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• W2078015812 abstract "The importance of PP2A in the regulation of Akt/PKB activity has long been recognized but the nature of the holoenzyme involved and the mechanisms controlling dephosphorylation are not yet known. We identified IEX-1, an early gene product with proliferative and survival activities, as a specific inhibitor of B56 regulatory subunit-containing PP2A. IEX-1 inhibits B56-PP2A activity by allowing the phosphorylation of B56 by ERK. This leads to sustained ERK activation. IEX-1 has no effect on PP2A containing other B family subunits. Thus, studying IEX-1 contribution to signaling should help the discovery of new pathways controlled by B56-PP2A. By using overexpression and RNA interference, we show here that IEX-1 increases Akt/PKB activity in response to various growth factors by preventing Akt dephosphorylation on both Thr308 and Ser473 residues. PP2A-B56β and γ subunits have the opposite effect and reverse IEX-1-mediated Akt activation. The effect of IEX-1 on Akt is ERK-dependent. Indeed: (i) a IEX-1 mutant deficient in ERK binding had no effect on Akt; (ii) ERK dominant-negative mutants reduced IEX-1-mediated increase in pAkt; (iii) a B56β mutant that cannot be phosphorylated in the ERK·IEX-1 complex showed an enhanced ability to compete with IEX-1. These results identify B56-containing PP2A holoenzymes as Akt phosphatases. They suggest that IEX-1 behaves as a general inhibitor of B56 activity, enabling the control of both ERK and Akt signaling downstream of ERK. The importance of PP2A in the regulation of Akt/PKB activity has long been recognized but the nature of the holoenzyme involved and the mechanisms controlling dephosphorylation are not yet known. We identified IEX-1, an early gene product with proliferative and survival activities, as a specific inhibitor of B56 regulatory subunit-containing PP2A. IEX-1 inhibits B56-PP2A activity by allowing the phosphorylation of B56 by ERK. This leads to sustained ERK activation. IEX-1 has no effect on PP2A containing other B family subunits. Thus, studying IEX-1 contribution to signaling should help the discovery of new pathways controlled by B56-PP2A. By using overexpression and RNA interference, we show here that IEX-1 increases Akt/PKB activity in response to various growth factors by preventing Akt dephosphorylation on both Thr308 and Ser473 residues. PP2A-B56β and γ subunits have the opposite effect and reverse IEX-1-mediated Akt activation. The effect of IEX-1 on Akt is ERK-dependent. Indeed: (i) a IEX-1 mutant deficient in ERK binding had no effect on Akt; (ii) ERK dominant-negative mutants reduced IEX-1-mediated increase in pAkt; (iii) a B56β mutant that cannot be phosphorylated in the ERK·IEX-1 complex showed an enhanced ability to compete with IEX-1. These results identify B56-containing PP2A holoenzymes as Akt phosphatases. They suggest that IEX-1 behaves as a general inhibitor of B56 activity, enabling the control of both ERK and Akt signaling downstream of ERK. Protein phosphorylation is central to signal transduction by growth factors and the maintenance of cellular homeostasis by controlling many cellular processes such as growth, survival, and differentiation. Deregulation of this activity is a key cause of cancer development. The net activation of a signaling pathway depends on the balance of enzymatic activities between kinases and phosphatases, suggesting that both activation and inactivation signals must be tightly controlled.Protein phosphatase 2A (PP2A) 2The abbreviations used are: PP2A, protein phosphatase 2A; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; GSK3, glycogen synthase kinase-3; IEX-1, immediate early response gene X-1; PI3K, phosphoinositide 3-kinase; TPO, thrombopoietin; WT, wild type; HA, hemagglutinin; shRNA, short hairpin RNA; GFP, green fluorescent protein; EPO, erythropoietin; CHO, Chinese hamster ovary; ER, endoplasmic reticulum; FCS, fetal calf serum; PDK1, phosphoinositide-dependent kinase-1. 2The abbreviations used are: PP2A, protein phosphatase 2A; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; GSK3, glycogen synthase kinase-3; IEX-1, immediate early response gene X-1; PI3K, phosphoinositide 3-kinase; TPO, thrombopoietin; WT, wild type; HA, hemagglutinin; shRNA, short hairpin RNA; GFP, green fluorescent protein; EPO, erythropoietin; CHO, Chinese hamster ovary; ER, endoplasmic reticulum; FCS, fetal calf serum; PDK1, phosphoinositide-dependent kinase-1. accounts for the majority of serine/threonine phosphatase activity in the cell (reviewed in Refs. 1Janssens V. Goris J. Biochem. J. 2001; 353: 417-439Crossref PubMed Scopus (1511) Google Scholar and 2Sontag E. Cell. Signal. 2001; 13: 7-16Crossref PubMed Scopus (287) Google Scholar)). The predominant form of PP2A is a heterotrimeric holoenzyme, composed of a scaffolding subunit (A), a catalytic subunit (C), and a variable regulatory subunit. The B subunits fall into three major unrelated families, B (PR55), B′ (B56/PR61), and B′′ (PR72). Each family comprises several isoforms that exhibit significant homology between them. With five different genes, generating at least 8 isoforms named B56 α, β, γ, δ, and ɛ, the B56 family is the most diverse. Many if not most of the components of signaling cascades are PP2A substrates. However, the function of the individual PP2A subunits in these pathways is only beginning to emerge. Interestingly, recent studies indicate that different PP2A holoenzymes regulate positively or negatively signaling by acting at different levels in a cascade. The best example of this type of regulation is the ERK/MAPK signaling pathway. Indeed, Adams et al. (3Adams D.G. Coffee Jr., R.L. Zhang H. Pelech S. Strack S. Wadzinski B.E. J. Biol. Chem. 2005; 280: 42644-42654Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar) showed that PP2As containing Bα and Bδ are required for activation of the MAPK kinase kinase Raf-1, whereas we demonstrated that the B56 family members shut down the signal by dephosphorylating ERK1 and ERK2 (4Letourneux C. Rocher G. Porteu F. EMBO J. 2006; 25: 727-738Crossref PubMed Scopus (158) Google Scholar). Likewise, the Wnt pathway has been shown to be differently regulated by B56-containing PP2As, which binds to axin and APC (5Seeling J.M. Miller J.R. Gil R. Moon R.T. White R. Virshup D.M. Science. 1999; 283: 2089-2091Crossref PubMed Scopus (363) Google Scholar) and also by PP2As containing the B′′ subunit acting at the level of Naked cuticle (6Creyghton M.P. Roel G. Eichhorn P.J. Hijmans E.M. Maurer I. Destree O. Bernards R. Genes Dev. 2005; 19: 376-386Crossref PubMed Scopus (61) Google Scholar). Unraveling the specific contribution of the various PP2A holoenzymes to signaling pathways, as well as the mechanisms regulating their function, is an important step to understand how kinase cascades are regulated and to find new tools to modulate their activities.The kinase Akt/PKB, a critical component of a pathway controlling growth and survival, is one of the major targets of PP2A enzymes. Akt exerts its function by phosphorylating several proteins involved in cell cycle regulation and apoptosis, including p21Cip/WAF1, BAD, the Forkhead family of transcription factors and glycogen synthase kinase-3 (GSK3) (reviewed in Ref. 7Brazil D.P. Yang Z.Z. Hemmings B.A. Trends Biochem. Sci. 2004; 29: 233-242Abstract Full Text Full Text PDF PubMed Scopus (714) Google Scholar). Activation of Akt occurs upon recruitment to the plasma membrane through binding of its plekstrin homology domain to phosphatidylinositol 3,4,5-triphosphates, which are produced by growth factor-activated phosphoinositide 3-kinase (PI3K). This allows phosphorylation of Akt at two key regulatory residues: Thr308 in the activation loop and Ser473 in a C-terminal hydrophobic motif. Phosphorylation of Thr308 is catalyzed by PDK1 (8Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar, 9Stephens L. Anderson K. Stokoe D. Erdjument-Bromage H. Painter G.F. Holmes A.B. Gaffney P.R. Reese C.B. McCormick F. Tempst P. Coadwell J. Hawkins P.T. Science. 1998; 279: 710-714Crossref PubMed Scopus (909) Google Scholar); that of Ser473 is mediated by the mTORC2 complex (10Sarbassov D.D. Guertin D.A. Ali S.M. Sabatini D.M. Science. 2005; 307: 1098-1101Crossref PubMed Scopus (5154) Google Scholar). Turn-off of Akt activity can be mediated by the tumor suppressor phosphatase tensin homologue, which dephosphorylates phosphatidylinositol 3,4,5-triphosphates. Although the importance of PP2A in the regulation of Akt activity has long been recognized (11Andjelkovic M. Jakubowicz T. Cron P. Ming X.F. Han J.W. Hemmings B.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5699-5704Crossref PubMed Scopus (426) Google Scholar, 12Resjo S. Goransson O. Harndahl L. Zolnierowicz S. Manganiello V. Degerman E. Cell. Signal. 2002; 14: 231-238Crossref PubMed Scopus (119) Google Scholar, 13Sato S. Fujita N. Tsuruo T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10832-10837Crossref PubMed Scopus (826) Google Scholar, 14Ugi S. Imamura T. Maegawa H. Egawa K. Yoshizaki T. Shi K. Obata T. Ebina Y. Kashiwagi A. Olefsky J.M. Mol. Cell. Biol. 2004; 24: 8778-8789Crossref PubMed Scopus (191) Google Scholar, 15Yamada T. Katagiri H. Asano T. Inukai K. Tsuru M. Kodama T. Kikuchi M. Oka Y. J. Biol. Chem. 2001; 276: 5339-5345Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar), the nature of the PP2A holoenzyme involved and the mechanisms controlling these dephosphorylation events have not yet been elucidated.IEX-1 (immediate early response gene X-1), also known as IER3, DIF2, or Gly96, is an ubiquitous early response gene product involved in cell proliferation and survival, which is rapidly induced by various growth factors, cytokines, chemical carcinogens, or viral infections (16Wu M.X. Apoptosis. 2003; 8: 11-18Crossref PubMed Scopus (104) Google Scholar). Conflicting data have been reported concerning the role of IEX-1 in apoptosis. Indeed, IEX-1 was found to increase apoptosis in response to UV radiation or upon serum deprivation (17Arlt A. Grobe O. Sieke A. Kruse M.L. Folsch U.R. Schmidt W.E. Schafer H. Oncogene. 2001; 20: 69-76Crossref PubMed Scopus (78) Google Scholar, 18Schilling D. Pittelkow M.R. Kumar R. Oncogene. 2001; 20: 7992-7997Crossref PubMed Scopus (61) Google Scholar) but it also plays a key role in cellular resistance to various apoptotic triggers and contributes to growth factor-mediated survival activities (19Garcia J. Ye Y. Arranz V. Letourneux C. Pezeron G. Porteu F. EMBO J. 2002; 21: 5151-5163Crossref PubMed Scopus (91) Google Scholar, 20Domachowske J.B. Bonville C.A. Mortelliti A.J. Colella C.B. Kim U. Rosenberg H.F. J. Infect. Dis. 2000; 181: 824-830Crossref PubMed Scopus (45) Google Scholar, 21Wu M.X. Ao Z. Prasad K.V. Wu R. Schlossman S.F. Science. 1998; 281: 998-1001Crossref PubMed Google Scholar, 22Zhang Y. Finegold M.J. Porteu F. Kanteti P. Wu M.X. Oncogene. 2003; 22: 6845-6851Crossref PubMed Scopus (30) Google Scholar, 23Shen L. Guo J. Santos-Berrios C. Wu M.X. J. Biol. Chem. 2006; 281: 15304-15311Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). These differential effects might be due to the fact that IEX-1 acquires its pro-survival function upon phosphorylation by ERK (19Garcia J. Ye Y. Arranz V. Letourneux C. Pezeron G. Porteu F. EMBO J. 2002; 21: 5151-5163Crossref PubMed Scopus (91) Google Scholar, 23Shen L. Guo J. Santos-Berrios C. Wu M.X. J. Biol. Chem. 2006; 281: 15304-15311Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). In addition to this function, we have demonstrated that IEX-1 behaves as an inhibitor of B56-containing PP2A enzymes (4Letourneux C. Rocher G. Porteu F. EMBO J. 2006; 25: 727-738Crossref PubMed Scopus (158) Google Scholar). IEX-1 binds to both B56 family members and to active ERKs, enhances the phosphorylation of B56 by ERK in this complex and triggers the dissociation of the A/C core from the B56 subunit. Inhibition of PP2A-B56 activity by IEX-1 was found to increase ERK signaling in response to various growth factors (4Letourneux C. Rocher G. Porteu F. EMBO J. 2006; 25: 727-738Crossref PubMed Scopus (158) Google Scholar). In the hematopoietic cell line UT7, the specific induction of the IEX-1 protein by thrombopoietin (TPO), correlates with the unique capacity of this cytokine to sustain the ERK signal and is required for this effect (4Letourneux C. Rocher G. Porteu F. EMBO J. 2006; 25: 727-738Crossref PubMed Scopus (158) Google Scholar, 19Garcia J. Ye Y. Arranz V. Letourneux C. Pezeron G. Porteu F. EMBO J. 2002; 21: 5151-5163Crossref PubMed Scopus (91) Google Scholar, 24Hamelin V. Letourneux C. Romeo P.H. Porteu F. Gaudry M. Blood. 2006; 107: 3106-3113Crossref PubMed Scopus (36) Google Scholar). IEX-1 has no effect on other B family of PP2As. Thus, studying IEX-1 contribution to signaling cascades should lead to the discovery of new pathways controlled by B56-containing PP2As. The functions of IEX-1 and its induction by various growth factors prompted us to analyze whether it can affect Akt activation. We show here that IEX-1 and PP2A-B56 regulate Akt activity in an opposite manner by controlling the phosphorylation state of both Thr308 and Ser473. These data add to our understanding of both PP2A and Akt regulatory pathways.MATERIALS AND METHODSPlasmids—Plasmids encoding IEX-1, WT, or deleted of its ERK binding site (IEX-1-ΔBD), were described previously (19Garcia J. Ye Y. Arranz V. Letourneux C. Pezeron G. Porteu F. EMBO J. 2002; 21: 5151-5163Crossref PubMed Scopus (91) Google Scholar). IEX-1 was inserted downstream of HA or His tags. pcDNA expression vectors for HA-B55α and 4xHA-B56β were generously provided by Dr. D. Virshup (University of Utah). pcDNA encoding HA-tagged B56γ was given by Dr. N. Nojima (Osaka university). B56β-S368A and B56γ1-S327A were previously characterized (4Letourneux C. Rocher G. Porteu F. EMBO J. 2006; 25: 727-738Crossref PubMed Scopus (158) Google Scholar) and were generated by the QuikChange site-directed mutagenesis kit (Stratagene). The insert was cloned downstream of HA4. Mouse ERK1 cDNA was cloned from reverse transcription of NIH3T3 mRNA. Mouse cDNA for ERK2 was a gift from Dr. M. Weber (University of Virginia Cancer Center). ERK1 and ERK2 dominant negative mutants were generated by mutating their Mg2+ binding pockets (consensus DFG in all kinases). The coding sequences were inserted into pcDNA3 to create plasmids pcDNA3-ERK1-D185A and pcDNA-ERK2-D165A. pcDNA-HA-Elk1 was a gift of R. Hipskind (Montpellier, France). For RNA interference, the target nucleotide sequences and the vectors that drive the expression of small hairpin shRNA for human IEX-1, B56β, or B56γ were described previously (4Letourneux C. Rocher G. Porteu F. EMBO J. 2006; 25: 727-738Crossref PubMed Scopus (158) Google Scholar, 25Chen J. St-Germain J.R. Li Q. Mol. Cell. Biol. 2005; 25: 525-532Crossref PubMed Scopus (32) Google Scholar, 26Chen W. Possemato R. Campbell K.T. Plattner C.A. Pallas D.C. Hahn W.C. Cancer Cell. 2004; 5: 127-136Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). Two shRNA targeting different nucleotides of human B56β sequence were used: pSM2c-shB56β-1 and pSM2c-shB56β-2 (Open Biosystems); pMKO-1-shB56γ was a gift of Dr. W. Hahn (Dana Farber Cancer Institute, Boston). The controls used were either sh0 (nucleotides 398–418 in the IEX-1 sequence), which was found to be ineffective at down-regulating IEX-1 expression, or shGFP (4Letourneux C. Rocher G. Porteu F. EMBO J. 2006; 25: 727-738Crossref PubMed Scopus (158) Google Scholar). The cassette encoding the H1 promoter-shIEX-1 or shRNA-control was introduced in a self-inactivating lentiviral/human immunodeficiency virus vector (TRIPΔU3). This vector also expresses GFP under the EF1α promoter. Production and titration of infectious particles were done as described (24Hamelin V. Letourneux C. Romeo P.H. Porteu F. Gaudry M. Blood. 2006; 107: 3106-3113Crossref PubMed Scopus (36) Google Scholar).Cell Cultures—Chinese hamster ovary cells expressing the erythropoietin (EPO) receptor (CHO-ER) cells were described previously (19Garcia J. Ye Y. Arranz V. Letourneux C. Pezeron G. Porteu F. EMBO J. 2002; 21: 5151-5163Crossref PubMed Scopus (91) Google Scholar). They were cultured in Dulbecco’s minimal essential medium nut Mix F-12 (Ham’s) medium containing 7% fetal calf serum (FCS). HEK-293 and HeLa cells were maintained in Dulbecco’s minimum essential medium supplemented with 10% FCS. UT7 cells (27Porteu F. Rouyez M.C. Cocault L. Benit L. Charon M. Picard F. Gisselbrecht S. Souyri M. Dusanter-Fourt I. Mol. Cell. Biol. 1996; 16: 2473-2482Crossref PubMed Scopus (81) Google Scholar) were grown in α-minimal essential medium supplemented with 10% FCS and 1 unit/ml EPO (Roche Molecular Biochemicals).Transfections and Viral Infections—CHO-ER and HeLa cells (60–80% confluence) were transiently transfected with 1–2 μg of DNA, using the Lipofectamine Plus reagent (Invitrogen), according to the manufacturer’s instructions. Stable clones of HeLa cells expressing the shRNAs targeting B56γ and B56β were previously described (4Letourneux C. Rocher G. Porteu F. EMBO J. 2006; 25: 727-738Crossref PubMed Scopus (158) Google Scholar). In some experiments, the vectors carrying shRNA were transiently introduced in HeLa cells and the cells were harvested 48 h later. HEK-293 and UT7 cells were incubated with TRIPΔU3-H1-shIEX-1 or control lentiviruses over a period of 36 h. The transduction efficiency, determined by the percentage of GFP positive cells by fluorescence-activated cell sorter analysis, usually reached 98%.Western Blotting, Immunoprecipitation, and Kinase Assays— Akt activation was assessed in total lysates by Western blotting with anti-pAkt-Ser473 or pAkt-Thr308 antibodies (Cell Signaling). To follow the kinetics of Akt inactivation, 24 h post-transfection the cells were either treated with the PI3K inhibitor LY294002 or washed 3 times in phosphate-buffered saline and loaded with serum-free medium. The cells were returned to 37 °C and harvested at various times. In some experiments, the cells were deprived of serum overnight, and stimulated with EPO or FCS for various times before lysis. For immunoprecipitation experiments, the cells were lysed in 50 mm Tris, pH 7.5, buffer containing 137 mm NaCl, 0.5% Nonidet P-40, 10% glycerol, 1 mm EDTA, 1 mm orthovanadate, 20 mm β-glycerophosphate, 20 mm NaF, 1 mm sodium pyrophosphate, and a protease inhibitor mixture (Roche). Gel electrophoresis, immunoblotting, and immunoprecipitation were carried out as previously described (4Letourneux C. Rocher G. Porteu F. EMBO J. 2006; 25: 727-738Crossref PubMed Scopus (158) Google Scholar). In vitro Akt kinase activity was assessed with the non-radioactive Akt kinase kit (Cell Signaling). The other antibodies used in this study were: anti-phospho: pERK, pGSK3, pFoxo3a, pan-Akt substrate, and pElk1 (Cell Signaling); anti-Akt (sc-9312), anti-ERK1 (sc-94), anti-ERK2 (sc-154), and anti-B56β from Santa Cruz Biotechnology; anti-HA (Roche). Anti-IEX-1 antiserum was as previously described (19Garcia J. Ye Y. Arranz V. Letourneux C. Pezeron G. Porteu F. EMBO J. 2002; 21: 5151-5163Crossref PubMed Scopus (91) Google Scholar). Rabbit anti-B56γ was a gift from Dr. M. Mumby (University of Texas).RESULTSIEX-1 Increases Akt Activation—IEX-1 can be induced by serum and various growth factors (4Letourneux C. Rocher G. Porteu F. EMBO J. 2006; 25: 727-738Crossref PubMed Scopus (158) Google Scholar, 16Wu M.X. Apoptosis. 2003; 8: 11-18Crossref PubMed Scopus (104) Google Scholar, 19Garcia J. Ye Y. Arranz V. Letourneux C. Pezeron G. Porteu F. EMBO J. 2002; 21: 5151-5163Crossref PubMed Scopus (91) Google Scholar, 24Hamelin V. Letourneux C. Romeo P.H. Porteu F. Gaudry M. Blood. 2006; 107: 3106-3113Crossref PubMed Scopus (36) Google Scholar, 28Charles C.H. Yoon J.K. Simske J.S. Lau L.F. Oncogene. 1993; 8: 797-801PubMed Google Scholar). To determine whether IEX-1 could affect other growth factor-induced signaling pathways in addition to ERK, we measured Akt activity in cells expressing IEX-1. Maximal Akt activation requires phosphorylation at the two activating residues Ser473 and Thr308 (8Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar, 9Stephens L. Anderson K. Stokoe D. Erdjument-Bromage H. Painter G.F. Holmes A.B. Gaffney P.R. Reese C.B. McCormick F. Tempst P. Coadwell J. Hawkins P.T. Science. 1998; 279: 710-714Crossref PubMed Scopus (909) Google Scholar, 10Sarbassov D.D. Guertin D.A. Ali S.M. Sabatini D.M. Science. 2005; 307: 1098-1101Crossref PubMed Scopus (5154) Google Scholar, 29Yang J. Cron P. Good V.M. Thompson V. Hemmings B.A. Barford D. Nat. Struct. Biol. 2002; 9: 940-944Crossref PubMed Scopus (426) Google Scholar). Thus, CHO cells were transiently transfected with HA-IEX-1 or empty vector and the cells were maintained in serum to activate Akt. Endogenous Akt activation was assessed both by Western blotting with Ser473 and Thr308 phosphospecific Akt antibodies and by immunoprecipitation kinase assays. As shown in Fig. 1A, Akt phosphorylation was greatly enhanced at both Ser473 and Thr308 in cells expressing IEX-1. To determine whether IEX-1 could affect Akt activation in response to growth factors, CHO cells expressing the EPO receptor (CHO-ER, (19Garcia J. Ye Y. Arranz V. Letourneux C. Pezeron G. Porteu F. EMBO J. 2002; 21: 5151-5163Crossref PubMed Scopus (91) Google Scholar)) were transfected with IEX-1 and analyzed after 3 h starvation in a serum-free medium and stimulation with EPO, under conditions leading to increased ERK activation in the presence of IEX-1 (Ref. 19Garcia J. Ye Y. Arranz V. Letourneux C. Pezeron G. Porteu F. EMBO J. 2002; 21: 5151-5163Crossref PubMed Scopus (91) Google Scholar and Fig. 1, B and C). No phosphorylation of Akt could be detected in non-stimulated starved cells whether they expressed IEX-1 or not. However, IEX-1 expression resulted in a great increase in EPO-induced Akt phosphorylation on both Ser473 and Thr308, at all time points and doses tested (Fig. 1, B and C). Endogenous Akt phosphorylation was also enhanced and prolonged in UT7 cells stably transfected with IEX-1, as compared with control cells (neo), upon stimulation with TPO (Fig. 1D).To examine IEX-1-dependent Akt activation further, we measured Akt kinase activity, by using an in vitro kinase assay on its substrate GSK3. Fig. 1E shows that the Akt kinase activity that could be immunoprecipitated from CHO cells expressing IEX-1 was increased, as compared with cells transfected with an empty vector. An increased and sustained in vivo GSK3 phosphorylation that paralleled pAkt levels was also detected in IEX-1-overexpressing cells (Fig. 1B). In addition, the phosphorylation levels of several Akt substrates, detected using an anti-pan phospho-Akt substrate antibody, were greatly increased in the presence of IEX-1 (Fig. 1B). Thus, expression of IEX-1 increases Akt activity and Akt downstream signaling.IEX-1 is an early response gene product that is induced by various growth factors. Thus Akt activity should be more sustained in response to growth factors triggering IEX-1 expression. To test this, we made use of the UT7 cell line. Indeed, whereas both EPO and TPO stimulate Akt activation in this cell line, only TPO can induce IEX-1 protein expression (Ref. 24Hamelin V. Letourneux C. Romeo P.H. Porteu F. Gaudry M. Blood. 2006; 107: 3106-3113Crossref PubMed Scopus (36) Google Scholar and Fig. 2A). As shown in Fig. 2A, Akt phosphorylation on both Ser473 and Thr308 was much higher and more sustained in cells stimulated with optimal doses of TPO, as compared with EPO. This increase in Akt phosphorylation translated into an increased Akt activity, as shown by the higher levels of phosphorylation of the Akt substrates GSK3 and Foxo3a observed upon TPO stimulation. Likewise, Western blotting with a pan-Akt substrate phospho-antibody shows that in TPO-treated, IEX-1 expressing cells, the global Akt-mediated phosphorylation is greatly increased, as compared with cells treated with EPO. Thus, the specific induction of the IEX-1 protein by TPO in UT7 cells correlates with the unique capacity of this cytokine to sustain Akt phosphorylation and Akt downstream signaling.FIGURE 2Endogenous IEX-1 expression is involved in sustained Akt activation induced by various growth factors. A, UT7 cells were starved overnight and stimulated for various times with either EPO (10 units/ml) or TPO (3 nm). IEX-1 expression and phosphorylation of Akt and its downstream substrates were analyzed by Western blotting with the indicated antibodies. B, UT7 cells were infected with lentiviral vectors encoding IEX-1 shRNA (shIEX-1) or control shRNA (shGFP and sh0). The effect of shRNA on endogenous IEX-1 expression was assessed on maximally induced IEX-1 after stimulation with 10 nm TPO in the presence of the proteasome inhibitor N-acetyl-l-leucinyl-l-leucinol-l-norleucinol for 2 h (left panel). Akt phosphorylation was analyzed upon stimulation with TPO alone for various times (right panel). C, HEK-293 cells expressing shGFP or shIEX-1 constructs were starved of serum overnight before being stimulated for various times with 2% serum. pAkt levels were quantified and normalized to total Akt levels and expressed relative to the levels of phosphorylation at the zero time point of control cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To further confirm the above results, we examined Akt activation in cells in which TPO-induced IEX-1 expression was impeded by the presence of IEX-1-specific shRNA. Thus, we generated UT7 cell lines expressing shRNA for IEX-1. Expression of endogenous IEX-1 upon TPO stimulation was severely inhibited in the presence of this shRNA (Fig. 2B, left panel). As a control, we used cell lines expressing either a shRNA designed from the IEX-1 nucleotide sequence (sh0, see “Materials and Methods”) but that was found unable to down-regulate endogenous IEX-1 levels (Fig. 2B), or a shRNA targeting GFP. As shown in Fig. 2B (right panel), TPO-mediated phosphorylation of both Akt and its downstream substrate GSK3 was greatly reduced in shRNA-IEX-1 expressing cells, as compared with control cells. This effect was particularly striking after 1.5 and 3 h TPO treatments, time points at which IEX-1 is maximally induced by the cytokine. The decreased Akt activation upon down-regulation of IEX-1 expression is not specific to TPO signaling or to the particular cell context of UT7 cells, as shown by the impaired serum-induced Akt phosphorylation at both Ser473 and Thr308 in HEK293 expressing IEX-1-shRNA (Fig. 2C). In these cells, basal expression of IEX-1 was observed but its level increased following serum stimulation. These results show that the induction of IEX-1 by various growth factors plays an important role in their capacity to control Akt activity.IEX-1 Prevents Akt Dephosphorylation—We next determined by which mechanism IEX-1 increased Akt activity. The marked ability of IEX-1 to sustain Akt signal at long stimulation time points (Fig. 1) suggested that IEX-1 may prevent Akt inactivation rather than activate upstream kinases. To test this possibility, CHO-ER cells, transfected or not with IEX-1, were first subjected to a growth factor pulse with an optimal dose of Epo (10 units/ml) to activate Akt before suppression of upstream activating signals by adding the PI3K inhibitor LY294002. pAkt levels were measured at different time points after inhibition. In agreement with previous reports (30Kim A.H. Sasaki T. Chao M.V. J. Biol. Chem. 2003; 278: 29830-29836Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 31Li L. Ren C.H. Tahir S.A. Ren C. Thompson T.C. Mol. Cell. Biol. 2003; 23: 9389-9404Crossref PubMed Scopus (256) Google Scholar), blocking PI3K activity after growth factor exposure led to a rapid decrease in Akt phosphorylation, as shown by the rapid drop of reactivity with both anti-pAkt-Ser473 and anti-pAkt-Thr308 5 min after addition of the inhibitor. This shows that Akt is rapidly dephosphorylated at its two activating residues by endogenous active phosphatases. By contrast, in cells expressing IEX-1, although the basal Akt activation after optimal EPO stimulation was unchanged, the rate of disappearance of pAkt upon addition of LY294002 was greatly delayed (Fig. 3A). This suggests that IEX-1 reduces the rate of Akt dephosphorylation rather than increases its activation.FIGURE 3IEX-1 inhibits Akt dephosphorylation at both Ser473 and Thr308. CHO-ER cells were transfected with either empty or HA-IEX-1 encoding plasmids. 24 h post-transfection, the cells were either stimulated for 7 min with 10 units/ml EPO prior to addition of 30 μm LY294002 (A) or with 10% FCS for 10 min and then starved for various times in serum-free medium (B). Akt phosphorylation and IEX-1 expression were analyzed by Western blotting of total lysates. Quantification of pAkt levels in control or IEX-1 expressing cells, normalized to total ERK (A) or Akt (B) levels, is expressed relative to the levels of phosphorylation at their zero time point.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To confirm these results, CHO cells were stimulated with FCS and Akt phosphorylation was measured after various times following arrest of Akt activation induced by FCS starvation. As above with th" @default.
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