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• W2080647545 abstract "PRAS40 has recently been identified as a protein that couples insulin/IGF signaling (IIS) to TORC1 activation in cell culture; however, the physiological function of PRAS40 is not known. In this study, we investigate flies lacking PRAS40. Surprisingly, we find both biochemically and genetically that PRAS40 couples IIS to TORC1 activation in a tissue-specific manner, regulating TORC1 activity in ovaries but not in other tissues of the animal. PRAS40 thereby regulates fertility but not growth of the fly, allowing distinct physiological functions of TORC1 to be uncoupled. We also show that the main function of PRAS40 in vivo is to regulate TORC1 activity, and not to act as a downstream target and effector of TORC1. Finally, this work sheds some light on the question of whether TORC1 activity is coupled to IIS in vivo. PRAS40 has recently been identified as a protein that couples insulin/IGF signaling (IIS) to TORC1 activation in cell culture; however, the physiological function of PRAS40 is not known. In this study, we investigate flies lacking PRAS40. Surprisingly, we find both biochemically and genetically that PRAS40 couples IIS to TORC1 activation in a tissue-specific manner, regulating TORC1 activity in ovaries but not in other tissues of the animal. PRAS40 thereby regulates fertility but not growth of the fly, allowing distinct physiological functions of TORC1 to be uncoupled. We also show that the main function of PRAS40 in vivo is to regulate TORC1 activity, and not to act as a downstream target and effector of TORC1. Finally, this work sheds some light on the question of whether TORC1 activity is coupled to IIS in vivo. PRAS40 mutant animals are normal in size PRAS40 does not regulate TORC1 activity in most of the body PRAS40 does regulate TORC1 activity in ovaries and affects fly fertility PRAS40 mutation rescues infertility of chico mutant flies TOR complex 1 (TORC1) and insulin/IGF signaling (IIS) are two highly conserved pathways that sense nutrient status in animals from flies to humans. IIS is responsive to hormonal cues, thereby integrating information about organismal nutrient status. In contrast TORC1 signaling, which is conserved in unicellular organisms, senses primarily cell-autonomous information such as cellular stress, energy, and nutrients (Avruch et al., 2006Avruch J. Hara K. Lin Y. Liu M. Long X. Ortiz-Vega S. Yonezawa K. Insulin and amino-acid regulation of mTOR signaling and kinase activity through the Rheb GTPase.Oncogene. 2006; 25: 6361-6372Crossref PubMed Scopus (264) Google Scholar, Kwiatkowski and Manning, 2005Kwiatkowski D.J. Manning B.D. Tuberous sclerosis: a GAP at the crossroads of multiple signaling pathways.Hum. Mol. Genet. 2005; 14 Spec No. 2: R251-R258Crossref PubMed Scopus (333) Google Scholar, Martin and Hall, 2005Martin D.E. Hall M.N. The expanding TOR signaling network.Curr. Opin. Cell Biol. 2005; 17: 158-166Crossref PubMed Scopus (445) Google Scholar, Shaw, 2009Shaw R.J. LKB1 and AMP-activated protein kinase control of mTOR signalling and growth.Acta Physiol. (Oxf.). 2009; 196: 65-80Crossref PubMed Scopus (498) Google Scholar). Both IIS and TORC1 integrate this information to regulate multiple physiological processes including carbohydrate metabolism, lipid metabolism, tissue growth, fertility and lifespan in a manner that is conserved from flies to mammals (Fontana et al., 2010Fontana L. Partridge L. Longo V.D. Extending healthy life span—from yeast to humans.Science. 2010; 328: 321-326Crossref PubMed Scopus (2115) Google Scholar, Goberdhan and Wilson, 2003Goberdhan D.C. Wilson C. The functions of insulin signaling: size isn't everything, even in Drosophila.Differentiation. 2003; 71: 375-397Crossref PubMed Scopus (109) Google Scholar, Grewal, 2009Grewal S.S. Insulin/TOR signaling in growth and homeostasis: a view from the fly world.Int. J. Biochem. Cell Biol. 2009; 41: 1006-1010Crossref PubMed Scopus (162) Google Scholar, Kozma and Thomas, 2002Kozma S.C. Thomas G. Regulation of cell size in growth, development and human disease: PI3K, PKB and S6K.Bioessays. 2002; 24: 65-71Crossref PubMed Scopus (256) Google Scholar, Nakae et al., 2001Nakae J. Kido Y. Accili D. Distinct and overlapping functions of insulin and IGF-I receptors.Endocr. Rev. 2001; 22: 818-835Crossref PubMed Scopus (357) Google Scholar). Of note, TORC1 is one of the most powerful anabolic signals in cells, regulating cellular growth via modulation of protein and lipid biosynthesis, leading to cellular mass accumulation. As a consequence TORC1 is hyperactivated in almost all cancers (Bjornsti and Houghton, 2004Bjornsti M.A. Houghton P.J. The TOR pathway: a target for cancer therapy.Nat. Rev. Cancer. 2004; 4: 335-348Crossref PubMed Scopus (1198) Google Scholar, Guertin and Sabatini, 2007Guertin D.A. Sabatini D.M. Defining the role of mTOR in cancer.Cancer Cell. 2007; 12: 9-22Abstract Full Text Full Text PDF PubMed Scopus (2415) Google Scholar). The connection between IIS and TORC1 has been a matter of debate. In human and Drosophila tissue culture cells, treatment with insulin leads to rapid TORC1 activation (Cai et al., 2006Cai S.L. Tee A.R. Short J.D. Bergeron J.M. Kim J. Shen J. Guo R. Johnson C.L. Kiguchi K. Walker C.L. Activity of TSC2 is inhibited by AKT-mediated phosphorylation and membrane partitioning.J. Cell Biol. 2006; 173: 279-289Crossref PubMed Scopus (280) Google Scholar, Hahn et al., 2010Hahn K. Miranda M. Francis V.A. Vendrell J. Zorzano A. Teleman A.A. PP2A regulatory subunit PP2A-B′ counteracts S6K phosphorylation.Cell Metab. 2010; 11: 438-444Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, Inoki et al., 2002Inoki K. Li Y. Zhu T. Wu J. Guan K.L. TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling.Nat. Cell Biol. 2002; 4: 648-657Crossref PubMed Scopus (2396) Google Scholar, Manning et al., 2002Manning B.D. Tee A.R. Logsdon M.N. Blenis J. Cantley L.C. Identification of the tuberous sclerosis complex-2 tumor suppressor gene product tuberin as a target of the phosphoinositide 3-kinase/akt pathway.Mol. Cell. 2002; 10: 151-162Abstract Full Text Full Text PDF PubMed Scopus (1274) Google Scholar, Potter et al., 2002Potter C.J. Pedraza L.G. Xu T. Akt regulates growth by directly phosphorylating Tsc2.Nat. Cell Biol. 2002; 4: 658-665Crossref PubMed Scopus (778) Google Scholar), indicating the two pathways are linked. Less clear is whether this link is functionally relevant for cells in an animal under physiological conditions. Studies in Drosophila have suggested this is not the case. One molecular link connecting IIS to TORC1 is the TSC1/2 complex. In cell culture, phosphorylation of Tsc2 by Akt inactivates it, thereby relieving its suppression of TORC1 (Cai et al., 2006Cai S.L. Tee A.R. Short J.D. Bergeron J.M. Kim J. Shen J. Guo R. Johnson C.L. Kiguchi K. Walker C.L. Activity of TSC2 is inhibited by AKT-mediated phosphorylation and membrane partitioning.J. Cell Biol. 2006; 173: 279-289Crossref PubMed Scopus (280) Google Scholar, Inoki et al., 2002Inoki K. Li Y. Zhu T. Wu J. Guan K.L. TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling.Nat. Cell Biol. 2002; 4: 648-657Crossref PubMed Scopus (2396) Google Scholar, Manning et al., 2002Manning B.D. Tee A.R. Logsdon M.N. Blenis J. Cantley L.C. Identification of the tuberous sclerosis complex-2 tumor suppressor gene product tuberin as a target of the phosphoinositide 3-kinase/akt pathway.Mol. Cell. 2002; 10: 151-162Abstract Full Text Full Text PDF PubMed Scopus (1274) Google Scholar, Potter et al., 2002Potter C.J. Pedraza L.G. Xu T. Akt regulates growth by directly phosphorylating Tsc2.Nat. Cell Biol. 2002; 4: 658-665Crossref PubMed Scopus (778) Google Scholar). However, in Drosophila in vivo, removal of the Akt phosphorylation sites on Tsc1 and Tsc2 leads to no defects in TOR activation and no physiological consequences (Dong and Pan, 2004Dong J. Pan D. Tsc2 is not a critical target of Akt during normal Drosophila development.Genes Dev. 2004; 18: 2479-2484Crossref PubMed Scopus (93) Google Scholar, Schleich and Teleman, 2009Schleich S. Teleman A.A. Akt phosphorylates both Tsc1 and Tsc2 in Drosophila, but neither phosphorylation is required for normal animal growth.PLoS ONE. 2009; 4: e6305Crossref PubMed Scopus (30) Google Scholar), suggesting that although this molecular link exists, it does not play an important functional role under physiological conditions. Although one explanation could be the presence of redundant molecular mechanisms linking IIS to TORC1, this appears not to be the case. In Drosophila larvae, reduction of IIS either via removal of the insulin receptor substrate chico, or via reduction of PI3K activity (Dp110A), has no effect on activity of S6K and hence TORC1 (Oldham et al., 2000Oldham S. Montagne J. Radimerski T. Thomas G. Hafen E. Genetic and biochemical characterization of dTOR, the Drosophila homolog of the target of rapamycin.Genes Dev. 2000; 14: 2689-2694Crossref PubMed Scopus (369) Google Scholar, Radimerski et al., 2002Radimerski T. Montagne J. Rintelen F. Stocker H. van der Kaay J. Downes C.P. Hafen E. Thomas G. dS6K-regulated cell growth is dPKB/dPI(3)K-independent, but requires dPDK1.Nat. Cell Biol. 2002; 4: 251-255Crossref PubMed Scopus (153) Google Scholar), raising the question whether TORC1 activation is at all linked to IIS in vivo under physiological conditions. Because of the central role TORC1 plays in regulating cellular growth and metabolism, a better understanding of the mechanisms regulating its activity would have implications for both normal and pathophysiological conditions such as cancer, metabolic disease, or infertility. Recently the protein PRAS40 has been proposed to link IIS to TORC1 in cell culture. Two reports showed that PRAS40 binds the TORC1 complex thereby inhibiting its activity, and that phosphorylation of PRAS40 by Akt relieves this inhibition (Nascimento et al., 2010Nascimento E.B. Snel M. Guigas B. van der Zon G.C. Kriek J. Maassen J.A. Jazet I.M. Diamant M. Ouwens D.M. Phosphorylation of PRAS40 on Thr246 by PKB/AKT facilitates efficient phosphorylation of Ser183 by mTORC1.Cell. Signal. 2010; 22: 961-967Crossref PubMed Scopus (64) Google Scholar, Sancak et al., 2007Sancak Y. Thoreen C.C. Peterson T.R. Lindquist R.A. Kang S.A. Spooner E. Carr S.A. Sabatini D.M. PRAS40 is an insulin-regulated inhibitor of the mTORC1 protein kinase.Mol. Cell. 2007; 25: 903-915Abstract Full Text Full Text PDF PubMed Scopus (994) Google Scholar, Vander Haar et al., 2007Vander Haar E. Lee S.I. Bandhakavi S. Griffin T.J. Kim D.H. Insulin signalling to mTOR mediated by the Akt/PKB substrate PRAS40.Nat. Cell Biol. 2007; 9: 316-323Crossref PubMed Scopus (927) Google Scholar). Three other studies, however, identified PRAS40 as a TORC1 substrate, suggesting that the apparent inhibitory effects of PRAS40 on the canonical TORC1 substrates 4EBP and S6K may reflect competition for substrate binding (Fonseca et al., 2007Fonseca B.D. Smith E.M. Lee V.H. MacKintosh C. Proud C.G. PRAS40 is a target for mammalian target of rapamycin complex 1 and is required for signaling downstream of this complex.J. Biol. Chem. 2007; 282: 24514-24524Crossref PubMed Scopus (204) Google Scholar, Oshiro et al., 2007Oshiro N. Takahashi R. Yoshino K. Tanimura K. Nakashima A. Eguchi S. Miyamoto T. Hara K. Takehana K. Avruch J. et al.The proline-rich Akt substrate of 40 kDa (PRAS40) is a physiological substrate of mammalian target of rapamycin complex 1.J. Biol. Chem. 2007; 282: 20329-20339Crossref PubMed Scopus (260) Google Scholar, Wang et al., 2007Wang L. Harris T.E. Roth R.A. Lawrence Jr., J.C. PRAS40 regulates mTORC1 kinase activity by functioning as a direct inhibitor of substrate binding.J. Biol. Chem. 2007; 282: 20036-20044Crossref PubMed Scopus (377) Google Scholar). This would place PRAS40 downstream, rather than upstream of TORC1. Indeed, as these studies point out, PRAS40 might function concomitantly as a TORC1 substrate and a TORC1 regulator, regulating mTORC1 activity via direct inhibition of substrate binding. These studies have led to several open questions: (1) does PRAS40 regulate TORC1 activity in vivo, as it does in cell culture? (2) does PRAS40 link IIS to TOR activation in vivo? and (3) is the main function of PRAS40 to act as a TOR substrate or as a TOR regulator? These two options can be distinguished in an animal context. If the main function of PRAS40 is to regulate TORC1 activity (i.e., it is genetically upstream of TORC1), then PRAS40 mutant phenotypes should be rescued by reducing activity of TORC1 or of a TORC1 target other than PRAS40. If, instead, PRAS40 functions mainly as a TOR substrate downstream of TORC1, then loss of PRAS40 cannot be rescued by manipulating TORC1. To our knowledge, no animal models for PRAS40 loss of function have yet been reported to address these questions. One physiological function of IIS and TORC1 of particular relevance to this present study is the regulation of fertility. In Drosophila, insulin-like peptides (DILPs) secreted by neurosecretory cells regulate the rate of germline stem cell division in the ovary (LaFever and Drummond-Barbosa, 2005LaFever L. Drummond-Barbosa D. Direct control of germline stem cell division and cyst growth by neural insulin in Drosophila.Science. 2005; 309: 1071-1073Crossref PubMed Scopus (248) Google Scholar). This links metabolic status to fertility, so that rich nutrient conditions cause high DILP secretion, leading to increased egg production. If IIS is abrogated in the ovary, as in the case of chico or InR mutants, egg production is completely blocked and the animals are sterile (Böhni et al., 1999Böhni R. Riesgo-Escovar J. Oldham S. Brogiolo W. Stocker H. Andruss B.F. Beckingham K. Hafen E. Autonomous control of cell and organ size by CHICO, a Drosophila homolog of vertebrate IRS1-4.Cell. 1999; 97: 865-875Abstract Full Text Full Text PDF PubMed Scopus (682) Google Scholar, Brogiolo et al., 2001Brogiolo W. Stocker H. Ikeya T. Rintelen F. Fernandez R. Hafen E. An evolutionarily conserved function of the Drosophila insulin receptor and insulin-like peptides in growth control.Curr. Biol. 2001; 11: 213-221Abstract Full Text Full Text PDF PubMed Scopus (918) Google Scholar, Drummond-Barbosa and Spradling, 2001Drummond-Barbosa D. Spradling A.C. Stem cells and their progeny respond to nutritional changes during Drosophila oogenesis.Dev. Biol. 2001; 231: 265-278Crossref PubMed Scopus (447) Google Scholar). The defect in chico mutant ovaries is ovary-autonomous because transplantation of chico mutant ovaries into wild-type hosts, containing normal levels of DILPS, does not rescue their defects (Richard et al., 2005Richard D.S. Rybczynski R. Wilson T.G. Wang Y. Wayne M.L. Zhou Y. Partridge L. Harshman L.G. Insulin signaling is necessary for vitellogenesis in Drosophila melanogaster independent of the roles of juvenile hormone and ecdysteroids: female sterility of the chico1 insulin signaling mutation is autonomous to the ovary.J. Insect Physiol. 2005; 51: 455-464Crossref PubMed Scopus (129) Google Scholar). At the cellular level, IIS and TORC1 regulate almost all aspects of oogenesis including the rate of proliferation of ovarian somatic and germline cells, germline stem cell maintenance, vitellogenesis, and oocyte loss (Drummond-Barbosa and Spradling, 2001Drummond-Barbosa D. Spradling A.C. Stem cells and their progeny respond to nutritional changes during Drosophila oogenesis.Dev. Biol. 2001; 231: 265-278Crossref PubMed Scopus (447) Google Scholar, Hsu and Drummond-Barbosa, 2009Hsu H.J. Drummond-Barbosa D. Insulin levels control female germline stem cell maintenance via the niche in Drosophila.Proc. Natl. Acad. Sci. USA. 2009; 106: 1117-1121Crossref PubMed Scopus (179) Google Scholar, Hsu et al., 2008Hsu H.J. LaFever L. Drummond-Barbosa D. Diet controls normal and tumorous germline stem cells via insulin-dependent and -independent mechanisms in Drosophila.Dev. Biol. 2008; 313: 700-712Crossref PubMed Scopus (128) Google Scholar, LaFever et al., 2010LaFever L. Feoktistov A. Hsu H.J. Drummond-Barbosa D. Specific roles of Target of rapamycin in the control of stem cells and their progeny in the Drosophila ovary.Development. 2010; 137: 2117-2126Crossref PubMed Scopus (107) Google Scholar, Sun et al., 2010Sun P. Quan Z. Zhang B. Wu T. Xi R. TSC1/2 tumour suppressor complex maintains Drosophila germline stem cells by preventing differentiation.Development. 2010; 137: 2461-2469Crossref PubMed Scopus (58) Google Scholar, Thomson et al., 2010Thomson T.C. Fitzpatrick K.E. Johnson J. Intrinsic and extrinsic mechanisms of oocyte loss.Mol. Hum. Reprod. 2010; 16: 916-927Crossref PubMed Scopus (33) Google Scholar). Interestingly, the roles of IIS and TORC1 in regulating fertility are highly conserved throughout evolution, regulating similar processes in Caenorhabditis elegans (Michaelson et al., 2010Michaelson D. Korta D.Z. Capua Y. Hubbard E.J. Insulin signaling promotes germline proliferation in C. elegans.Development. 2010; 137: 671-680Crossref PubMed Scopus (143) Google Scholar) and in mammals. As in flies, reduction of IIS via knockout of IGF-1 or IRS-2 causes infertility in mice (Baker et al., 1993Baker J. Liu J.P. Robertson E.J. Efstratiadis A. Role of insulin-like growth factors in embryonic and postnatal growth.Cell. 1993; 75: 73-82Abstract Full Text PDF PubMed Scopus (2061) Google Scholar, Burks et al., 2000Burks D.J. Font de Mora J. Schubert M. Withers D.J. Myers M.G. Towery H.H. Altamuro S.L. Flint C.L. White M.F. IRS-2 pathways integrate female reproduction and energy homeostasis.Nature. 2000; 407: 377-382Crossref PubMed Scopus (397) Google Scholar). As in flies, normal TORC1 in mice prevents oocyte loss (Thomson et al., 2010Thomson T.C. Fitzpatrick K.E. Johnson J. Intrinsic and extrinsic mechanisms of oocyte loss.Mol. Hum. Reprod. 2010; 16: 916-927Crossref PubMed Scopus (33) Google Scholar) and hyperactivation of IIS or TORC1 leads to premature activation of all primordial follicles, resulting in premature follicular depletion (Reddy et al., 2010Reddy P. Zheng W. Liu K. Mechanisms maintaining the dormancy and survival of mammalian primordial follicles.Trends Endocrinol. Metab. 2010; 21: 96-103Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, Sun et al., 2010Sun P. Quan Z. Zhang B. Wu T. Xi R. TSC1/2 tumour suppressor complex maintains Drosophila germline stem cells by preventing differentiation.Development. 2010; 137: 2461-2469Crossref PubMed Scopus (58) Google Scholar). In sum, IIS and TORC1 play critical roles in regulating fertility in an evolutionarily conserved manner. We present here a PRAS40 loss-of-function animal model. By generating PRAS40 knockout Drosophila, we study the in vivo function of PRAS40, as well as the connection between IIS and TORC1. We show that PRAS40 does function to link IIS to TORC1 in the animal. Unexpectedly, however, we find that it does so in a tissue-specific manner, influencing TORC1 activity predominantly in the fly ovary, but not in other tissues of the animal. As a result, PRAS40 regulates development of the ovary, but not growth or proliferation of somatic tissues, thereby influencing animal fertility but not animal growth. Because PRAS40 is present in all tissues of the fly, this indicates PRAS40 is a link between IIS and TORC1 that can be switched on and off in a tissue-specific manner. Furthermore, we find that PRAS40 knockout phenotypes can be rescued by inhibiting TORC1 or by reducing S6K gene dosage, indicating that PRAS40 functions mainly as a TORC1 inhibitor in vivo. Finally, this work sheds light on the conundrum whether the IIS and TORC1 signaling pathways are linked under normal physiological conditions, showing that they are indeed linked, but only in particular tissues. A BLAST search of the Drosophila proteome using human PRAS40 protein sequence identifies CG10109 as the top hit (E = 10−4). Conversely, BLASTing the human proteome with CG10109 identifies hPRAS40 as a top hit, establishing an orthology relationship between hPRAS40 and CG10109, in agreement with previous reports (Sancak et al., 2007Sancak Y. Thoreen C.C. Peterson T.R. Lindquist R.A. Kang S.A. Spooner E. Carr S.A. Sabatini D.M. PRAS40 is an insulin-regulated inhibitor of the mTORC1 protein kinase.Mol. Cell. 2007; 25: 903-915Abstract Full Text Full Text PDF PubMed Scopus (994) Google Scholar). The CG10109 coding sequence was previously associated with a mutant phenotype called Lobe (Chern and Choi, 2002Chern J.J. Choi K.W. Lobe mediates Notch signaling to control domain-specific growth in the Drosophila eye disc.Development. 2002; 129: 4005-4013Crossref PubMed Google Scholar, Wang and Huang, 2009Wang Y.H. Huang M.L. Reduction of Lobe leads to TORC1 hypoactivation that induces ectopic Jak/STAT signaling to impair Drosophila eye development.Mech. Dev. 2009; 126: 781-790Crossref PubMed Scopus (23) Google Scholar). Lobe alleles cause preferential loss of the ventral eye domain due to aberrant Notch and JAK/STAT signaling (Chern and Choi, 2002Chern J.J. Choi K.W. Lobe mediates Notch signaling to control domain-specific growth in the Drosophila eye disc.Development. 2002; 129: 4005-4013Crossref PubMed Google Scholar). Although the Lobe alleles were mapped to the 51A2-B1 genomic region, which also contains CG10109, to our knowledge they were not molecularly mapped to the CG10109 gene. Although Lobe loss-of-function alleles such as Lrev6-3 are lethal (Chern and Choi, 2002Chern J.J. Choi K.W. Lobe mediates Notch signaling to control domain-specific growth in the Drosophila eye disc.Development. 2002; 129: 4005-4013Crossref PubMed Google Scholar), we found they are rescued to viability without any obvious phenotype when put in trans to a deficiency uncovering the CG10109 locus, Df(2R)ED2354, suggesting Lobe does not map genetically to CG10109. Furthermore, we tried to rescue viability of the Lrev6-3 allele using a UAS-CG10109 transgene but were unable to do so. Because these results raise the possibility that Lobe might not correspond to CG10109, we undertook a de novo analysis of CG10109 function, which we rename here dPRAS40. We first tested whether dPRAS40 has the biochemical characteristics previously described for human PRAS40—that it binds Raptor and is phosphorylated on T246 in response to insulin stimulation. A protein alignment of dPRAS40 and hPRAS40 reveals that both the Akt phosphorylation motif containing Thr246 and the Raptor binding motif (TOS) are conserved (RPRLRS and FDLED, respectively). We expressed HA-tagged dPRAS40, immunoprecipitated (IP) it from Kc cells, and tested its phosphorylation status using a “phospho Akt substrate” antibody recognizing the phosphorylated R-x-R-x-x-p(S/T) motif. This revealed that insulin stimulation causes a marked induction in PRAS40 phosphorylation (Figure 1A , lanes 3 and 4). This phosphorylation is abrogated if Ser558, the equivalent site to Thr246 in hPRAS40, is mutated to alanine (Figure 1A, lanes 5 and 6), indicating that the phospho-signal is specific for S558. We next tested whether dPRAS40 can bind dRaptor by co-IP in S2 cells, and this was indeed the case (Figure 1B). We next studied the physiological consequences of dPRAS40 overexpression. Overexpression of PRAS40 specifically in the posterior half of the fly wing using engrailed-GAL4 caused a marked reduction in size of the posterior compartment (Figure 1C). This reduced tissue size was associated with reduced cell size (Figure 1D) and no defects in patterning of the veins or of the wing margin. The reduced size of the posterior compartment could not be rescued by concomitant expression of the apoptosis inhibitor p35 (Figure 1E), indicating it is not due to apoptosis. In sum, the observed phenotypes of PRAS40 overexpression are consistent with an effect on TORC1, which regulates tissue size mainly via regulation of cell size. Consistent with this, overexpression of Rheb, an upstream activator of TORC1, was able to partially rescue the undergrowth caused by PRAS40 overexpression (Figure 1F). These gain-of-function effects were not specific for the wing, as overexpression in the eye caused reduced eye size (see Figure S1A available online) and ubiquitous overexpression with actin-GAL4 caused reduced size of the entire animal and pupal lethality (Figure S1B). To study the function of endogenous PRAS40 in Drosophila, we generated a CG10109 loss-of-function allele by homologous recombination-mediated gene knockout (PRAS40KO, Figure 2A ). We knocked-out 500 bp of coding sequence from the second exon, replacing it with the mini white gene, causing the remainder of the gene to be out of frame, yielding a predicted null allele. Indeed, PRAS40− animals had no detectable PRAS40 protein (Figure S2A), and their phenotypes were not exacerbated by placing the mutation in trans to a deficiency uncovering the locus (data not shown). We backcrossed female flies harboring the PRAS40KO mutation to w1118 flies for five generations, obtaining two stocks with similar genetic backgrounds but differing by presence or absence of the PRAS40 knockout. The resulting stocks were used for all experiments described here, and we refer to them as “PRAS40 mutant” flies, and the w1118 stock as “controls.” Surprisingly, PRAS40 mutant flies are viable to adulthood, fertile, and have no obvious eye patterning defects (Figure 2B). This was surprising because mutations in other known regulators of TOR such as Tsc1, Tsc2, or Rheb, are all lethal. Also contrary to our expectation, PRAS40 knockouts are normal in size. The weight of both male and female flies is indistinguishable to those of controls (Figure 2C). Quantification of wing and cell size in PRAS40 mutants also revealed no difference compared to controls (Figure 2D). This was unexpected from the proposed role of PRAS40 as a TOR regulator, and given that PRAS40 overexpression has clear effects on tissue size (above). The pupation rate of PRAS40− flies also did not differ significantly from that of controls (Figure 2E), indicating that PRAS40 mutants have a normal rate of growth. One possible explanation for the lack of a size effect in PRAS40− animals would be that PRAS40 and the Tsc1/Tsc2 complex form redundant mechanisms connecting IIS to TORC1. To test this, we generated flies in which both Tsc1 and Tsc2 were replaced with mutant versions that could not be phosphorylated by Akt (Dong and Pan, 2004Dong J. Pan D. Tsc2 is not a critical target of Akt during normal Drosophila development.Genes Dev. 2004; 18: 2479-2484Crossref PubMed Scopus (93) Google Scholar, Schleich and Teleman, 2009Schleich S. Teleman A.A. Akt phosphorylates both Tsc1 and Tsc2 in Drosophila, but neither phosphorylation is required for normal animal growth.PLoS ONE. 2009; 4: e6305Crossref PubMed Scopus (30) Google Scholar), simultaneously harboring the PRAS40− mutation (“PRAS40−, Tsc1(A), Tsc2(4A)” flies). These flies were also viable and normal in size (Figure S2B), and quantification of their wing size revealed no differences compared to controls (Figure 2F). Because manipulation of TORC1 activity during Drosophila development causes clear and dramatic effects on final organismal size, these data suggested that removal of PRAS40 does not reduce TORC1 activity during development. To test this, we assayed phosphorylation levels of S6K on Thr398, an established readout for TORC1 activity, in third instar larvae by western blot analysis (Figure 2G). Consistent with a lack of size phenotypes in PRAS40 knockouts, we could detect no reduction in phospho-S6K levels in the mutant larvae (Figure 2G). In sum, these data suggest PRAS40 does not regulate TORC1 activity in Drosophila during growth of the animal. We next tested whether TORC1 activity is aberrant in PRAS40− adults. Contrary to the situation in larvae, phosphorylation of the two canonical TORC1 targets 4E-BP and S6K was mildly elevated in PRAS40− adults compared to controls (Figure 3A ). Interestingly, the increase in TORC1 activity appeared to be of larger magnitude in females compared to males (Figure 3A, top). One difference between female and male flies is that a substantial fraction of the female body is comprised of ovaries. We therefore tested whether TORC1 activity is elevated in the ovaries of PRAS40 knockouts. We separated ovaries from the rest of the female body and performed western blot analysis on these two fractions. Surprisingly, phospho-S6K levels were dramatically increased in ovaries of PRAS40 mutants (Figure 3B, lanes 1 and 2), whereas there was no detectable difference in phospho-S6K levels in the remainder of the female body compared to controls (Figure 3B, lanes 5 and 6). This unexpected result indicates that the observable difference in TORC1 activity levels in adult females derives mainly from elevated TORC1 activity in ovaries. Furthermore, it indicates that endogenous PRAS40 represses TORC1 in ovaries but not in the rest of the female body. In cell culture, PRAS40 links IIS activation to TORC1 activation (Nascimento et al., 2010Nascimento E.B. Snel M. Guigas B. van der Zon G.C. Kriek J. Maassen J.A. Jazet I.M. Diamant M. Ouwens D.M. Phosphorylation of PRAS40 on Thr246 by PKB/AKT facilitates efficient phosphorylation of Ser183 by mTORC1.Cell. Signal. 2010; 22: 961-967Crossref PubMed Scopus (64) Google Scholar, Sancak et al., 2007Sancak Y. Thoreen C.C. Peterson T.R. Lindquist R.A. Kang S.A. Spooner E. Carr S.A. Sabatini D.M. PRAS40 is an insulin-regulated inhibitor of the mTORC1 protein kinase.Mol. Cell. 2007; 25: 903-915Abstract Full Text Full Text PDF PubMed Scopus (994) Google Scholar, Vander Haar et al., 2007Vander Haar E. Lee S.I. Bandhakavi S. Griffin T.J. Kim D.H. Insulin signalling to mTOR mediated by the Akt/PKB substrate PRAS40.Nat. Cell Biol. 2007; 9: 316-323Crossref PubMed Scopus (927) Google Scholar). We tested whether this is also the case in vivo in the fly, in the adult ova" @default.
• W2080647545 created "2016-06-24" @default.
• W2080647545 creator A5031106458 @default.
• W2080647545 creator A5042507409 @default.
• W2080647545 creator A5052778454 @default.
• W2080647545 date "2012-01-01" @default.
• W2080647545 modified "2023-09-26" @default.
• W2080647545 title "Tissue-Specific Coupling between Insulin/IGF and TORC1 Signaling via PRAS40 in Drosophila" @default.
• W2080647545 cites W1546970785 @default.
• W2080647545 cites W1965965763 @default.
• W2080647545 cites W1970212288 @default.
• W2080647545 cites W1973769866 @default.
• W2080647545 cites W1975861025 @default.
• W2080647545 cites W1977893138 @default.
• W2080647545 cites W1978625554 @default.
• W2080647545 cites W1979612739 @default.
• W2080647545 cites W1983880033 @default.
• W2080647545 cites W1984494721 @default.
• W2080647545 cites W2005066107 @default.
• W2080647545 cites W2007636825 @default.
• W2080647545 cites W2009971361 @default.
• W2080647545 cites W2011239505 @default.
• W2080647545 cites W2011282245 @default.
• W2080647545 cites W2015651900 @default.
• W2080647545 cites W2017018900 @default.
• W2080647545 cites W2017392540 @default.
• W2080647545 cites W2017949036 @default.
• W2080647545 cites W2022751526 @default.
• W2080647545 cites W2023834045 @default.
• W2080647545 cites W2029577022 @default.
• W2080647545 cites W2046660052 @default.
• W2080647545 cites W2055248589 @default.
• W2080647545 cites W2057697250 @default.
• W2080647545 cites W2061893502 @default.
• W2080647545 cites W2062767343 @default.
• W2080647545 cites W2062856997 @default.
• W2080647545 cites W2065978566 @default.
• W2080647545 cites W2066659565 @default.
• W2080647545 cites W2068605060 @default.
• W2080647545 cites W2069290496 @default.
• W2080647545 cites W2071203725 @default.
• W2080647545 cites W2072352903 @default.
• W2080647545 cites W2076551678 @default.
• W2080647545 cites W2076684078 @default.
• W2080647545 cites W2080094749 @default.
• W2080647545 cites W2080100938 @default.
• W2080647545 cites W2080483991 @default.
• W2080647545 cites W2083850113 @default.
• W2080647545 cites W2094524167 @default.
• W2080647545 cites W2094941511 @default.
• W2080647545 cites W2097000222 @default.
• W2080647545 cites W2103180230 @default.
• W2080647545 cites W2106097347 @default.
• W2080647545 cites W2113288593 @default.
• W2080647545 cites W2130327741 @default.
• W2080647545 cites W2131844826 @default.
• W2080647545 cites W2136676465 @default.
• W2080647545 cites W2137712918 @default.
• W2080647545 cites W2149576862 @default.
• W2080647545 cites W2152170899 @default.
• W2080647545 cites W2153963805 @default.
• W2080647545 cites W2159540848 @default.
• W2080647545 cites W2160082612 @default.
• W2080647545 cites W2160479695 @default.
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