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• W2061893502 abstract "The insulin/TOR signaling pathway plays a crucial role in animal homeostasis, sensing nutrient status to regulate organismal growth and metabolism. We identify here the Drosophila B′ regulatory subunit of PP2A (PP2A-B′) as a novel, conserved component of the insulin pathway that specifically targets the PP2A holoenzyme to dephosphorylate S6K. PP2A-B′ knockout flies have elevated S6K phosphorylation and exhibit phenotypes typical of elevated insulin signaling such as reduced total body triglycerides and reduced longevity. We show that PP2A-B′ interacts with S6K both physically and genetically. The human homolog of PP2A-B′, PPP2R5C, also counteracts S6K1 phosphorylation, indicating a conserved mechanism in mammals. Since S6K affects development of cancer and metabolic disease, our data identify PPP2R5C as a novel factor of potential medical relevance. The insulin/TOR signaling pathway plays a crucial role in animal homeostasis, sensing nutrient status to regulate organismal growth and metabolism. We identify here the Drosophila B′ regulatory subunit of PP2A (PP2A-B′) as a novel, conserved component of the insulin pathway that specifically targets the PP2A holoenzyme to dephosphorylate S6K. PP2A-B′ knockout flies have elevated S6K phosphorylation and exhibit phenotypes typical of elevated insulin signaling such as reduced total body triglycerides and reduced longevity. We show that PP2A-B′ interacts with S6K both physically and genetically. The human homolog of PP2A-B′, PPP2R5C, also counteracts S6K1 phosphorylation, indicating a conserved mechanism in mammals. Since S6K affects development of cancer and metabolic disease, our data identify PPP2R5C as a novel factor of potential medical relevance. The B′ subunit of protein phosphatase 2A (PP2A-B′) targets the holoenzyme to S6K PP2A-B′ knockout flies have elevated S6K phosphorylation PP2A-B′ mutants are lean and short-lived phenotypes of elevated S6K activity PP2A-B′ function is conserved in human cells Insulin signaling is an evolutionarily conserved pathway crucial for the proper regulation of growth, metabolism, and longevity in animals (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 (103) Google Scholar, Taguchi and White, 2008Taguchi A. White M.F. Insulin-like signaling, nutrient homeostasis, and life span.Annu. Rev. Physiol. 2008; 70: 191-212Crossref PubMed Scopus (240) Google Scholar). Insulin signaling is highly conserved from mammals to flies, both molecularly and in terms of the physiological processes that it regulates (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 (147) Google Scholar). Insulin signaling integrates inputs from growth factors and nutrients to regulate cellular metabolic pathways so that the proper organismal balance of nutrient/energy expenditure versus storage is achieved. Dysregulation of insulin signaling in humans contributes to disease, as hyperactivation of the pathway is observed in almost all cancers (Hafen, 2004Hafen E. Cancer, type 2 diabetes, and ageing: news from flies and worms.Swiss Med. Wkly. 2004; 134: 711-719PubMed Google Scholar), and reduced activation is associated with diabetes and obesity (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 (103) Google Scholar). (Although there are multiple connections between insulin and TOR signaling, the significance of these connections is currently an issue of debate. We use here the term “insulin signaling” in a broad sense, denoting insulin signaling and TOR signaling combined.) In order to maintain organismal energy homeostasis, insulin signaling needs to respond to changing nutrient conditions, both rapidly increasing and decreasing its state of activation as necessary. The insulin pathway contains a large number of kinases that activate each other through a relay of phosphorylation events upon binding of insulin to its receptor. This gives a molecular explanation for activation of the pathway; however, the fact that the pathway can also be inactivated suggests phosphatases exist to counteract the various kinases. S6K is an important effector of the insulin/TOR pathway inducing ribosome biogenesis, enhancing translation of mRNAs containing structured 5′UTRs and inhibiting apoptosis (Hay and Sonenberg, 2004Hay N. Sonenberg N. Upstream and downstream of mTOR.Genes Dev. 2004; 18: 1926-1945Crossref PubMed Scopus (3245) Google Scholar). As a consequence, S6K promotes cell growth and proliferation, contributing to the development of cancer (Jastrzebski et al., 2007Jastrzebski K. Hannan K.M. Tchoubrieva E.B. Hannan R.D. Pearson R.B. Coordinate regulation of ribosome biogenesis and function by the ribosomal protein S6 kinase, a key mediator of mTOR function.Growth Factors. 2007; 25: 209-226Crossref PubMed Scopus (173) Google Scholar), and plays an important role in the regulation of organismal metabolism (Um et al., 2004Um S.H. Frigerio F. Watanabe M. Picard F. Joaquin M. Sticker M. Fumagalli S. Allegrini P.R. Kozma S.C. Auwerx J. Thomas G. Absence of S6K1 protects against age- and diet-induced obesity while enhancing insulin sensitivity.Nature. 2004; 431: 200-205Crossref PubMed Scopus (1282) Google Scholar). Thus, the precise regulation of S6K activity is critical for normal animal growth and homeostasis. Activation of S6K has been extensively studied and occurs via phosphorylation by TOR and PDK1, thus integrating nutrient availability and growth factor signaling (Jastrzebski et al., 2007Jastrzebski K. Hannan K.M. Tchoubrieva E.B. Hannan R.D. Pearson R.B. Coordinate regulation of ribosome biogenesis and function by the ribosomal protein S6 kinase, a key mediator of mTOR function.Growth Factors. 2007; 25: 209-226Crossref PubMed Scopus (173) Google Scholar). Dephosphorylation of S6K is less well understood. Inhibition of TOR causes S6K to become dephosphorylated within minutes, despite S6K protein levels remaining unchanged (Bielinski and Mumby, 2007Bielinski V.A. Mumby M.C. Functional analysis of the PP2A subfamily of protein phosphatases in regulating Drosophila S6 kinase.Exp. Cell Res. 2007; 313: 3117-3126Crossref PubMed Scopus (31) Google Scholar), suggesting that a phosphatase dephosphorylates S6K. We identify here PP2A-B′ (CG7913), encoding a regulatory B′ subunit of PP2A, as a specific negative regulator of S6K phosphorylation. PP2A-B′ knockout flies have elevated levels of S6K phosphorylation. They display significant metabolic aberrations, which are rescued by reducing S6K gene dosage. We show that PP2A-B′ mutants lose robustness to nutritional variation, highlighting an important role of PP2A-B′ in the maintenance of organismal homeostasis. Knockdown of the human PP2A-B′ homolog PPP2R5C in cells also affects S6K phosphorylation, implying functional conservation of this regulatory subunit in humans. Although the catalytic subunit of PP2A has been implicated in the dephosphorylation of S6K in cell culture (Ballou et al., 1988Ballou L.M. Jeno P. Thomas G. Protein phosphatase 2A inactivates the mitogen-stimulated S6 kinase from Swiss mouse 3T3 cells.J. Biol. Chem. 1988; 263: 1188-1194Abstract Full Text PDF PubMed Google Scholar, Bielinski and Mumby, 2007Bielinski V.A. Mumby M.C. Functional analysis of the PP2A subfamily of protein phosphatases in regulating Drosophila S6 kinase.Exp. Cell Res. 2007; 313: 3117-3126Crossref PubMed Scopus (31) Google Scholar), its function is highly pleiotropic, accounting for a large fraction of phosphatase activity in eukaryotic cells (Millward et al., 1999Millward T.A. Zolnierowicz S. Hemmings B.A. Regulation of protein kinase cascades by protein phosphatase 2A.Trends Biochem. Sci. 1999; 24: 186-191Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar). The discovery of a regulatory subunit targeting PP2A activity specifically to S6K opens the possibility of using it for diagnostic or therapeutic purposes. To study the function of PP2A-B′, we created PP2A-B′ knockout flies by replacing the first exon common to all splice variants with the miniwhite gene by homologous recombination (Figure 1A ) (Gong and Golic, 2003Gong W.J. Golic K.G. Ends-out, or replacement, gene targeting in Drosophila.Proc. Natl. Acad. Sci. USA. 2003; 100: 2556-2561Crossref PubMed Scopus (298) Google Scholar). This leads to premature disruption of the coding sequence and a shift in the reading frame of remaining exons, generating a null mutation. To clean the genetic background of knockout flies, females heterozygous for the knockout mutation were backcrossed to w1118 flies, an inbred but not isogenic laboratory stock. After four generations of backcrossing, stocks were established from single males, yielding two independent knockout lines, KO1 and KO2. KO1 flies are viable to adulthood (Figure 1B), normal in developmental rate (Figure 1C), and almost normal in size (Figure 1D, see Figures S1A and S1B online). To test whether PP2A-B′ affects fly metabolism, we measured total body triglycerides, normalized to total body protein, of KO1 and w1118 control flies grown under controlled nutrient and density conditions. PP2A-B′ mutants are strikingly lean, containing 28% of the triglyceride levels of w1118 controls (Figure 1E). This leanness is specific for loss of PP2A-B′, as it is significantly rescued by expression of PP2A-B′ from a transgene (Figure 1E). PP2A-B′ mutants also contain roughly half as much total body glycogen as control flies (Figure S1C). We also asked whether PP2A-B′ affects longevity and found that KO1 flies have significantly reduced life span compared to controls (Figure 1F). We studied the effect of PP2A-B′ overexpression by generating UAS-PP2A-B′ transgenic lines and crossing them to GAL4 drivers. Overexpression of PP2A-B′ with en-GAL4 caused lethality with most UAS lines (data not shown). One weak UAS line, however, gave viable adults with wing posterior compartments—where en-GAL4 is expressed—that were normally patterned but significantly reduced in size by 12% (Figure 1G). Overexpression of PP2A-B′ in the head and eye using eyeless-Gal4 also caused a reduction in tissue size, ranging in strength from mild to strong (Figures 1H and 1H′). In summary, PP2A-B′ overexpression causes a reduction in tissue size or lethality. The phenotypes described above suggested PP2A-B′ might play a repressive role in insulin signaling. Flies with elevated insulin signaling in the whole body by mutation of PTEN have reduced lipid levels (Oldham et al., 2002Oldham S. Stocker H. Laffargue M. Wittwer F. Wymann M. Hafen E. The Drosophila insulin/IGF receptor controls growth and size by modulating PtdInsP(3) levels.Development. 2002; 129: 4103-4109PubMed Google Scholar), similar to PP2A-B′ mutants. Insulin signaling also regulates longevity (Taguchi and White, 2008Taguchi A. White M.F. Insulin-like signaling, nutrient homeostasis, and life span.Annu. Rev. Physiol. 2008; 70: 191-212Crossref PubMed Scopus (240) Google Scholar), and overactivation of S6K leads to reduced life span (Kapahi et al., 2004Kapahi P. Zid B.M. Harper T. Koslover D. Sapin V. Benzer S. Regulation of lifespan in Drosophila by modulation of genes in the TOR signaling pathway.Curr. Biol. 2004; 14: 885-890Abstract Full Text Full Text PDF PubMed Scopus (946) Google Scholar), similar to PP2A-B′ mutants. Finally, inhibition of insulin signaling leads to reduced tissue growth (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 (103) Google Scholar), analogous to what we observe with PP2A-B′ overexpression. Therefore, we asked whether PP2A-B′ affects insulin signaling by assaying phosphorylation of S6K, a direct target of TOR Complex 1 (TOR-C1) (Hay and Sonenberg, 2004Hay N. Sonenberg N. Upstream and downstream of mTOR.Genes Dev. 2004; 18: 1926-1945Crossref PubMed Scopus (3245) Google Scholar). Immunoblotting of protein extracts from KO1 and control w1118 animals revealed that KO1 flies have significantly elevated levels of phosphorylated S6K (Figure 2A , lanes 1 and 2). This effect was reversed by ubiquitous expression of PP2A-B′ from a transgene in the KO1 mutant background (Figure 2A, lane 3). Total levels of dERK are shown as loading control, since work in our laboratory with other genes of the insulin pathway has shown that tubulin levels can vary when insulin signaling is modulated (data not shown). We asked at what level of the insulin pathway PP2A-B′ might be functioning by detecting the state of activation of other components of the pathway. We assayed phosphorylation of Akt on the site for which antibodies are available (S505) and found that it was unaffected in KO1 animals compared to controls (Figure 2B). We also assayed activity of the transcription factor FOXO, which is regulated via phosphorylation by Akt and is a sensitive readout for Akt activation (Puig et al., 2003Puig O. Marr M.T. Ruhf M.L. Tjian R. Control of cell number by Drosophila FOXO: downstream and feedback regulation of the insulin receptor pathway.Genes Dev. 2003; 17: 2006-2020Crossref PubMed Scopus (443) Google Scholar). Quantitative RT-PCR on RNA extracted from KO1 and control w1118 animals revealed no difference in mRNA levels of a well-characterized FOXO target, 4E-BP (Figure 2C) (Teleman et al., 2005Teleman A.A. Chen Y.W. Cohen S.M. 4E-BP functions as a metabolic brake used under stress conditions but not during normal growth.Genes Dev. 2005; 19: 1844-1848Crossref PubMed Scopus (190) Google Scholar). These data indicate that activation of the pathway at the level of Akt is not increased in PP2A-B′ mutants. If anything, we observed slightly reduced Akt phosphorylation on dSer505/mSer473 in PP2A-B′ loss-of-function conditions in vivo and in cell culture (Figure 2D and Figure 4E, lanes 3 and 4). The only kinase known to phosphorylate S6K on Thr398 in Drosophila is TOR-C1. Therefore, increased phosphorylation of S6K in PP2A-B′ knockout animals could be due to increased TOR-C1 activity. To test this, we assayed phosphorylation of another direct target of TOR-C1, 4E-BP. 4E-BP phosphorylation was not elevated in PP2A-B′ knockouts (Figure 2B), indicating that elevated levels of S6K phosphorylation in KO1 animals are not due to increased TOR-C1 activity. Together, these results suggest PP2A-B′ acts specifically at the level of S6K. Since the insulin pathway is highly conserved from flies to humans, we asked whether the human homologs of dPP2A-B′ also affect S6K phosphorylation. Alignment of human and Drosophila PP2A-B′ regulatory subunits reveals that dPP2A-B′ is most related to PPP2R5C and PPP2R5D (Figure S2A). Therefore, we tested both PPP2R5C and PPP2R5D. While siRNA-mediated knockdown of PPP2R5D had no consistent effect on S6K1 phosphorylation, knockdown of PPP2R5C with two independent siRNAs increased the amount of phosphorylated S6K protein in HeLa cells (Figures 2D and 2E and Figure S2B). Total levels of S6K1 protein were also elevated, perhaps as a secondary consequence of S6K1 stabilization (Wang et al., 2008Wang M.L. Panasyuk G. Gwalter J. Nemazanyy I. Fenton T. Filonenko V. Gout I. Regulation of ribosomal protein S6 kinases by ubiquitination.Biochem. Biophys. Res. Commun. 2008; 369: 382-387Crossref PubMed Scopus (29) Google Scholar) (Figures 2D and 2E). As in Drosophila, phosphorylation of 4E-BP and Akt were not increased (Figure 2D), indicating a specific effect on S6K1. The specific effect of PP2A-B′ on S6K raised the possibility that PP2A-B′ might be acting directly on S6K. The regulatory subunits of PP2A such as PP2A-B′ are thought to provide specificity to the PP2A holoenzyme by recruiting it to specific substrates (Mumby, 2007Mumby M. PP2A: unveiling a reluctant tumor suppressor.Cell. 2007; 130: 21-24Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). To test this, we asked whether PP2A-B′ and S6K interact physically. We expressed FLAG-tagged PP2A-B′ and HA-tagged S6K in S2 cells and precipitated PP2A-B′ with anti-FLAG antibody. S6K could be detected in the FLAG immunoprecipitate (IP), suggesting PP2A-B′ and S6K interact physically (Figure 3A ). S6K could not be detected in the FLAG IP in the absence of FLAG-PP2A-B′, demonstrating specificity of the pull-down (Figure 3A, lane 1). The S6K protein that coimmunoprecipitated with PP2A-B′ was found to be dephosphorylated (Figure S3A), consistent with the PP2A holoenzyme having acted upon it. In line with PP2A-B′ and S6K interacting physically, the two genes are coexpressed ubiquitously in fly tissues (Figures S3B and S3C). To test whether the PP2A-B′-containing holoenzyme can dephosphorylate S6K directly in vitro, we expressed HIS-tagged S6K in Drosophila S2 cells, purified it on a nickel column, and eluted it with imidazole, yielding soluble, phosphorylated S6K that could be used as substrate for in vitro dephosphorylation assays. PP2A-B′ containing PP2A holoenzyme was purified by immunoprecipitation with anti-FLAG antibody from flies expressing FLAG-tagged PP2A-B′. As a control, we performed in parallel an anti-FLAG immunoprecipitation from flies not expressing FLAG-PP2A-B′. Whereas incubation of phospho-S6K with IP from control animals caused no dephosphorylation of S6K (Figure 3B, lanes 1 and 2), incubation with IP from FLAG-PP2A-B′-expressing animals caused strong and obvious dephosphorylation of S6K (Figure 3B, lane 3). We tested whether PP2A-B′ and S6K interact genetically in vivo. If the phenotypes of PP2A-B′ knockouts are due to elevated S6K activity, they should be rescued by removing one copy of S6K (KO1, S6K+/−). Indeed, triglyceride and glycogen levels of KO1, S6K+/− flies were significantly rescued compared to KO1 flies (Figure 3C and Figure S1C). Removing one copy of S6K also partially rescued the decreased life span of PP2A-B′ knockouts (Figure 3D). In summary, these results indicate that PP2A-B′ and S6K interact physically and genetically. While studying the PP2A-B′ knockout flies, we realized that one of the PP2A-B′ knockout lines, KO2, is semilethal when grown on standard laboratory food, which is rich in carbohydrates. Eighty-eight percent of KO2 animals die as pharate adults (Figure 4A ). Both KO1 and KO2 lines were generated by backcrossing knockout flies for four generations to the w1118 stock, which is an inbred but not isogenic reference stock in the lab. The fact that KO2 is semilethal and KO1 is viable suggests the two lines differ in their genetic backgrounds. Indeed, the semilethality of KO2 flies could be completely rescued by changing the second chromosomes in the KO2 stock (Figure S4). This indicates the semilethality is caused by genetic interaction between the PP2A-B′ mutation on the third chromosome and genetic elements on the second chromosome. It is becoming increasingly clear that genetic background plays an important role in modulation of complex phenotypes and in development of diseases such as diabetes or cancer (Adamo and Tesson, 2008Adamo K.B. Tesson F. Gene-environment interaction and the metabolic syndrome.Novartis Found. Symp. 2008; 293: 103-119, discussion 119–127Crossref PubMed Scopus (3) Google Scholar). This is well known in mice, for instance, where mutation of leptin (Lepob) in the C57BL/6J background causes transient hyperglycemia, subsiding 14–16 weeks of age, whereas the same mutation in the C57BLKS/J background causes severe diabetes with regression of islets and early death (Davis et al., 2005Davis R.C. Schadt E.E. Cervino A.C. Peterfy M. Lusis A.J. Ultrafine mapping of SNPs from mouse strains C57BL/6J, DBA/2J, and C57BLKS/J for loci contributing to diabetes and atherosclerosis susceptibility.Diabetes. 2005; 54: 1191-1199Crossref PubMed Scopus (30) Google Scholar). Since one genetic background is not more “correct” than another, we decided to also study the KO2 flies. We first tested whether KO2 flies have the same metabolic and biochemical phenotypes as KO1 flies. KO2 animals, like KO1 animals, are lean (Figure 4B) and have elevated levels of S6K phosphorylation (Figure 4C). These results show that KO2 animals have the same metabolic and biochemical phenotypes as KO1 animals, except that they exhibit poor survival, indicating a more aggravated phenotype. If the poor survival rate of KO2 flies is due to a combined effect of elevated S6K activation and other genetic background effects, reducing S6K phosphorylation by reducing insulin signaling should rescue them. We tested this by rearing animals on normal food (100% food) or on food diluted 1:5 with PBS/agarose to make it less rich (20% food). Animals growing on 20% food take several additional days to pupate compared to animals on 100% food, reflecting the lower richness of 20% food (data not shown), and accordingly have reduced expression of nutrient-responsive insulin-like peptides 2 and 5 (Figure 4D) (Ikeya et al., 2002Ikeya T. Galic M. Belawat P. Nairz K. Hafen E. Nutrient-dependent expression of insulin-like peptides from neuroendocrine cells in the CNS contributes to growth regulation in Drosophila.Curr. Biol. 2002; 12: 1293-1300Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar, Teleman et al., 2008Teleman A.A. Hietakangas V. Sayadian A.C. Cohen S.M. Nutritional control of protein biosynthetic capacity by insulin via Myc in Drosophila.Cell Metab. 2008; 7: 21-32Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). Consistent with this, animals growing on 20% food have reduced intracellular insulin signaling compared to animals on 100% food, assayed by phosphorylation of Akt and S6K (Figure 4E). On 20% food, levels of phospho-S6K in KO2 animals are still higher than in control flies (Figure 4E, lanes 3 and 4), consistent with phospho-S6K levels reflecting a balance of dephosphorylation by the PP2A/PP2A-B′ holoenzyme and nutrient-responsive phosphorylation by TOR. We then determined the survival rate of control and KO2 animals reared on 100% and 20% food. Intriguingly, on 20% food the survival of KO2 flies was significantly improved (Figure 4F). These results indicate that, unlike control flies, KO2 flies are sensitive to the nutritional overload of our rich laboratory food. This is particularly striking since 20% food is normally a stress condition for flies but results in improved viability here. One reason phosphatases have been difficult to study is that they often act pleiotropically. Blocking their activity usually leads to multiple concurrent molecular effects and often lethality. The phosphatase PP2A is a case in point; it accounts for a large fraction of phosphatase activity in eukaryotic cells (Millward et al., 1999Millward T.A. Zolnierowicz S. Hemmings B.A. Regulation of protein kinase cascades by protein phosphatase 2A.Trends Biochem. Sci. 1999; 24: 186-191Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar), and mutation of the PP2A catalytic subunit leads to lethality (Gotz et al., 1998Gotz J. Probst A. Ehler E. Hemmings B. Kues W. Delayed embryonic lethality in mice lacking protein phosphatase 2A catalytic subunit Calpha.Proc. Natl. Acad. Sci. USA. 1998; 95: 12370-12375Crossref PubMed Scopus (175) Google Scholar). The PP2A active core dimer associates with a large number of possible B subunits that give it specificity by mediating interaction with specific targets (Mumby, 2007Mumby M. PP2A: unveiling a reluctant tumor suppressor.Cell. 2007; 130: 21-24Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). Therefore, studies of the individual regulatory B subunits are critical for teasing apart the various functions of PP2A. We discover here the regulatory subunit of PP2A, PP2A-B′/PPP2R5C, as a novel component of the insulin pathway, which specifically directs PP2A to dephosphorylate S6K (Figure 2). The phenotypes we observe in PP2A-B′ knockout flies—reduced longevity and reduced nutrient stores—are insulin gain-of-function phenotypes in the fly (Kapahi et al., 2004Kapahi P. Zid B.M. Harper T. Koslover D. Sapin V. Benzer S. Regulation of lifespan in Drosophila by modulation of genes in the TOR signaling pathway.Curr. Biol. 2004; 14: 885-890Abstract Full Text Full Text PDF PubMed Scopus (946) Google Scholar, Oldham et al., 2002Oldham S. Stocker H. Laffargue M. Wittwer F. Wymann M. Hafen E. The Drosophila insulin/IGF receptor controls growth and size by modulating PtdInsP(3) levels.Development. 2002; 129: 4103-4109PubMed Google Scholar) and are rescued by removal of one copy of S6K (Figures 3C and 3D and Figure S1C). Recently, the other B′ regulatory subunit, widerborst, and its corresponding homolog pptr-1 in C. elegans were shown to target PP2A to Akt (Padmanabhan et al., 2009Padmanabhan S. Mukhopadhyay A. Narasimhan S.D. Tesz G. Czech M.P. Tissenbaum H.A. A PP2A regulatory subunit regulates C. elegans insulin/IGF-1 signaling by modulating AKT-1 phosphorylation.Cell. 2009; 136: 939-951Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, Vereshchagina et al., 2008Vereshchagina N. Ramel M.C. Bitoun E. Wilson C. The protein phosphatase PP2A-B′ subunit Widerborst is a negative regulator of cytoplasmic activated Akt and lipid metabolism in Drosophila.J. Cell Sci. 2008; 121: 3383-3392Crossref PubMed Scopus (44) Google Scholar), indicating that both B′ subunits are dedicated to keeping activation of the insulin signaling pathway under control. S6K also regulates tissue growth (Hay and Sonenberg, 2004Hay N. Sonenberg N. Upstream and downstream of mTOR.Genes Dev. 2004; 18: 1926-1945Crossref PubMed Scopus (3245) Google Scholar). Consistent with this, overexpression of PP2A-B′ both in the wing and in the eye leads to a reduction in tissue size (Figures 1G and 1H), phenocopying S6K loss of function (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 (253) Google Scholar). Unexpectedly, PP2A-B′ knockout flies are not larger than controls. One reason may be that S6K gain of function in the fly gives surprisingly mild overgrowth (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 (149) Google Scholar), suggesting S6K activity may not be limiting for fly growth under normal circumstances. Another reason may be that larval and imaginal tissues compete for nutrients during development and therefore the relative balance of PP2A-B′ activity in various tissues might be important. Future work may shed more light on this issue. Flies mutant for PP2A-B′, called well-rounded, were recently reported with mild defects in the number and size of boutons at neuromuscular junctions (Viquez et al., 2006Viquez N.M. Li C.R. Wairkar Y.P. DiAntonio A. The B′ protein phosphatase 2A regulatory subunit well-rounded regulates synaptic growth and cytoskeletal stability at the Drosophila neuromuscular junction.J. Neurosci. 2006; 26: 9293-9303Crossref PubMed Scopus (35) Google Scholar). TOR and S6K play a variety of roles in neuronal growth, differentiation, and dentritic growth and arborization. Whether repression of S6K phosphorylation by PP2A-B′ plays a role in this context remains to be investigated. Signaling through the insulin pathway is both activated and inactivated in a tightly controlled and rapid fashion, assuring adequate and timely responses to varying nutritional conditions. We show here that phosphatases, and in particular PP2A-B′, play a key role in maintaining this balance, as PP2A-B′ mutants can become sensitive to nutritional status. The viability of KO2 flies varied dramatically depending on the richness of their food (Figure 4F), in contrast to wild-type animals, which were viable under a wide range of genetic backgrounds and nutritional conditions. Drosophila PP2A-B′ has two mammalian homologs, PPP2R5C and PPP2R5D. Knockdown of PPP2R5C resulted in elevated levels of phosphorylated S6K protein in human cells (Figures 2D and 2E), suggesting PPP2R5C is the human functional homolog of PP2A-B′. Aberrant insulin and S6K signaling have been implicated in the etiology of a number of human diseases, including diabetes, obesity, and cancer (Hafen, 2004Hafen E. Cancer, type 2 diabetes, and ageing: news from flies and worms.Swiss Med. Wkly. 2004; 134: 711-719PubMed 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 (253) Google Scholar). Therefore, it will be interesting to investigate whether PPP2R5C, as a novel regulator of S6K phosphorylation, plays a role in development of these diseases. Our data suggest dPP2A-B′ is required for the organism to respond properly to elevated nutrient conditions. Diseases such as diabetes and obesity could be considered situations in which the ability of insulin signaling to compensate for elevated nutrient supply has been exceeded. Furthermore, S6K1, as one of the principal effectors of TOR-mediated tissue growth, has been linked to a number of cancers (Mamane et al., 2006Mamane Y. Petroulakis E. LeBacquer O. Sonenberg N. mTOR, translation initiation and cancer.Oncogene. 2006; 25: 6416-6422Crossref PubMed Scopus (496) Google Scholar). This raises the possibility that PPP2R5C might also act as a tumor suppressor by inhibiting S6K1 phosphorylation. Indeed, PPP2R5C was identified in a screen for genes downregulated in malignant melanoma (Deichmann et al., 2001Deichmann M. Polychronidis M. Wacker J. Thome M. Naher H. The protein phosphatase 2A subunit Bgamma gene is identified to be differentially expressed in malignant melanomas by subtractive suppression hybridization.Melanoma Res. 2001; 11: 577-585Crossref PubMed Scopus (27) Google Scholar). Future work will shed further light on links between PPP2R5C and diseases involving insulin signaling, such as cancer and diabetes/obesity. Detailed description of plasmid construction, oligos, and metabolic analysis is found in the Supplemental Information. HA-S6K expression construct was a kind gift from Duojia Pan. hs-Gal4, en-Gal4, eye-Gal4, and S6K07084 flies were from Bloomington Stock Center. A detailed description of cell-culturing conditions, IP, and antibodies is in the Supplemental Information. Gene knockdown in HeLa cells was performed in 6-well dishes using 5 μl of Dharmafect reagent (Dharmacon) and 0.1 μM of siRNAs (Dharmacon L-009433-00-005, L-009799-00-0005) or 0.07 μM of siRNA (Ambion P/N 4390824) per 1.5 × 105 cells/well, for 3 or 2.5 days, respectively. mRNA levels of target genes were reduced by 80%, quantified by Q-PCR. We thank Stephen Cohen for comments on this manuscript. This work was supported by a Helmholtz Young Investigator grant. We apologize to the many authors whose work could not be cited due to space constraints. Download .pdf (.4 MB) Help with pdf files Document S1. Four Figures, Supplemental Experimental Procedures, and Supplemental References" @default.
• W2061893502 created "2016-06-24" @default.
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• W2061893502 date "2010-05-01" @default.
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• W2061893502 title "PP2A Regulatory Subunit PP2A-B′ Counteracts S6K Phosphorylation" @default.
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