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• W2045852899 abstract "Akt is a protein serine/threonine kinase that is involved in the regulation of diverse cellular processes. Phosphorylation of Akt at regulatory residues Thr-308 and Ser-473 leads to its full activation. The protein phosphatase 2A (PP2A) has long been known to negatively regulate Akt activity. The PP2A holoenzyme consists of the structural subunit (A), catalytic subunit (C), and a variable regulatory subunit (B). Here we report the identification of the specific B regulatory subunit that targets the PP2A holoenzyme to Akt. We found endogenous association of PP2A AB55C holoenzymes with Akt by co-immunoprecipitation analyses in pro-lymphoid FL5.12 cells. Akt was shown to associate with ectopically expressed B55α subunit in NIH3T3 cells. The direct interaction between B55α subunit and Akt was confirmed using in vitro pulldown analyses. Intriguingly, we found that overexpression of B55α subunit significantly impaired phosphorylation at Thr-308, but to a lesser extent at Ser-473 of Akt in both FL5.12 and NIH3T3 cells. Concomitantly, phosphorylation of a subset of Akt substrates, including FoxO3a, was substantially decreased by B55α overexpression in these cells. Silencing of B55α expression markedly increased phosphorylation at Thr-308 but not at Ser-473 in both FL5.12 cells and NIH3T3 cells. Consistently, PP2A AB55αC holoenzymes preferentially dephosphorylated phospho-Thr-308 rather than phospho-Ser-473 in in vitro dephosphorylation assays. Furthermore, B55α overexpression retarded proliferation of NIH3T3 cells, and knockdown of B55α expression increased survival of FL5.12 cells upon interleukin-3 deprivation. Together, our data demonstrate that B55α-dependent targeting of the PP2A holoenzyme to Akt selectively regulates Akt phosphorylation at Thr-308 to regulate cell proliferation and survival. Akt is a protein serine/threonine kinase that is involved in the regulation of diverse cellular processes. Phosphorylation of Akt at regulatory residues Thr-308 and Ser-473 leads to its full activation. The protein phosphatase 2A (PP2A) has long been known to negatively regulate Akt activity. The PP2A holoenzyme consists of the structural subunit (A), catalytic subunit (C), and a variable regulatory subunit (B). Here we report the identification of the specific B regulatory subunit that targets the PP2A holoenzyme to Akt. We found endogenous association of PP2A AB55C holoenzymes with Akt by co-immunoprecipitation analyses in pro-lymphoid FL5.12 cells. Akt was shown to associate with ectopically expressed B55α subunit in NIH3T3 cells. The direct interaction between B55α subunit and Akt was confirmed using in vitro pulldown analyses. Intriguingly, we found that overexpression of B55α subunit significantly impaired phosphorylation at Thr-308, but to a lesser extent at Ser-473 of Akt in both FL5.12 and NIH3T3 cells. Concomitantly, phosphorylation of a subset of Akt substrates, including FoxO3a, was substantially decreased by B55α overexpression in these cells. Silencing of B55α expression markedly increased phosphorylation at Thr-308 but not at Ser-473 in both FL5.12 cells and NIH3T3 cells. Consistently, PP2A AB55αC holoenzymes preferentially dephosphorylated phospho-Thr-308 rather than phospho-Ser-473 in in vitro dephosphorylation assays. Furthermore, B55α overexpression retarded proliferation of NIH3T3 cells, and knockdown of B55α expression increased survival of FL5.12 cells upon interleukin-3 deprivation. Together, our data demonstrate that B55α-dependent targeting of the PP2A holoenzyme to Akt selectively regulates Akt phosphorylation at Thr-308 to regulate cell proliferation and survival. Akt/protein kinase B (PKB), 2The abbreviations used are: PKBprotein kinase BPP2Aprotein phosphatase 2APP2A/APP2A structural subunitPP2A/BPP2A B regulatory subunitPP2A/CPP2A catalytic subunitPP2A-B55αPP2A AB55αC holoenzymemTORmammalian target of rapamycinMAPKmitogen-activated protein kinaseshRNAshort hairpin RNAPI3Kphosphatidylinositide 3′-OH kinaseILinterleukinGSTglutathione S-transferaseHAhemagglutininPMSFphenylmethylsulfonyl fluorideOAokadaic acidBSbovine serumPHLPPpleckstrin homology domain leucine-rich repeat protein phosphataseERKextracellular signal-regulated kinase. also named RAC kinase, is involved in the regulation of diverse cellular processes, including glucose metabolism, cell growth, cell proliferation, angiogenesis, and apoptosis (1Jones P.F. Jakubowicz T. Pitossi F.J. Maurer F. Hemmings B.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4171-4175Crossref PubMed Scopus (443) Google Scholar, 2Bellacosa A. Testa J.R. Staal S.P. Tsichlis P.N. 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Drug Discov. 2005; 4: 988-1004Crossref PubMed Scopus (1774) Google Scholar, 8Nicholson K.M. Anderson N.G. Cell. Signal. 2002; 14: 381-395Crossref PubMed Scopus (1388) Google Scholar). Akt is a crucial signal transducer downstream of phosphatidylinositide 3′-OH kinase (PI3K) that is activated upon ligand and receptor engagement on the cell surface. The Akt family consists of three major isoforms, Akt1/PKBα, Akt2/PKBβ, and Akt3/PKBγ, that have conserved functional domains, including an N-terminal pleckstrin homology domain, a central kinase domain, and a C-terminal regulatory domain. Recruitment of Akt to the plasma membrane upon activation of PI3K results in a conformational change, and phosphorylation of Thr-308 and Ser-473 after membrane recruitment of Akt leads to full activation of Akt (6Hanada M. Feng J. Hemmings B.A. Biochim. Biophys. Acta. 2004; 1697: 3-16Crossref PubMed Scopus (623) Google Scholar, 9Scheid M.P. Woodgett J.R. FEBS Lett. 2003; 546: 108-112Crossref PubMed Scopus (346) Google Scholar). Akt activation is tightly controlled by counteraction of phosphatases, and tensin homologue deleted on chromosome 10 (PTEN) (10Maehama T. Dixon J.E. J. Biol. Chem. 1998; 273: 13375-13378Abstract Full Text Full Text PDF PubMed Scopus (2601) Google Scholar), protein phosphatase 2A (PP2A) (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 (430) Google Scholar, 12Meier R. Thelen M. Hemmings B.A. EMBO J. 1998; 17: 7294-7303Crossref PubMed Scopus (147) Google Scholar, 13Chen D. Fucini R.V. Olson A.L. Hemmings B.A. Pessin J.E. Mol. Cell. Biol. 1999; 19: 4684-4694Crossref PubMed Google Scholar, 14Sato S. Fujita N. Tsuruo T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10832-10837Crossref PubMed Scopus (840) Google Scholar, 15Ivaska J. Nissinen L. Immonen N. Eriksson J.E. Kahari V.M. Heino J. Mol. Cell. 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It has long been known that PP2A negatively regulates Akt activity in various systems. PP2A holoenzymes consist of a dimeric core enzyme, which includes the catalytic subunit C, the structural subunit A, and a variable regulatory subunit B (18Janssens V. Goris J. Biochem. J. 2001; 353: 417-439Crossref PubMed Scopus (1542) Google Scholar, 19Sontag E. Cell. Signal. 2001; 13: 7-16Crossref PubMed Scopus (288) Google Scholar, 20Janssens V. Goris J. Van Hoof C. Curr. Opin. Genet. Dev. 2005; 15: 34-41Crossref PubMed Scopus (365) Google Scholar). Multiple families and isoforms of the regulatory subunit B increase the diversity of the PP2A holoenzymes. There are at least four subfamilies of the regulatory subunit, including B (B55 or PR55) (21Mayer R.E. Hendrix P. Cron P. Matthies R. Stone S.R. Goris J. Merlevede W. Hofsteenge J. Hemmings B.A. Biochemistry. 1991; 30: 3589-3597Crossref PubMed Scopus (171) Google Scholar, 22Healy A.M. Zolnierowicz S. Stapleton A.E. Goebl M. DePaoli-Roach A.A. Pringle J.R. Mol. Cell. Biol. 1991; 11: 5767-5780Crossref PubMed Scopus (228) Google Scholar, 23Zolnierowicz S. Csortos C. Bondor J. Verin A. Mumby M.C. DePaoli-Roach A.A. Biochemistry. 1994; 33: 11858-11867Crossref PubMed Scopus (95) Google Scholar, 24Zolnierowicz S. Biochem. Pharmacol. 2000; 60: 1225-1235Crossref PubMed Scopus (173) Google Scholar), B′ (B56 or PR61) (25McCright B. Virshup D.M. J. Biol. Chem. 1995; 270: 26123-26128Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 26Csortos C. Zolnierowicz S. Bako E. Durbin S.D. DePaoli-Roach A.A. J. Biol. Chem. 1996; 271: 2578-2588Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar), B″ (PR72) (27Tanabe O. Nagase T. Murakami T. Nozaki H. Usui H. Nishito Y. Hayashi H. Kagamiyama H. Takeda M. FEBS Lett. 1996; 379: 107-111Crossref PubMed Scopus (49) Google Scholar), and B′′′ (PR93/PR110) (28Moreno C.S. Park S. Nelson K. Ashby D. Hubalek F. Lane W.S. Pallas D.C. J. Biol. Chem. 2000; 275: 5257-5263Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). It is thought that the diverse regulatory subunits dictate substrate specificity and subcellular localization of PP2A. Signaling modules composed of PP2A and variable kinases have been identified and are believed to increase the fidelity of signaling transduction process. The kinases that have been found to interact with PP2A include calcium-calmodulin-dependent protein kinase IV (29Westphal R.S. Anderson K.A. Means A.R. Wadzinski B.E. Science. 1998; 280: 1258-1261Crossref PubMed Scopus (223) Google Scholar), p70 S6 kinase (30Westphal R.S. Coffee Jr., R.L. Marotta A. Pelech S.L. Wadzinski B.E. J. Biol. Chem. 1999; 274: 687-692Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 31Peterson R.T. Desai B.N. Hardwick J.S. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4438-4442Crossref PubMed Scopus (429) Google Scholar), p21-activated kinases (30Westphal R.S. Coffee Jr., R.L. Marotta A. Pelech S.L. Wadzinski B.E. J. Biol. Chem. 1999; 274: 687-692Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar), p38 kinase (32Lee T. Kim S.J. Sumpio B.E. J. Cell. Physiol. 2003; 194: 349-355Crossref PubMed Scopus (44) Google Scholar), casein kinase II (33Heriche J.K. Lebrin F. Rabilloud T. Leroy D. Chambaz E.M. Goldberg Y. Science. 1997; 276: 952-955Crossref PubMed Scopus (250) Google Scholar), IκB kinases (34Fu D.X. Kuo Y.L. Liu B.Y. Jeang K.T. Giam C.Z. J. Biol. Chem. 2003; 278: 1487-1493Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 35Kray A.E. Carter R.S. Pennington K.N. Gomez R.J. Sanders L.E. Llanes J.M. Khan W.N. Ballard D.W. Wadzinski B.E. J. Biol. Chem. 2005; 280: 35974-35982Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), mitogen-activated protein kinase (MAPK) (36Ory S. Zhou M. Conrads T.P. Veenstra T.D. Morrison D.K. Curr. Biol. 2003; 13: 1356-1364Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar, 37Liu Q. Hofmann P.A. Am. J. Physiol. 2004; 286: H2204-H2212Crossref PubMed Scopus (142) Google Scholar), and Akt (15Ivaska J. Nissinen L. Immonen N. Eriksson J.E. Kahari V.M. Heino J. Mol. Cell. Biol. 2002; 22: 1352-1359Crossref PubMed Scopus (154) Google Scholar, 38Yellaturu C.R. Bhanoori M. Neeli I. Rao G.N. J. Biol. Chem. 2002; 277: 40148-40155Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 39Liu W. Akhand A.A. Takeda K. Kawamoto Y. Itoigawa M. Kato M. Suzuki H. Ishikawa N. Nakashima I. Cell Death Differ. 2003; 10: 772-781Crossref PubMed Scopus (81) Google Scholar, 40Borgatti P. Martelli A.M. Tabellini G. Bellacosa A. Capitani S. Neri L.M. J. Cell. Physiol. 2003; 196: 79-88Crossref PubMed Scopus (58) Google Scholar, 41Trotman L.C. Alimonti A. Scaglioni P.P. Koutcher J.A. Cordon-Cardo C. Pandolfi P.P. Nature. 2006; 441: 523-527Crossref PubMed Scopus (332) Google Scholar). Although it remains unclear whether B regulatory subunits are involved in the interactions between these kinases and PP2A, several reports using overexpression and RNA interference approaches have demonstrated the roles of the B55 regulatory subunits (42Silverstein A.M. Barrow C.A. Davis A.J. Mumby M.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4221-4226Crossref PubMed Scopus (228) Google Scholar, 43Van Kanegan M.J. Adams D.G. Wadzinski B.E. Strack S. J. Biol. Chem. 2005; 280: 36029-36036Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 44Adams 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 (115) Google Scholar) and the B56 subunits (45Letourneux C. Rocher G. Porteu F. EMBO J. 2006; 25: 727-738Crossref PubMed Scopus (163) Google Scholar) in the MAPK signaling pathway in multiple systems. Two reports have suggested the involvement of the B56 family in regulating Akt phosphorylation (43Van Kanegan M.J. Adams D.G. Wadzinski B.E. Strack S. J. Biol. Chem. 2005; 280: 36029-36036Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 46Rocher G. Letourneux C. Lenormand P. Porteu F. J. Biol. Chem. 2007; 282: 5468-5477Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Intriguingly, cells expressing a mutant Aα subunit with selective B56 subunit binding deficiency showed decreased phosphorylation of Akt (43Van Kanegan M.J. Adams D.G. Wadzinski B.E. Strack S. J. Biol. Chem. 2005; 280: 36029-36036Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). IEX, an early response gene involved in survival and proliferation, was shown to increase Akt phosphorylation by inhibiting B56-containing PP2A holoenzymes via an extracellular signal-regulated kinase (ERK)-dependent manner (46Rocher G. Letourneux C. Lenormand P. Porteu F. J. Biol. Chem. 2007; 282: 5468-5477Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Here we report the identification of B55α as an Akt-targeting regulatory subunit of PP2A. Importantly, we demonstrate that B55α preferentially targets the PP2A holoenzyme to dephosphorylate Akt at Thr-308 rather than Ser-473 both in cells and in vitro, indicating substrate (site) specificity determined by the specific PP2A B regulatory subunit at the intramolecular level. Furthermore, consistent with the regulatory activity on ERKs and Akt signaling pathways, we demonstrate that B55α plays roles in regulating cell proliferation and survival. Antibodies and Reagents—Antibodies employed include the following: anti-phospho-Akt Ser-473, anti-phospho-Akt Thr-308, anti-phospho-ERK1/2 (Thr-202/Tyr-204), anti-HA, pan-anti-phospho-(Ser/Thr) Akt substrate, anti-phospho-FoxO1/3a (Thr-24/Thr-32), and anti-PP2A/B from Cell Signaling; and anti-PP2A/A from Santa Cruz Biotechnology; anti-PP2A/B clone 2G9 from Upstate; anti-Akt and anti-PP2A/C from BD Transduction Laboratories; anti-GSK from Santa Cruz Biotechnology; anti-GST from GE Healthcare; and anti-B56γ3 from Novus. Serine/threonine phosphatase inhibitors and okadaic acid (OA) were from Alexis Biochemicals, and microcystin-LR was from Calbiochem. Protease inhibitors, phenylmethylsulfonyl fluoride (PMSF), aprotinin, leupeptin, bacterial protease inhibitor mixture, anti-β-actin, anti-FLAG M2 antibodies, and anti-FLAG M2-agarose were from Sigma. The specific anti-B55δ antibody was kindly provided by Dr. Brian Wadzinski (Vanderbilt University, Nashville, TN). DNA Constructs and Retrovirus Preparation—The full-length human Akt1 cDNA with a C-terminal FLAG tag was prepared by PCR using a template of human Akt1 (kindly provided by Dr. P. C. Lin, Vanderbilt University, Nashville, TN) and was subsequently cloned into the bacterial expression vector pQE30 and the retroviral pMSCVpuro vector (Clontech). The C-terminal HA-tagged rat B55α and human B56γ3 were prepared by PCR using the pCEP4B55α and pCEP4B56γ3, respectively, as a template (kindly provided by Dr. David Virshup, University of Utah, Salt Lake City) and were subsequently cloned into the retroviral pMSCVpuro vector and the bacterial expression pGEX4T-1 vector (GE Healthcare). The sequence encoding short hairpin RNA (shRNA) targeting B55α was synthesized as two complementary oligonucleotides as described before (44Adams 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 (115) Google Scholar), which were subsequently phosphorylated, annealed, and ligated into the pSUPERretropuro vector (47Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3968) Google Scholar). The retroviral expression vectors, pMSCVpuro and pSUPERretropuro, harboring Akt cDNA, B55α cDNA, B56γ3 cDNA, or B55α shRNA-encoding sequence, were transfected into BOSC23 cells by the calcium phosphate method, and viral supernatants were prepared by collecting the BOSC23 culture media 48 h after transfection and centrifugation at 1,500 rpm for 5 min. Cell Culture—FL5.12 cells were cultured in Iscove's modified Dulbecco's medium supplemented with 10% fetal bovine serum and interleukin 3 (IL-3) as described before (48Chiang C.W. Harris G. Ellig C. Masters S.C. Subramanian R. Shenolikar S. Wadzinski B.E. Yang E. Blood. 2001; 97: 1289-1297Crossref PubMed Scopus (131) Google Scholar). NIH3T3 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% bovine serum (BS). BOSC23 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. In serum stimulation experiments, NIH3T3 cells were grown in medium containing 0.5% BS for 14–16 h and were stimulated with 10% BS-containing medium. In IL-3 deprivation experiments, FL5.12 cells were grown in medium without IL-3 for 3 h and then stimulated with medium containing IL-3. In IL-3-dependent survival assay, cells were deprived of IL-3 for 24 h. In proliferation assays, NIH3T3 cells stably carrying vector or expressing B55αHA were seeded onto 24-well plates, and cell numbers were directly counted. Viability was measured by trypan blue exclusion. Selection of Cells Stably Expressing FLAG-tagged Akt, FLAG-tagged B55α, HA-tagged B55α, HA-tagged B56γ3, B55α shRNA, or Vector Only—The cells stably expressing genes of interest were created by retroviral infection and subsequent drug selection. Cells were infected with retroviruses for 2 days and then selected with puromycin at 5 μg/ml. Cells stably expressing FLAG-tagged Akt, FLAG-tagged B55α, HA-tagged B55α, HA-tagged B56γ3, B55α shRNA, or vector only were isolated after a 2-day drug selection and characterized by immunoblotting for specific gene expression. Immunoblotting and Immunoprecipitation—Cell lysates for immunoblotting were prepared in radioimmunoprecipitation assay buffer (10 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.1% SDS, 50 mm Tris-Cl, pH 8.0, and 1 mm EDTA) containing 1 mm PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 5 mm EGTA, 5 mm EDTA, 10 mm NaF, 1 μm microcystin, and 1 mm Na3VO4. Cell lysates for immunoprecipitation were prepared as described before (49McCright B. Rivers A.M. Audlin S. Virshup D.M. J. Biol. Chem. 1996; 271: 22081-22089Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar) with some modifications. Cells were lysed in hypotonic buffer A (10 mm Tris-Cl, pH 7.9, 1.5 mm MgCl2, 10 mm KCl, 1 mm EDTA, 1 mm EGTA) supplemented with 1 mm PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, followed by sonication. The lysates were then subjected to centrifugation at 1000 × g for 5 min. The supernatant was then adjusted to isotonic osmolarity by adding 1/10 volume of buffer B (1.4 m KCl, 30 mm MgCl2). Immunoprecipitation assays were carried out using control IgG or a specific antibody in isotonic buffer, as described above, and precipitated using protein A/G-Sepharose. The immune complexes were then washed with wash buffer (isotonic buffer plus 0.2% Triton X-100) three times and were subjected to SDS-PAGE and immunoblotting. Immunoblots were developed using enhanced chemiluminescence. Results were quantified by densitometry using Alpha Innotech AlphaImager. In Vitro Akt Dephosphorylation Assay—NIH3T3 cells expressing FLAG-tagged Akt were serum-starved for 16 h and stimulated with 10% BS for 5 min. Cells were lysed in the immunoprecipitation buffer as described above without phosphatase inhibitors, and FLAG-tagged Akt was immunoprecipitated using anti-FLAG-agarose. The PP2A-B55α holoenzymes were prepared from NIH3T3 cells expressing FLAG-tagged B55α by immunoprecipitating FLAG-tagged B55α immunocomplexes using anti-FLAG-agarose. The FLAG-tagged Akt or B55α immunocomplexes were then resuspended in the phosphatase assay buffer (20 mm imidazole, 150 mm NaCl, 14.4 mm β-mercaptoethanol, 1 mm PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin). Aliquots of Akt immunocomplexes were then mixed with various amounts of B55α immunocomplexes preincubated with or without 100 nm OA for 10 min at 4 °C and incubated at 30 °C for 30 min. Dephosphorylation reactions were terminated by adding 4× SDS sample buffer and boiling for 5 min. Results were quantified by densitometry using Alpha Innotech AlphaImager. Percentages of reduction of phosphorylation were calculated by subtracting the relative phosphorylation of reactions containing PP2A-B55α from 100 (control reactions with no addition of PP2A-B55α complexes were set as 100%), and the values were further normalized by relative amounts of pulled down C subunits (Thr(P)-308 versus Ser(P)-473) in individual reactions within a set input of PP2A-B55α complexes. Recombinant Proteins—Bacterial expression constructs employed include pQE30-His6-Akt-FLAG, pGEX-4T-1-B55-HA, pGEX-4T-PP2A/A (a gift from Dr. Kerry Campbell, Fox Chase Cancer Center, Philadelphia), and pGEX-4T-1-PP2A/C. Each individual expression construct was transformed into Escherichia coli BL21 cells. Appropriate antibiotics were applied to select bacteria carrying the expression constructs. Isopropyl β-d-thiogalactopyranoside was added at various concentrations (0.2 to 1 mm) to induce the optimal expression of the recombinant proteins. The bacterial culture was pelleted and resuspended in lysis buffer (phosphate-buffered saline with 1% Triton X-100 and 1% Tween 20 for GST fusion clones, and 50 mm Na2HPO4, 300 mm NaCl, 10 mm imidazole for His-tagged clones) containing 1 mg/ml lysozyme, and a bacterial protease inhibitor mixture (Sigma). GST fusion protein was purified with glutathione-Sepharose 4B (GE Healthcare) under native conditions following the manufacturer's protocols. His-tagged protein was purified with nickel-nitrilotriacetic acid resin (Qiagen) under native conditions following the manufacturer's protocols. In Vitro Pulldown Analysis—For analyzing in vitro interactions of individual PP2A subunits and Akt, 2 μg of purified recombinant GST-PP2A/A, GST-PP2A/B, or GST-PP2A/C protein were incubated with 2 μg of purified recombinant His-tagged Akt for 4 h at 4 °C. GST fusion proteins were pulled down by glutathione-Sepharose 4B, washed three times with wash buffer (20 mm Tris-Cl, pH 7.5, 140 mm NaCl, 1 mm CaCl2, 0.2% Triton X-100), fractionated by SDS-PAGE, and analyzed by immunoblotting with anti-Akt or anti-GST antibody. Akt Is Associated with PP2A AB55αC Holoenzyme Complexes—We addressed the role of B regulatory subunits in bridging the association between PP2A and Akt in the signaling module by exploiting two systems, serum-dependent NIH3T3 cells and interleukin-3 (IL-3)-dependent FL5.12 lymphoid cells. NIH3T3 cells are highly transfectable and easily activate Akt by serum stimulation. The IL-3-dependent FL5.12 lymphoid cell represents one of cytokine-dependent hematopoietic cells in which the PI3K/Akt pathway is crucial to cellular survival (50Songyang Z. Baltimore D. Cantley L.C. Kaplan D.R. Franke T.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11345-11350Crossref PubMed Scopus (322) Google Scholar, 51Yamaguchi H. Wang H.G. Oncogene. 2001; 20: 7779-7786Crossref PubMed Scopus (340) Google Scholar). In FL5.12 cells, treatment with the protein phosphatase inhibitor OA at 250 nm that was shown to selectively inhibit PP2A (48Chiang C.W. Harris G. Ellig C. Masters S.C. Subramanian R. Shenolikar S. Wadzinski B.E. Yang E. Blood. 2001; 97: 1289-1297Crossref PubMed Scopus (131) Google Scholar) resulted in significantly increased phosphorylation of Akt at Thr-308, and to a lesser extent at Ser-473 (Fig. 1A), suggesting that PP2A constantly regulated phosphorylation of Akt in FL5.12 cells. We then explored the possible involvement of a specific PP2A holoenzyme in regulating Akt activity in FL5.12 cells. The B55/PR55 family was shown to be widely expressed and displaced from the PP2A holoenzyme by the DNA tumor virus SV40 small T antigen (52Sontag E. Fedorov S. Kamibayashi C. Robbins D. Cobb M. Mumby M. Cell. 1993; 75: 887-897Abstract Full Text PDF PubMed Scopus (461) Google Scholar, 53Chen 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 (281) Google Scholar), which activates Akt (54Yuan H. Veldman T. Rundell K. Schlegel R. J. Virol. 2002; 76: 10685-10691Crossref PubMed Scopus (78) Google Scholar, 55Zhao J.J. Gjoerup O.V. Subramanian R.R. Cheng Y. Chen W. Roberts T.M. Hahn W.C. Cancer Cell. 2003; 3: 483-495Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 56Arroyo J.D. Hahn W.C. Oncogene. 2005; 24: 7746-7755Crossref PubMed Scopus (194) Google Scholar). We immunoprecipitated endogenous B55 by a pan-anti-B55 antibody from the FL5.12 cells and examined the association of Akt in the immunoprecipitated complexes. Significantly, Akt was co-immunoprecipitated with B55 subunits (Fig. 1B), and as expected, A and C subunits were also co-immunoprecipitated with B55 subunits (Fig. 1B). Because the pan-anti-B55 antibody preferentially reacts with the α isoform, we next investigated the possible association of PP2A AB55αC holoenzyme (hereafter referred to as PP2A-B55α holoenzyme) with Akt. HA-tagged B55α (B55αHA) was ectopically expressed in NIH3T3 cells and subsequently pulled down by anti-HA antibody. Co-immunoprecipitation analyses of B55αHA-associated complexes revealed that endogenous Akt was co-immunoprecipitated with PP2A-B55α holoenzymes in cells expressing B55αHA but not in parental NIH3T3 cells (Fig. 1C). In addition, the endogenous A (PP2A/A) and C subunits (PP2A/C) were co-immunoprecipitated with the exogenous B55αHA (Fig. 1C), indicating that the B55αHA associates with endogenous AC core enzyme and forms an AB55αHA holoenzyme complex. Furthermore, it was not clear whether B55α has the ability to directly associate with Akt and targets PP2A holoenzyme to Akt. We addressed this issue with in vitro interaction assays using bacterially expressed recombinant proteins. Recombinant GST, GST-fused PP2A/Aα, PP2A/B55α, or PP2A/Cα proteins were incubated with recombinant His-tagged Akt proteins and pulled down using glutathione-Sepharose. The results (Fig. 2) showed that His-tagged Akt proteins were pulled down with GST-fused B55α proteins by glutathione-Sepharose but not with control GST, GST-PP2A/Aα, or GST-PP2A/Cα proteins. These data demonstrate that B55α subunit directly targets the PP2A-B55α holoenzyme to interact with Akt.FIGURE 2Recombinant PP2A B55α regulatory subunit interacts with recombinant Akt in vitro. In vitro GST pulldown analyses were performed by mixing 3 μg of recombinant GST, GST-PP2A/Aα, GST-PP2A/Cα, or GST-PP2A/B55α with 2 μg of recombinant His-Akt and incubating at 4 °C. GST pulldowns were then analyzed by SDS-PAGE and immunoblotting by specific antibodies for GST and Akt. Asterisks indicate the intact full-length GST-PP2A subunit fusion proteins. Fifty percent of input recombinant His-Akt proteins (1 μg) were loaded in parallel and detected as a control. WB, Western blot.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The PP2A-B55α Holoenzyme Preferentially Regulates the Phosphorylation of Akt at Thr-308 Downstream of IL-3 Stimulation in FL5.12 Cells—Given that the PP2A-B55α holoenzyme interacts with Akt, we investigated the impact of this association on Akt phosphorylation. Akt achieves its full activity through phosphorylation at both Thr-308 and Ser-473 upon stimulation by growth factors or cytokines (57Alessi D" @default.
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• W2045852899 date "2008-01-01" @default.
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• W2045852899 title "Regulation of Phosphorylation of Thr-308 of Akt, Cell Proliferation, and Survival by the B55α Regulatory Subunit Targeting of the Protein Phosphatase 2A Holoenzyme to Akt" @default.
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