, owing to its divergent eIF4E-binding motif (Miron, 2001).
4E-BP1 is hyperphosphorylated in response to insulin in many cell types. To test whether this response operates in Drosophila, Schneider-2 (S2) cells were deprived of serum and treated with insulin. Increasing levels of a slower migrating form of d4E-BP (d4E-BP) were observed consequent to insulin treatment. To determine whether the ß-form corresponds to phosphorylated d4E-BP, extracts from insulin-stimulated S2 cells were treated with either calf intestine alkaline phosphatase (CIP) or protein phosphatase 2A (PP2A). Untreated extracts (or extracts kept on ice) contain both the faster migrating alpha- and the slower migrating ß-forms. In contrast, phosphatase-treated extracts contained only the alpha-form (Miron, 2001).
LY294002 and rapamycin inhibit PI(3)K and target of rapamycin (TOR) activity, respectively, and block the insulin-induced hyperphosphorylation of 4E-BP1. Similarly, exposure of serum-deprived S2 cells to either drug before treatment with insulin, results in a dose-dependent decrease in d4E-BP phosphorylation. To determine whether phosphorylation of d4E-BP prevents its binding to deIF4E, m7GDP-agarose precipitation was performed. The alpha form is present primarily in the bound fraction, whereas the ß-form is found exclusively in the unbound fraction. These results show that d4E-BP is a downstream target of the PI(3)K pathway, and that the binding of d4E-BP to deIF4E is modulated by its phosphorylation state (Miron, 2001).
Assembly of eIF4F is essential for translational control, and overexpression of eIF4E in mammalian cells results in malignant transformation. To investigate whether eIF4F is also linked to growth control, eIF4F assembly was perturbed in Drosophila. UAS transgenic fly lines were generated that express wild-type d4E-BP or the mutant d4E-BP that binds deIF4E most strongly, d4E-BP(LL). Expression of d4E-BP was targeted to the wing-imaginal disc using MS1096-GAL4. The size and cell number of wings from males were measured. Expression of wild-type d4E-BP has no effect on wing size or pattern. However, expression of d4E-BP(LL) from one line [d4E-BP(LL)w] causes a marked reduction of wing size without affecting cell number. Another line, [d4E-BP(LL)s], which expresses d4E-BP(LL) more strongly, causes a larger reduction, which is partly due to a decrease in cell number. Since direct inhibition of cellular proliferation increases, rather than decreases, cell size, it is possible that d4E-BP(LL) also affects cell size directly, and cell proliferation as a consequence. This is supported by analysis of the effects of d4E-BP(LL) expression in larval-wing discs. Although discs from the d4E-BP(wt) and d4E-BP(LL)w lines are indistinguishable from control discs, d4E-BP(LL)s discs are 52% smaller. d4E-BP(LL)s males also required 1-2 days longer to eclose, which would account for the smaller decrease in adult wings (Miron, 2001).
Acridine-orange staining shows that d4E-BP(LL)s discs contain many apoptotic cells. Co-expression of p35, the baculovirus inhibitor of apoptosis, with d4E-BP(LL)s partially rescues the size of adult wings. To distinguish between apoptosis and decreased proliferation, cell clones expressing d4E-BP(LL), with or without p35, and co-expressing green fluorescent protein (GFP), were induced 72 h after egg deposition in developing wing discs using the flip-out technique. Clones expressing d4E-BP(LL)w contain fewer cells than wild-type clones, but co-expression of p35 with d4E-BP(LL)w does not affect the number of cells per clone, indicating that decreased proliferation, but not increased apoptosis, is the cause of reduction. Few clones expressing d4E-BP(LL)s are recovered, and they usually contain 1-2 cells. Co-expression of p35 greatly increases the number of clones recovered, but only marginally increases the number of cells per clone (1-4 cells) (Miron, 2001).
Direct interference with cell proliferation using string mutants results in increased cell size. To help distinguish effects on size from effects on proliferation, cell size was evaluated by flow cytometry (FACS). Mean forward-light scatter values for GFP-positive cells that expressed d4E-BP(LL) were reduced by 6%-8%. Because cells that expressed d4E-BP(LL) are smaller and proliferate more slowly than their wild-type counterparts, it is conceivable that d4E-BP(LL) directly affects cell growth and consequently retards proliferation, which would lead to reduced viability and ultimately apoptosis. Similar results were observed in dTOR mutants, and interpreted as a primary defect in cellular growth coupled with a consequent decrease in cell proliferation. The possibility that growth and proliferation are affected independently by d4E-BP(LL) expression cannot be excluded (Miron, 2001).
To exclude proliferation effects, the growth and viability of d4E-BP(LL) cells were examined in a post-mitotic tissue. Polyploid fat-body cells undergo successive rounds of DNA synthesis without mitoses. Cells that express d4E-BP(LL)s, induced 48 h after egg deposition in the fat body, are 45%-70% smaller than neighboring wild-type cells, but their frequency is much higher than in mitotically active tissues, such as the wing-imaginal disc. Thus, viability of cells that express d4E-BP(LL) is maintained in the absence of mitogenic signals, indicating that proliferation of wild-type neighboring cells is necessary to cause the disappearance of cells expressing d4E-BP(LL). In support of this notion is the finding that when d4E-BP(LL)s clones are induced during development of eye-imaginal discs, only the clones that are generated posterior to the morphogenetic furrow survive; the clones generated anterior to the furrow (that is, in mitotically active cells), are eliminated (Miron, 2001).
To study the possible role of d4E-BP as an effector of cell growth through the PI(3)K signaling pathway, potential interactions between d4E-BP and relevant signaling genes of this pathway were examined. Expression of wild-type d4E-BP in eye-imaginal discs, using GMR-GAL4, does not engender any discernible phenotype, whereas expression of dAkt1 results in an enlarged eye. However, co-expression of wild-type d4E-BP and dAkt1 partially suppresses the enlarged-eye phenotype, and fully suppresses the roughness induced by expression of dAkt1. Since d4E-BP by itself has no effect on eye size but is able to suppress the dAkt1 phenotype, there is a genuine epistatic relationship between d4E-BP and dAkt1 (Miron, 2001).
Other components of the PI(3)K pathway were also examined for potential epistatic interactions with d4E-BP in the wing, using dpp-GAL4 and 4E-BP(LL)s. Ectopic expression of Dp110 and dAkt1 causes an enlargement of the region encompassed by the third and fourth longitudinal veins, the anterior crossvein and wing margin. In contrast, expression of a dominant-negative mutant form of PI(3)K (Dp110D954A) or d4E-BP(LL)s results in reduction of the size of this region. Co-expression of d4E-BP(LL)s with Dp110 or dAkt1 suppresses the growth enhancement engendered by expression of these kinases, whereas co-expression of d4E-BP(LL)s with Dp110D954A results in further size reduction. Flies that lacked a copy of the gene encoding the adaptor protein p60 [the Drosophila homolog of mammalian PI(3)K subunit p85] are also reduced in size when d4E-BP(LL)s is co-expressed. These results provide genetic evidence that d4E-BP is a downstream effector of the PI(3)K pathway (Miron, 2001).
Null mutants of d4E-BP are viable and although their immune response is compromised (Bernal, 2000), they do not exhibit increased growth. Furthermore, ectopic expression of Drosophila eIF4E in a wild-type or d4E-BP null background fails to produce a growth-related phenotype. Therefore, an increase in eIF4E activity alone is not sufficient to promote cell growth in Drosophila imaginal discs. This is consistent with data in primary mouse-embryo fibroblasts, in which eIF4E overexpression leads only to oncogenic transformation when co-expressed with c-myc or E1A. Attempts were made to rescue the d4E-BP(LL)-induced growth defects in imaginal discs by co-expressing deIF4E. Unexpectedly, growth is further reduced. Thus, endogenous eIF4E expression levels are optimal for cell growth and proliferation, and in the absence of activation of the PI(3)K pathway, a further increase in eIF4E expression is either without effect or deleterious (Miron, 2001).
Many studies have shown that PI(3)K and TOR-mediated signaling is important for normal cell growth and proliferation. However, one downstream target of this pathway, S6K, regulates cell size but not proliferation. Constitutive expression of dS6K in dTOR mutants only partially suppresses the dTOR phenotype, indicating that S6K-independent targets operate downstream of dTOR. Regulation of eIF4E activity, independent of S6K, contributes to the control of cell size. In Drosophila, the activity of eIF4E is modulated through 4E-BP. Phosphorylation of eIF4E is correlated with increased translation rates. Mutation of the phosphorylation site in Drosophila eIF4E causes a cell size reduction. In summary, the results presented here show that d4E-BP acts as an important downstream effector of PI(3)K in the regulation of cell proliferation and growth, independent of S6K, and further underline the importance of translation initiation in the latter process (Miron, 2001).
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 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).
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).
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).
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).
Signaling through the PI3K/Akt/FOXO pathway plays an important role in vertebrates in protecting cells from programmed cell death. PI3K and Akt have been similarly shown to be involved in survival signaling in Drosophila. However, it is not known whether PI3K and Akt execute this function by controlling a pro-apoptotic activity of Drosophila FOXO. This study shows that elevated signaling through PI3K and Akt can prevent developmentally controlled death in the salivary glands of the fruit fly. Drosophila FOXO is not required for normal salivary gland death and the rescue of salivary gland death by PI3K occurs independent of FOXO. These results give support to the notion that FOXOs have acquired pro-apoptotic functions after separation of the vertebrate and invertebrate lineages (Liu, 2006).
To determine whether elevated signaling through PI3K can rescue normal salivary gland death, the catalytic subunit of PI3K, Dp110, was expressed in late-prepupal glands using P{UAS-Dp110} and a heat-shock GAL4 driver. Most of the pupae expressing the subunit still possessed intact salivary glands 20 h APF, i.e., ∼6 h after the glands are normally destroyed. This result shows that a high level of PI3K activity can overcome the stimuli that normally lead to the destruction of the salivary glands in early pupae. Moreover, it suggests that the PI3K pathway is normally inactive or, at least, strongly downregulated in dying salivary glands (Liu, 2006).
If the effect of PI3K is mediated by the canonical PI3K/Akt pathway, an elevated activity of Akt should have the same or a similar effect as an elevated PI3K activity. To test this prediction, both wild-type Akt and a constitutively active form of Drosophila Akt, Daktmyr, were expressed in late-prepupal salivary glands. Daktmyr carries a myristoylated amino terminus that targets the protein to the cell membrane. Under normal conditions, Akt is recruited to the membrane by the PI3K product PIP3 and subsequently activated by phosphorylation through PDK1. The expression of both UAS-Dakt myr and UAS-akt was driven by heat-shock GAL4. The constitutively active Daktmyr led to a complete rescue of salivary gland death, whereas unmodified Akt had no effect. It is concluded that only membrane-associated Akt can rescue salivary gland death. This is consistent with the normal mechanism of Akt activation that requires PI3K-induced recruitment of Akt to the cell membrane. The inhibition of death by active Akt, but not by inactive Akt, confirms that PI3K activity is limited in late-prepupal salivary glands and underscores the specificity of the observed effect (Liu, 2006).
In summary, the results show that the survival function of PI3K/Akt does not depend on the inactivation of dFOXO and that dFOXO has no apparent role in the activation of PCD in the salivary glands. Moreover, they indicate that an intact PI3K/Akt signaling pathway is not required for salivary gland survival. However, downregulation of the pathway may be required for salivary gland death, because elevated signaling through the pathway can rescue the salivary glands. Importantly, this study on the role of PI3K/Akt/dFOXO signaling in salivary gland death did not reveal a pro-apoptotic role of dFOXO, further strengthening the assumption that the functions of FOXOs in apoptosis are a late evolutionary acquisition in the vertebrate lineage (Liu, 2006).
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Atk1:
Biological Overview
| Developmental Biology
| Effects of Mutation
| References
Society for Developmental Biology's Web server.