Inhibition of Insulin-induced Activation of Akt by a Kinase-deficient Mutant of the ε Isozyme of Protein Kinase C
2001; Elsevier BV; Volume: 276; Issue: 17 Linguagem: Inglês
10.1074/jbc.m011093200
ISSN1083-351X
AutoresMichihiro Matsumoto, Wataru Ogawa, Yasuhisa Hino, Kensuke Furukawa, Yoshitaka Ono, Mikiko Takahashi, Motoi Ohba, Toshio Kuroki, Masato Kasuga,
Tópico(s)Metabolism, Diabetes, and Cancer
ResumoAkt, also known as protein kinase B, is a protein-serine/threonine kinase that is activated by growth factors in a phosphoinositide (PI) 3-kinase-dependent manner. Although Akt mediates a variety of biological activities, the mechanisms by which its activity is regulated remain unclear. The potential role of the ε isozyme of protein kinase C (PKC) in the activation of Akt induced by insulin has now been examined. Expression of a kinase-deficient mutant of PKCε (εKD), but not that of wild-type PKCε or of kinase-deficient mutants of PKCα or PKCλ, with the use of adenovirus-mediated gene transfer inhibited the phosphorylation and activation of Akt induced by insulin in Chinese hamster ovary cells or L6 myotubes. Whereas the εKD mutant did not affect insulin stimulation of PI 3-kinase activity, the phosphorylation and activation of Akt induced by a constitutively active mutant of PI 3-kinase were inhibited by εKD, suggesting that εKD affects insulin signaling downstream of PI 3-kinase. PDK1 (3′-phosphoinositide-dependent kinase 1) is thought to participate in Akt activation. Overexpression of PDK1 with the use of an adenovirus vector induced the phosphorylation and activation of Akt; εKD inhibited, whereas wild-type PKCε had no effect on, these actions of PDK1. These results suggest that εKD inhibits the insulin-induced phosphorylation and activation of Akt by interfering with the ability of PDK1 to phosphorylate Akt. Akt, also known as protein kinase B, is a protein-serine/threonine kinase that is activated by growth factors in a phosphoinositide (PI) 3-kinase-dependent manner. Although Akt mediates a variety of biological activities, the mechanisms by which its activity is regulated remain unclear. The potential role of the ε isozyme of protein kinase C (PKC) in the activation of Akt induced by insulin has now been examined. Expression of a kinase-deficient mutant of PKCε (εKD), but not that of wild-type PKCε or of kinase-deficient mutants of PKCα or PKCλ, with the use of adenovirus-mediated gene transfer inhibited the phosphorylation and activation of Akt induced by insulin in Chinese hamster ovary cells or L6 myotubes. Whereas the εKD mutant did not affect insulin stimulation of PI 3-kinase activity, the phosphorylation and activation of Akt induced by a constitutively active mutant of PI 3-kinase were inhibited by εKD, suggesting that εKD affects insulin signaling downstream of PI 3-kinase. PDK1 (3′-phosphoinositide-dependent kinase 1) is thought to participate in Akt activation. Overexpression of PDK1 with the use of an adenovirus vector induced the phosphorylation and activation of Akt; εKD inhibited, whereas wild-type PKCε had no effect on, these actions of PDK1. These results suggest that εKD inhibits the insulin-induced phosphorylation and activation of Akt by interfering with the ability of PDK1 to phosphorylate Akt. Akt, also known as protein kinase B, is activated by growth factors such as platelet-derived growth factor (PDGF)1 and insulin (1Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1884) Google Scholar, 2Franke T.F. Yang S.I. Chan T.O. Datta K. Kazluskas A. Morrison D.K. Kaplan D.R. Tsichlis P.N. Cell. 1995; 81: 727-736Abstract Full Text PDF PubMed Scopus (1829) Google Scholar, 3Kohn A.D. Kovacina K.S. Roth R.A. EMBO J. 1995; 14: 4288-4295Crossref PubMed Scopus (320) Google Scholar), by cytokines (4de Peso L. Gonzalez-Galcia M. Page C. Herrera R. Nunez G. 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The activation of Akt by these stimuli contributes to a variety of their biological effects, including promotion of cell survival and protection from apoptosis (4de Peso L. Gonzalez-Galcia M. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1988) Google Scholar, 9Dudek H. Datta S.R. Franke T.F. Birnbaum M.J. Yao R. Cooper G.M. Segal R.A. Kaplan D.R. Greenberg M.E. Science. 1997; 275: 661-667Crossref PubMed Scopus (2222) Google Scholar), induction of meiosis in oocytes (10Andersen C.B. Roth R.A. Conti M. J. Biol. Chem. 1998; 273: 18705-18708Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), regulation of vascular contractility (8Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3056) Google Scholar, 11Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. 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Kuroda S. Kotani K. Klip A. Gingras A.-C. Sonenberg N. Kasuga M. J. Biol. Chem. 1999; 274: 20611-20618Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Activation of Akt is mediated by phosphorylation of a threonine residue in the kinase activation loop (Thr308 in Akt1) and a serine residue in the COOH-terminal region (Ser473 in Akt1) (18Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2530) Google Scholar). Akt mutants in which these residues are replaced by neutral amino acids are not activated in cells (15Kitamura T. Ogawa W. Sakaue H. Hino Y. Kuroda S. Takata M. Matsumoto M. Maeda T. Konishi H. Kikkawa U. Kasuga M. Mol. Cell. Biol. 1998; 18: 3708-3717Crossref PubMed Scopus (296) Google Scholar, 18Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2530) Google Scholar). The phosphorylation and activation of Akt induced by growth factors are blocked by pharmacological or molecular biological inhibitors of phosphoinositide (PI) 3-kinase (1Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1884) Google Scholar, 2Franke T.F. Yang S.I. Chan T.O. Datta K. Kazluskas A. Morrison D.K. Kaplan D.R. Tsichlis P.N. Cell. 1995; 81: 727-736Abstract Full Text PDF PubMed Scopus (1829) Google Scholar, 3Kohn A.D. Kovacina K.S. Roth R.A. EMBO J. 1995; 14: 4288-4295Crossref PubMed Scopus (320) Google Scholar, 15Kitamura T. Ogawa W. Sakaue H. Hino Y. Kuroda S. Takata M. Matsumoto M. Maeda T. Konishi H. Kikkawa U. Kasuga M. Mol. Cell. Biol. 1998; 18: 3708-3717Crossref PubMed Scopus (296) Google Scholar), indicating that PI 3-kinase is required for the phosphorylation of Thr308 and Ser473 and activation of Akt in response to growth factors. PDK1 (3′-phosphoinositide-dependent kinase 1), originally identified as a kinase that selectively phosphorylates Thr308 of Akt in vitro, is thought to contribute to Akt activation (19Alessi D.R. Deak M. Casamayor A. Caudwell F.B. Morrice N. Norman D.G. Gaffney P. Reese C.B. MacDougall C.N. Harbison D. Ashworth A. Bownes M. Curr. Biol. 1997; 7: 776-789Abstract Full Text Full Text PDF PubMed Scopus (623) Google Scholar, 20Alessi 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, 21Stephens 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 (915) Google Scholar). However, the mechanism by which PDK1 phosphorylates Akt in intact cells remains unclear. Although the phosphorylation of Akt by PDK1 is stimulated in the presence of 3′-phosphoinositides in vitro (19Alessi D.R. Deak M. Casamayor A. Caudwell F.B. Morrice N. Norman D.G. Gaffney P. Reese C.B. MacDougall C.N. Harbison D. Ashworth A. Bownes M. Curr. Biol. 1997; 7: 776-789Abstract Full Text Full Text PDF PubMed Scopus (623) Google Scholar, 20Alessi 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, 21Stephens 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 (915) Google Scholar), the kinase activity of PDK1 immunoprecipitated from cells is not affected by prior treatment of the cells with either growth factors or pharmacological inhibitors of PI 3-kinase (22Dong L.Q. Zhang R.-B. Langlais P. He H. Matthew C. Zhu L. Liu F. J. Biol. Chem. 1999; 274: 8117-8122Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), suggesting that PDK1 is constitutively active in cells. Given that Akt translocates from the cytosol to the membrane fraction of cells in response to growth factors (23Andjelkovic M. Alessi D.R. Meier R. Fernandez A. Lamb N.J. Frech M. Cron P. Cohen P. Lucocq J.M. Hemmings B.A. J. Biol. Chem. 1997; 272: 31515-31524Abstract Full Text Full Text PDF PubMed Scopus (904) Google Scholar), and that membrane-targeted mutants of Akt are constitutively active in quiescent cells (23Andjelkovic M. Alessi D.R. Meier R. Fernandez A. Lamb N.J. Frech M. Cron P. Cohen P. Lucocq J.M. Hemmings B.A. J. Biol. Chem. 1997; 272: 31515-31524Abstract Full Text Full Text PDF PubMed Scopus (904) Google Scholar, 24Kohn A.D. Takeuchi F. Roth R.A. J. Biol. Chem. 1996; 271: 21920-21926Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar), it is thought that membrane-associated Akt is phosphorylated by PDK1. Mutational analysis has revealed that phosphorylation of both Thr308 and Ser473 is required for Akt activation (18Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2530) Google Scholar). Although a kinase that phosphorylates Ser473 of Akt has been tentatively designated PDK2, its nature remains unclear. Balendran et al. (25Balendran A. Casamayor A. Deak M. Paterson A. Gaffney P. Currie R. Downes C.P. Alessi D.R. Curr. Biol. 1999; 9: 393-404Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar) recently showed that PDK1 phosphorylates Ser473 of Akt in vitro only in the presence of a glutathioneS-transferase (GST) fusion protein that contains the COOH-terminal portion of protein kinase C (PKC)-related kinase 2 (PRK2) or of synthetic peptides corresponding to this region of PRK2. In contrast, the phosphorylation of p70 S6 kinase by PDK1 was inhibited in the presence of the same GST fusion protein or peptides (26Balendran A. Currie R. Armstrong C.G. Avruch J. Alessi D.R. J. Biol. Chem. 1999; 274: 37400-37406Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Moreover, when overexpressed in intact cells, this same region of PRK2 prevented ligand-induced activation of p70 S6 kinase (26Balendran A. Currie R. Armstrong C.G. Avruch J. Alessi D.R. J. Biol. Chem. 1999; 274: 37400-37406Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). These observations suggest that the interactions of PDK1 with its substrates, at least those with Akt and p70 S6 kinase, are regulated by another kinase. PKCε is a member of the novel subfamily of PKC isozymes (27Ono Y. Fujii T. Ogita K. Kikkawa U. Igarashi K. Nishizuka Y. J. Biol. Chem. 1988; 263: 6927-6932Abstract Full Text PDF PubMed Google Scholar, 28Mellor H. Parker P.J. Biochem. J. 1998; 332: 281-292Crossref PubMed Scopus (1361) Google Scholar). When expressed in fibroblasts also expressing various mutant PDGF receptors, PKCε contributed to the transactivation of the TPA (12-O-tetradecanoylphorbol 13-acetate)-responsive element induced by PDGF in a PI 3-kinase-dependent manner (29Moriya S. Kazlauskas A. Akimoto K. Hirai S. Mizuno K. Takenawa T. Fukui Y. Watanabe Y. Ozaki S. Ohno S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 151-155Crossref PubMed Scopus (167) Google Scholar), suggesting that PKCε participates in signaling downstream of PI 3-kinase. We have therefore now investigated whether PKCε plays a role in activation of Akt. We examined the effects of overexpression of wild-type or a kinase-deficient mutant of PKCε on Akt activation induced by insulin, heat shock, or hydrogen peroxide. Expression of the kinase-deficient mutant of PKCε inhibited the phosphorylation and activation of Akt in response to all three stimuli, probably by affecting the ability of PDK1 to phosphorylate Akt. L6 myoblasts were maintained and induced to differentiate into myotubes as described previously (30Mitsumoto Y. Liu Z. Klip A. Endocrine J. 1993; 1: 307-315Google Scholar). We amplified a full-length mouse serum- and glucocorticoid-regulated protein kinase (SGK) 2 cDNA (31Kobayashi T. Deak M. Morrice N. Cohen P. Biochem. J. 1999; 344: 189-197Crossref PubMed Scopus (335) Google Scholar) by the polymerase chain reaction (PCR) with cDNA synthesized from RNA extracted from mouse liver as template. The amplified SGK2 cDNA was tagged with the HA epitope at its NH2 terminus with the use of PCR. To establish CHO-IR cells that express (in addition to insulin receptors) FLAG epitope-tagged Akt1 or HA epitope-tagged SGK2 (CHO-IR/Akt cells and CHO-IR/SGK2 cells, respectively), we transfected CHO-IR cells with pSV40-hgh (which confers resistance to hygromycin) and a PECE vector encoding FLAG-tagged rat Akt1 (RAC-PKα) (6Konishi H. Matsuzaki H. Tanaka M. Ono Y. Tokunaga C. Kuroda S. Kikkawa U. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7639-7643Crossref PubMed Scopus (189) Google Scholar) or a pCMV4 vector encoding HA-tagged mouse SGK2. Transfected cells were selected and cloned as described previously (32Kotani K. Ogawa W. Matsumoto M. Kitamura T. Sakaue H. Hino Y. Miyake K. Sano W. Akimoto K. Ohno S. Kasuga M. Mol. Cell. Biol. 1998; 18: 6971-6982Crossref PubMed Google Scholar). CHO-IR cells that express phosphodiesterase 3B (PDE3B), designated CHO-IR/PDE3B-WT cells, were previously described (16Kitamura T. Kitamura Y. Kuroda S. Hino Y. Ando M. Kotani K. Konishi H. Matsuzaki H. Kikkawa U. Ogawa W. Kasuga M. Mol. Cell. Biol. 1999; 19: 6286-6296Crossref PubMed Scopus (311) Google Scholar). Polyclonal antibodies to Akt, to mitogen-activated protein (MAP) kinase, and to PDE3B were generated as described previously (15Kitamura T. Ogawa W. Sakaue H. Hino Y. Kuroda S. Takata M. Matsumoto M. Maeda T. Konishi H. Kikkawa U. Kasuga M. Mol. Cell. Biol. 1998; 18: 3708-3717Crossref PubMed Scopus (296) Google Scholar, 16Kitamura T. Kitamura Y. Kuroda S. Hino Y. Ando M. Kotani K. Konishi H. Matsuzaki H. Kikkawa U. Ogawa W. Kasuga M. Mol. Cell. Biol. 1999; 19: 6286-6296Crossref PubMed Scopus (311) Google Scholar, 33Sakaue H. Ogawa W. Takata M. Kuroda S. Kotani K. Matsumoto M. Sakaue M. Nishio S. Ueno H. Kasuga M. Mol. Endocrinol. 1997; 11: 1552-1562Crossref PubMed Scopus (116) Google Scholar). Polyclonal antibodies to PKCε as well as monoclonal antibodies to PKCα and to PKCλ were obtained from Santa Cruz Biotechnology and Transduction Laboratories, respectively. Monoclonal antibodies to the HA epitope tag (12CA5) or to the FLAG epitope tag were obtained from Roche Molecular Biochemicals. Polyclonal antibodies specific for phospho-Thr308 or phospho-Ser473 forms of Akt or for the phosphorylated form of MAP kinase were obtained from New England BioLabs. Adenovirus vectors encoding a kinase-deficient mutant of PKCλ in which Lys273 is replaced by glutamate (AxCAλKD) (32Kotani K. Ogawa W. Matsumoto M. Kitamura T. Sakaue H. Hino Y. Miyake K. Sano W. Akimoto K. Ohno S. Kasuga M. Mol. Cell. Biol. 1998; 18: 6971-6982Crossref PubMed Google Scholar), or a constitutively active mutant of PI 3-kinase (AxCAMyr-p110) (16Kitamura T. Kitamura Y. Kuroda S. Hino Y. Ando M. Kotani K. Konishi H. Matsuzaki H. Kikkawa U. Ogawa W. Kasuga M. Mol. Cell. Biol. 1999; 19: 6286-6296Crossref PubMed Scopus (311) Google Scholar, 34Kotani K. Ogawa W. Hino Y. Kitamura T. Ueno H. Sano W. Sutherland C. Granner D.K. Kasuga M. J. Biol. Chem. 1999; 274: 21305-21312Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) were described previously. Complementary DNAs for wild-type PKCε (εWT, Ref. 27Ono Y. Fujii T. Ogita K. Kikkawa U. Igarashi K. Nishizuka Y. J. Biol. Chem. 1988; 263: 6927-6932Abstract Full Text PDF PubMed Google Scholar) and a kinase-deficient mutant of PKCε in which Lys437 is replaced by methionine (εKD, Ref. 35Kuroda S. Tokunaga C. Kiyohara Y. Higuchi O. Konishi H. Mizuno K. Kikkawa U. J. Biol. Chem. 1996; 271: 31029-31032Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar) were modified with the use of PCR to encode the T7 or FLAG epitope tags at the NH2 termini of the respective proteins. A kinase-deficient mutant of PKCε in which Thr566 is replaced by alanine (εT566A) was as described (36Takahashi M. Mukai H. Oishi K. Isagawa T. Ono Y. J. Biol. Chem. 2000; 275: 34592-34596Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). A constitutively active mutant of PKCε in which Ala159 is replaced by glutamate (εA159E) was constructed from the cDNA that encodes FLAG epitope-tagged εWT with the use of a QuickChange site-directed mutagenesis kit (Stratagene). Complementary DNAs encoding T7-tagged εWT, the FLAG-tagged εKD, εT566A, FLAG-tagged εA159E, Myc epitope-tagged PDK1 (21Stephens 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 (915) Google Scholar), kindly provided by L. Stephens (The Babraham Institute), or a kinase-deficient mutant of PKCα in which Lys368 is replaced with arginine (37Ohno S. Konno Y. Akita Y. Yano Y. Suzuki K. J. Biol. Chem. 1990; 265: 6296-6300Abstract Full Text PDF PubMed Google Scholar) were subcloned into pAxCAwt (38Miyake S. Makimura M. Kanegae Y. Harada S. Sato Y. Takamori K. Tokuda C. Saito I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1320-1324Crossref PubMed Scopus (788) Google Scholar), and adenoviral vectors containing these cDNAs were generated with the use of an adenovirus expression kit (Takara, Tokyo, Japan) as described previously (15Kitamura T. Ogawa W. Sakaue H. Hino Y. Kuroda S. Takata M. Matsumoto M. Maeda T. Konishi H. Kikkawa U. Kasuga M. Mol. Cell. Biol. 1998; 18: 3708-3717Crossref PubMed Scopus (296) Google Scholar, 16Kitamura T. Kitamura Y. Kuroda S. Hino Y. Ando M. Kotani K. Konishi H. Matsuzaki H. Kikkawa U. Ogawa W. Kasuga M. Mol. Cell. Biol. 1999; 19: 6286-6296Crossref PubMed Scopus (311) Google Scholar). The resultant adenovirus vectors were termed AxCAεWT, AxCAεKD, AxCAεT566A, AxCAεA159E, AxCAPDK1, and AxCAαKD, respectively. CHO-IR/Akt cells or differentiated L6 myotubes were infected with adenovirus vectors at the indicated multiplicity of infection (MOI), expressed in plaque-forming units (PFU) per cell, as described previously (33Sakaue H. Ogawa W. Takata M. Kuroda S. Kotani K. Matsumoto M. Sakaue M. Nishio S. Ueno H. Kasuga M. Mol. Endocrinol. 1997; 11: 1552-1562Crossref PubMed Scopus (116) Google Scholar). The cells were subjected to experiments 24–48 h after infection. L6 myotubes or CHO-IR/Akt cells were deprived of serum for 16–20 h, incubated in the absence or presence of 100 nm insulin for the indicated time, and then immediately frozen with liquid nitrogen. The assay for MAP kinase activity was performed with immunoprecipitates prepared with antibodies to MAP kinase, as described previously (33Sakaue H. Ogawa W. Takata M. Kuroda S. Kotani K. Matsumoto M. Sakaue M. Nishio S. Ueno H. Kasuga M. Mol. Endocrinol. 1997; 11: 1552-1562Crossref PubMed Scopus (116) Google Scholar). For assay of PI 3-kinase activity, cells were lysed and subjected to immunoprecipitation with antibodies to phosphotyrosine (PY20; Transduction Laboratories); the resulting immunoprecipitates were washed and PI 3-kinase activity in the washed precipitates was assayed as described previously (33Sakaue H. Ogawa W. Takata M. Kuroda S. Kotani K. Matsumoto M. Sakaue M. Nishio S. Ueno H. Kasuga M. Mol. Endocrinol. 1997; 11: 1552-1562Crossref PubMed Scopus (116) Google Scholar). For assay of Akt activity, cells were lysed and subjected to immunoprecipitation with polyclonal antibodies to Akt as described (15Kitamura T. Ogawa W. Sakaue H. Hino Y. Kuroda S. Takata M. Matsumoto M. Maeda T. Konishi H. Kikkawa U. Kasuga M. Mol. Cell. Biol. 1998; 18: 3708-3717Crossref PubMed Scopus (296) Google Scholar). The immunoprecipitates were then mixed with 30 μl of kinase reaction mixture containing 50 mm Tris-HCl, pH 7.5, 10 mm MgCl2, 1 mm dithiothreitol, 1 mm of the specific peptide inhibitor of cAMP-dependent protein kinase (PKI), 5 μmnonradioactive ATP, 2 μCi of [γ-32P]ATP (4000 Ci/mmol), and 5 μm Crosstide peptide (GRPRTSSFAEG) (39Cross D.A.E. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4393) Google Scholar) and then incubated for 30 min at 30 °C. Assay of SGK activity was performed essentially as described (40Kobayashi T. Cohen P. Biochem. J. 1999; 339: 319-328Crossref PubMed Scopus (530) Google Scholar) with the following modifications. Cells were lysed in a solution containing 50 mm Tris-HCl, pH 7.5, 120 mm NaCl, 1% Triton X-100, 1 mm benzamidine, 25 mm NaF, 40 mm β-glycerophosphate, 1 mmphenylmethylsulfonyl fluoride, and leupeptin (10 μg/ml). The lysate was centrifuged, and the resulting supernatant was subjected to immunoprecipitation with antibodies to HA. The immunoprecipitates were washed once with the lysis buffer containing 500 mm NaCl and with the lysis buffer and then twice with 50 mmTris-HCl, pH 7.5 containing 0.1% (v/v) of β-mercaptoethanol. The immunoprecipitates were then mixed with 30 μl of kinase reaction mixture containing 60 mm Tris-HCl, pH 7.5, 12 mm MgCl2, 0.12 mm EDTA, 0.12% (v/v) β-mercaptoethanol, 3 μg/ml of PKI, 5 μmnonradioactive ATP, 4 μCi of [γ-32P]ATP (4000 Ci/mmol), and 36 μm Crosstide peptide, and then incubated for 60 min at 30 °C. The kinase recation mixture for Akt or SGK was spotted onto a P81 phosphocellulose filter (Whatman), the filters were washed three times with 0.5% (w/v) orthophosphoric acid, and the radioactivity remaining on the filters was measured. The in vivophosphorylation of PDE3B was assayed as described previously (16Kitamura T. Kitamura Y. Kuroda S. Hino Y. Ando M. Kotani K. Konishi H. Matsuzaki H. Kikkawa U. Ogawa W. Kasuga M. Mol. Cell. Biol. 1999; 19: 6286-6296Crossref PubMed Scopus (311) Google Scholar). In brief, CHO-IR/PDE3B-WT cells, previously infected (or not) with AxCAεKD, were labeled with [32P]orthophosphate, incubated in the absence or presence of 100 nm insulin for 15 min, and lysed. Cell lysates were subjected to immunoprecipitation with polyclonal antibodies to PDE3B, the resulting precipitates were separated by SDS-polyacrylamide gel electrophoresis on a 7% gel, and the incorporation of radioactivity into PDE3B was visualized and quantitated with a Fuji BAS2000 image analyzer. Infection of CHO-IR/Akt cells, which stably express both human insulin receptors and rat Akt1, with an adenovirus vector that encodes a kinase-deficient mutant of PKCε (AxCAεKD) resulted in a dose-dependent increase in the amount of PKCε protein as assessed by immunoblot analysis; the amount of PKCε protein in cells infected at an MOI of 10 PFU/cell was ∼10–20 times that of endogenous PKCε (Fig.1 A). Exposure of noninfected CHO-IR/Akt cells to insulin resulted in a >30-fold increase in the amount of Akt activity measured in immunoprecipitates prepared with antibodies to Akt (Fig. 1 A). Expression of the kinase-deficient mutant of PKCε (εKD) in the cells inhibited insulin-induced activation of Akt in a dose-dependent manner. Infection of CHO-IR/Akt cells with AxCAεKD also inhibited insulin-induced phosphorylation of both Thr308 and Ser473 of Akt, as assessed by immunoblot analysis with antibodies specific for either phospho-Thr308 or phospho-Ser473 forms of Akt (Fig. 1 A). Expression of a structurally distinct kinase deficient mutant of PKCε (εT566A) also exerted similar inhibitory effects on insulin-induced activity (Fig. 1 B) and phosphorylation (data not shown) of Akt in CHO-IR/Akt cells. We next investigated whether εKD exerted a similar inhibitory effect on Akt activation in physiological target cells of insulin. Insulin induced an approximately 6-fold increase in Akt activity in L6 myotubes (Fig. 1 C). Expression of εKD inhibited insulin-induced phosphorylation and activation of Akt in a dose-dependent manner (Fig. 1 C). In contrast, overexpression of wild-type PKCε in L6 myotubes did not affect insulin-induced phosphorylation and activation of Akt (Fig. 1 D). Expression of wild-type PKCε also had no effect on the activation of Akt in response to insulin in CHO-IR/Akt cells, and expression of either wild-type PKCε or a constitutively active mutant of PKCε (εA159E), of which kinase activity was ∼10-fold higher than that of wild-type PKCε (data not shown), inconsistent with a previous report (41Genot E.M. Parker P.J. Cantrell D.A. J. Biol. Chem. 1995; 270: 9833-9839Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar), did not induce Akt activation in the absence of insulin in CHO/IR Akt cells or in L6 myotubes (data not shown). These results suggest that εKD inhibits insulin-induced activation of Akt by preventing the phosphorylation of Thr308 and Ser473, and that signal mediated through PKCε alone is not sufficient to activate Akt in both CHO-IR/Akt cells and L6 myotubes. We investigated whether kinase-deficient mutants of other PKC isozymes also affected the activation of Akt by insulin. Infection of L6 myotubes with adenovirus vectors encoding εKD, wild-type PKCε, or kinase-deficient mutants of PKCα (αKD) or PKCλ (λKD) resulted in marked expression of the encoded proteins (Fig.2 A); at an MOI of 20 PFU/cell; the amount of each recombinant protein was at least 10 times that of the corresponding endogenous PKC isoform. Whereas expression of εKD inhibited insulin-induced activation of Akt, αKD had no effect on this action of insulin (Fig. 2 A). The expression of λKD resulted in a slight enhancement of the effect of insulin on Akt activity, consistent with previous observations (42Doornbos R.P. Theelen M. van der Hoeven P.C. van Blitterswijk W.J. Verkleij A.J. van Bergen en Henegouwen P.M. J. Biol. Chem. 1999; 274: 8589-8596Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Akt is also activated by heat shock and by hydrogen peroxide (6Konishi H. Matsuzaki H. Tanaka M. Ono Y. Tokunaga C. Kuroda S. Kikkawa U. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7639-7643Crossref PubMed Scopus (189) Google Scholar, 7Morag S. Cohen P. Alessi D.R. Biochem. J. 1998; 336: 241-246Crossref PubMed Scopus (241) Google Scholar). These stimuli induced ∼10- and 20-fold increases in Akt activity, respectively, in CHO-IR/Akt cells (Fig. 2 B). Expression of εKD inhibited the activation of Akt in response to either heat shock or hydrogen peroxide. The phosphorylation of Akt on Thr308 and Ser473 induced by these stimuli was also inhibited by expression of εKD (Fig. 2 B). PDE3B was recently identified as a direct substrate of Akt (16Kitamura T. Kitamura Y. Kuroda S. Hino Y. Ando M. Kotani K. Konishi H. Matsuzaki H. Kikkawa U. Ogawa W. Kasuga M. Mol. Cell. Biol. 1999; 19: 6286-6296Crossref PubMed Scopus (311) Google Scholar). In CHO-IR cells expressing PDE3B, insulin induced an approximately 2-fold increase in the extent of phosphorylation of PDE3B (Fig. 3). Infection of the cells with AxCAεKD at an MOI of 3 PFU/cell, a virus dose that inhibited insulin-induced activation of Akt by ∼50% in CHO-IR/Akt cells (Fig. 1 A), resulted in ∼50% inhibition of insulin-induced phosphorylation of PDE3B. This observation suggests that inhibition by εKD of Akt activation attenuates signaling downstream of Akt. We further attempted to identify the step of the insulin signaling pathway leading to activation of Akt that is affected by εKD. Overexpression of εKD had no effect on the insulin-induced increase in the activity of PI 3-kinase immunoprecipitated from L6 myotubes with antibodies to phosphotyrosine (Fig. 4 A), suggesting that εKD affects insulin-induced activation of Akt at a step downstream of PI 3-kinase. To verify this conclusion, we examined the effect of εKD on the activation of Akt by a constitutively active mutant of PI 3-kinase, Myr-p110, which comprises the catalytic subunit of PI 3-kinase ligated to a myristoylation signal sequence at its NH2 terminus (16Kitamura T. Kitamura Y. Kuroda S. Hino Y. Ando M. Kotani K. Konishi H. Matsuzaki H. Kikkawa U. Ogawa W. Kasuga M. Mol. Cell. Biol. 1999; 19: 6286-6296Crossref PubMed Scopus (311) Google Scholar, 34Kotani K. Ogawa W. Hino Y. Kitamura T. Ueno H. Sano W. Sutherland C. Granner D.K. Kasuga M. J. Biol. Chem. 1999; 274: 21305-21312Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Infection of L6 myotubes with an adenovirus vector encoding Myr-p110 (AxCAMyr-p110) induced both the phosphorylation and activation of Akt (Fig. 4 B). Coexpression of εKD inhibited in a dose-dependent manner the phosphorylation and activation of Akt induced by Myr-p110 (Fig.4 B); expression of εKD did not affect the amount Myr-p110 protein as assessed by immunoblot analysis (data not shown). These observations are thus consistent with the notion that εKD inhibits insulin-induced activation of Akt by affecting signaling downstream of PI 3-kinase. Expression of εKD did not affect insulin-induced phosphorylation and activation of MAP kinase (Fig. 4 C), indicating that early events of insulin signaling, such as activation of the insulin receptor kinase and phosphorylation of its substrates, are not prevented by εKD. PDK1 is a serine/threonine kinase that phosphorylates and activates Akt in vitro (19Alessi D.R. Deak M. Casamayor A. Caudwell F.B. Morrice N. Norman D.G. Gaffney P. Reese C.B. MacDougall C.N. Harbison D. Ashworth A. Bownes M. Curr. Biol. 1997; 7: 776-789Abstract Full Text Full Text PDF PubMed Scopus (623) Google Scholar, 20Alessi 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, 21Stephens 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 (915) Google Scholar). We thus investigated the effect of εKD on PDK1-induced activation of Akt. Infection of CHO-IR/Akt cells with an adenovirus vector encoding PDK1 (AxCAPDK1) induced the phosphorylation of Akt on both Thr308 and Ser473 as well as the activation of this enzyme (Fig. 5). Coexpression of εKD with PDK1 resulted in inhibition of the PDK1-induced phosphorylation and activation of Akt, with no effect on the amount of PDK1 protein. In contrast, expression of wild-type PKCε had no effect on the phosphorylation and activation of Akt induced by PDK1. These results suggest that εKD inhibited the ability of PDK1 to phosphorylate and activate Akt. PDK1 has recently been shown to contribute to the phosphorylation and the activation of SGK (31Kobayashi T. Deak M. Morrice N. Cohen P. Biochem. J. 1999; 344: 189-197Crossref PubMed Scopus (335) Google Scholar, 40Kobayashi T. Cohen P. Biochem. J. 1999; 339: 319-328Crossref PubMed Scopus (530) Google Scholar, 43Park J. Leong M.L. Buse P. Maiyar A.C. Firestone G.L. Hemmings B.A. EMBO J. 1999; 18: 3024-3033Crossref PubMed Scopus (482) Google Scholar). We finally investigated the effect of εKD on insulin-induced activation of SGK. CHO-IR/SGK2 cells, which stably express both human insulin receptors and HA-tagged mouse SGK2, were incubated in the absence or presence of insulin for 10 min, lysed, and immunoprecipitated with antibodies to HA, and then SGK kinase activity toward Crosstide was assayed in the immunoprecipitates. Insulin induced ∼6-fold increase in the activity of SGK, and overexpression of εKD in the cells inhibited insulin-induced activation of SGK in a dose-dependent manner (Fig. 6). We have shown that overexpression of kinase-deficient mutants of PKCε (εKD or εT566A) with the use of adenovirus-mediated gene transfer inhibited the phosphorylation and activation of Akt induced by insulin. An adenovirus vector encoding wild-type PKCε had no effect on the insulin-induced increase in Akt activity, and the virus encoding εKD had no effect on insulin-induced activation of either MAP kinase or PI 3-kinase, suggesting that the inhibition of Akt activation by εKD is due neither to nonspecific effects of viral infection nor to general inhibition of insulin signaling. The phosphorylation and activation of Akt induced by heat shock or hydrogen peroxide were also inhibited by εKD, suggesting that this mutant protein affects a common component of the Akt activation pathways triggered by various extracellular stimuli. PI 3-kinase acts as an upstream mediator of Akt activation induced by several stimuli (1Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1884) Google Scholar, 2Franke T.F. Yang S.I. Chan T.O. Datta K. Kazluskas A. Morrison D.K. Kaplan D.R. Tsichlis P.N. Cell. 1995; 81: 727-736Abstract Full Text PDF PubMed Scopus (1829) Google Scholar, 3Kohn A.D. Kovacina K.S. Roth R.A. EMBO J. 1995; 14: 4288-4295Crossref PubMed Scopus (320) Google Scholar, 5Murga C. Laguinge L. Wetzker R. Cuadrado A.M. Gutkind J.S. J. Biol. Chem. 1998; 273: 19080-19085Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 7Morag S. Cohen P. Alessi D.R. Biochem. J. 1998; 336: 241-246Crossref PubMed Scopus (241) Google Scholar). However, our observations that εKD both did not inhibit insulin-induced activation of PI 3-kinase and prevented Akt activation by a constitutively active mutant of PI 3-kinase indicate that εKD affects a signaling component that acts downstream of PI 3-kinase. PDK1 was originally identified as a kinase that phosphorylates Thr308 of Akt in vitro (20Alessi 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, 21Stephens 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 (915) Google Scholar), and an unidentified kinase that phosphorylates Ser473 of Akt has been termed PDK2. We have now shown that overexpression of PDK1 induced the phosphorylation of Akt on both Thr308 and Ser473 in intact cells. PDK1 has been shown to phosphorylate Ser473 of Akt in vitro in the presence of a GST fusion protein containing the COOH-terminal region of PRK2 or of synthetic peptides that encompass this region of PRK2 (25Balendran A. Casamayor A. Deak M. Paterson A. Gaffney P. Currie R. Downes C.P. Alessi D.R. Curr. Biol. 1999; 9: 393-404Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar). It is thus possible that, in intact cells, PDK1 phosphorylates both Thr308 and Ser473 of Akt, which would explain why overexpression of PDK1 alone resulted in the activation of Akt. We cannot, however, exclude the possibility that expression of PDK1 resulted in the activation of PDK2, and that PDK1 and PDK2 coordinately phosphorylate and activate Akt. We have shown that εKD inhibited PDK1-induced phosphorylation and activation of Akt, suggesting that εKD affects the insulin signaling pathway at a step downstream of PDK1 action. PDK1 has been shown, at least in vitro, to phosphorylate not only Akt but various other kinases including p70 S6 kinase (44Pullen N. Dennis P.B. Andjelkovic M. Dufner A. Kozma S.C. Hemmings B. Thomas G. Science. 1998; 279: 707-710Crossref PubMed Scopus (731) Google Scholar), SGK (31Kobayashi T. Deak M. Morrice N. Cohen P. Biochem. J. 1999; 344: 189-197Crossref PubMed Scopus (335) Google Scholar, 40Kobayashi T. Cohen P. Biochem. J. 1999; 339: 319-328Crossref PubMed Scopus (530) Google Scholar, 43Park J. Leong M.L. Buse P. Maiyar A.C. Firestone G.L. Hemmings B.A. EMBO J. 1999; 18: 3024-3033Crossref PubMed Scopus (482) Google Scholar), p90RSK (45Jensen C.J. Buch M.B. Krag T.O. Hemmings B.A. Gammeltoft S. Frodin M. J. Biol. Chem. 1999; 274: 27168-27176Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar), cAMP-dependent protein kinase (46Cheng X. Ma Y. Moore M. Hemmings B.A. Taylor S.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9848-9854Google Scholar), and PKC isozymes including PKCζ and PKCδ (47Le Good J.A. Ziegler W.H. Parekh D.B. Alessi D.R. Cohen P. Parker P.J. Science. 1998; 281: 2042-2045Crossref PubMed Scopus (976) Google Scholar), all of which belong to the AGC family of protein kinases. Thus, the observation that εKD inhibited insulin-induced activity of SGK is also consistent with this hypothesis that εKD interfere with the insulin signaling at a step downstream of PDK1. Given that PKCε is a member of this family of kinases, it also might serve as a substrate for PDK1. However, it is not likely that εKD inhibits insulin-induced activation of Akt simply by competing with Akt for PDK1, because expression of wild-type PKCε did not inhibit this effect of insulin. The mechanism by which εKD inhibits the ability of PDK1 to activate Akt remains unclear. It is possible that endogenous PKCε phosphorylates an unidentified substrate that is important for the interaction between PDK1 and Akt, and that εKD exerts a dominant-negative effect on endogenous PKCε. Our observation that overexpression of wild-type PKCε or a constitutively active form of PKCε alone did not increase Akt activity in the absence of insulin may be explained if such a substrate is constitutively phosphorylated in cells, so that overexpression of the wild-type or a constitutively active enzyme would not alter the phosphorylation state of the substrate. The COOH-terminal region of PRK2, which includes a consensus sequence for a PDK2 phosphorylation site similar to that present in Akt with the exception that the residue equivalent to Ser473 is aspartic acid, modulates PDK1 activity (25Balendran A. Casamayor A. Deak M. Paterson A. Gaffney P. Currie R. Downes C.P. Alessi D.R. Curr. Biol. 1999; 9: 393-404Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar, 26Balendran A. Currie R. Armstrong C.G. Avruch J. Alessi D.R. J. Biol. Chem. 1999; 274: 37400-37406Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Analysis of the three-dimensional structure of PDK1 suggests the presence in the kinase domain of a hydrophobic pocket that interacts with this region of PRK2 (48Biondi R.M. Cheung P.C. Casamayor A. Deak M. Currie R.A. Alessi D.R. EMBO J. 2000; 19: 979-988Crossref PubMed Scopus (251) Google Scholar). Given that PKCε also contains a sequence similar to the COOH-terminal region of PRK2, it is possible that εKD affects the ability of PDK1 to phosphorylate Akt by interacting with the hydrophobic pocket of PDK1. Whereas serine and threonine residues in the COOH-terminal region of wild-type PKCβII are phosphorylated in intact cells, those of a kinase-deficient mutant of the enzyme are not (49Behn-Krappa A. Newton A.C. Curr. Biol. 1999; 9: 728-737Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). It is thus possible that the phosphorylation status of wild-type PKCε and εKD differ and that the difference in the abilities of these molecules to affect Akt activation might be because of such a difference in phosphorylation status. platelet-derived growth factor phosphoinositide 3′-phosphoinositide-dependent kinase glutathioneS-transferase protein kinase C PKC-related kinase 2 serum- and glucocorticoid-regulated protein kinase polymerase chain reaction phosphodiesterase 3B mitogen-activated protein multiplicity of infection plaque-forming unit Chinese hamster ovary wild-type
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