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• W2039604857 abstract "Activation of Akt/PKB by growth factors requires multiple phosphorylation events. Phosphorylation of Thr308 and Ser473 of Akt by its upstream kinase(s) or autophosphorylation is critical for optimal activation of its kinase activity. Here, we present evidence that tyrosine phosphorylation is required for Akt activation. Epidermal growth factor treatment induces tyrosine phosphorylation of Akt in COS1 and PC3M cells, which is abrogated by PP2, a selective inhibitor for Src family tyrosine kinases. Elevated Akt activity is observed in v-Src transformed NIH3T3 cells, which is accompanied with increased tyrosine phosphorylation of Akt. Akt activity induced by growth factors is significantly reduced in SYF cells lacking Src, Yes, and Fyn, which can be restored by introducing c-Src, but not the kinase-inactive Src, back to these cells. Furthermore, we have identified two tyrosine residues near the activation loop of Akt that are important for its activation. Substitution of these residues with phenylalanine abolishes Akt kinase activity stimulated by growth factors. These two YF mutants fail to block Forkhead transcription factor activity in 293 cells and are unable to prevent apoptosis induced by matrix detachment. Our data suggest that, in addition to phosphorylation of Thr308 and Ser473, tyrosine phosphorylation of Akt may be essential for its biological function. Activation of Akt/PKB by growth factors requires multiple phosphorylation events. Phosphorylation of Thr308 and Ser473 of Akt by its upstream kinase(s) or autophosphorylation is critical for optimal activation of its kinase activity. Here, we present evidence that tyrosine phosphorylation is required for Akt activation. Epidermal growth factor treatment induces tyrosine phosphorylation of Akt in COS1 and PC3M cells, which is abrogated by PP2, a selective inhibitor for Src family tyrosine kinases. Elevated Akt activity is observed in v-Src transformed NIH3T3 cells, which is accompanied with increased tyrosine phosphorylation of Akt. Akt activity induced by growth factors is significantly reduced in SYF cells lacking Src, Yes, and Fyn, which can be restored by introducing c-Src, but not the kinase-inactive Src, back to these cells. Furthermore, we have identified two tyrosine residues near the activation loop of Akt that are important for its activation. Substitution of these residues with phenylalanine abolishes Akt kinase activity stimulated by growth factors. These two YF mutants fail to block Forkhead transcription factor activity in 293 cells and are unable to prevent apoptosis induced by matrix detachment. Our data suggest that, in addition to phosphorylation of Thr308 and Ser473, tyrosine phosphorylation of Akt may be essential for its biological function. phosphatidylinositol protein kinase C Dulbecco's modified Eagle's medium mitogen-activated protein kinase hemagglutinin Madin-Darby canine kidney poly-(2-hydroxyethyl methacrylate) green fluorescent protein epidermal growth factor phosphotyrosine phosphatase in vitro kinase Akt/PKB, originally identified as an oncogene, is one of major downstream effectors of PI13-kinase and plays a pivotal role in the regulation of various biological processes, including apoptosis, proliferation, differentiation, and intermediary metabolism (for reviews, see Refs.1Chan T.O. Rittenhouse S.E. Tsichlis P.N. Annu. Rev. Biochem. 1999; 68: 965-1014Crossref PubMed Scopus (871) Google Scholar, 2Galetic I. Andjelkovic M. Meier R. Brodbeck D. Park J. Hemmings B.A. Pharmacol. Ther. 1999; 82: 409-425Crossref PubMed Scopus (95) Google Scholar, 3Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1381) Google Scholar). Activation of Akt by extracellular stimuli is a multistep process involving translocation and phosphorylation. Two phosphorylation sites, Thr308 and Ser473, have been identified to be critical for activation of Akt induced by growth factors (4Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2476) Google Scholar, 5Stokoe D. Stephens L.R. Copeland T. Gaffney P.R. Reese C.B. Painter G.F. Holmes A.B. McCormick F. Hawkins P.T. Science. 1997; 277: 567-570Crossref PubMed Scopus (1043) Google Scholar, 6Stephens 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 (907) Google Scholar). Phosphorylation of Thr308 in the activation loop by PDK1 is essential for Akt activation, and phosphorylation of Ser473 at the C-terminal tail by either autophosphorylation or by an as yet unidentified PDK2 is required for maximal activation of the kinase activity. Recent studies (7Li H.L. Davis W.W. Whiteman E.L. Birnbaum M.J. Pure E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6890-6895Crossref PubMed Scopus (56) Google Scholar, 8Craxton A. Jiang A. Kurosaki T. Clark E.A. J. Biol. Chem. 1999; 274: 30644-30650Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 9Wong B.R. Besser D. Kim N. Arron J.R. Vologodskaia M. Hanafusa H. Choi Y. Mol. Cell. 1999; 4: 1041-1049Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar, 10Xing L. Venegas A.M. Chen A. Garrett-Beal L. Boyce B.F. Varmus H.E. Schwartzberg P.L. Genes Dev. 2001; 15: 241-253Crossref PubMed Scopus (97) Google Scholar, 11Datta K. Bellacosa A. Chan T.O. Tsichlis P.N. J. Biol. Chem. 1996; 271: 30835-30839Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 12Nguyen K.T. Wang W.J. Chan J.L. Wang L.H. Oncogene. 2000; 19: 5385-5397Crossref PubMed Scopus (43) Google Scholar) suggest that tyrosine kinases may also play an important role in activation of Akt. Several groups reported that Syk and Btk are required for B cell antigen receptor-mediated activation of Akt in B lymphocytes (7Li H.L. Davis W.W. Whiteman E.L. Birnbaum M.J. Pure E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6890-6895Crossref PubMed Scopus (56) Google Scholar,8Craxton A. Jiang A. Kurosaki T. Clark E.A. J. Biol. Chem. 1999; 274: 30644-30650Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Akt activity is reduced in osteoclasts isolated from c-Src-deficient mice (9Wong B.R. Besser D. Kim N. Arron J.R. Vologodskaia M. Hanafusa H. Choi Y. Mol. Cell. 1999; 4: 1041-1049Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar, 10Xing L. Venegas A.M. Chen A. Garrett-Beal L. Boyce B.F. Varmus H.E. Schwartzberg P.L. Genes Dev. 2001; 15: 241-253Crossref PubMed Scopus (97) Google Scholar). Akt activity is up-regulated in Src-transformed cells and is activated by v-Src in a coexpression system in both insect Sf9 cells and mammalian cells (11Datta K. Bellacosa A. Chan T.O. Tsichlis P.N. J. Biol. Chem. 1996; 271: 30835-30839Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 12Nguyen K.T. Wang W.J. Chan J.L. Wang L.H. Oncogene. 2000; 19: 5385-5397Crossref PubMed Scopus (43) Google Scholar). However, the mechanisms by which these tyrosine kinases regulate Akt activity is not understood yet. Since tyrosine phosphorylation of Thr/Ser kinases such as PKCs has been reported to be important for its activation (13Li W. Mischak H., Yu, J.C. Wang L.M. Mushinski J.F. Heidaran M.A. Pierce J.H. J. Biol. Chem. 1994; 269: 2349-2352Abstract Full Text PDF PubMed Google Scholar, 14Konishi H. Tanaka M. Takemura Y. Matsuzaki H. Ono Y. Kikkawa U. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11233-11237Crossref PubMed Scopus (531) Google Scholar), we investigated the role of tyrosine phosphorylation in the regulation of Akt activity. Here, we present evidence that tyrosine phosphorylation of Akt is required for Akt activation in response to growth factors. Mutation of two tyrosine residues near the activation loop of Akt completely abolishes its kinase activity induced by growth factors. Interestingly, these two tyrosine residues are conserved in many Ser/Thr kinases, and phosphorylation of the corresponding residues Tyr512 and Tyr523 in PKCδ has been shown to be critical for PKCδ activation in response to H2O2 (14Konishi H. Tanaka M. Takemura Y. Matsuzaki H. Ono Y. Kikkawa U. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11233-11237Crossref PubMed Scopus (531) Google Scholar), suggesting that tyrosine phosphorylation of these two tyrosine residues may be a general mechanism by which these kinases are regulated. 293, COS1, and NIH3T3 cells (from ATCC) were maintained in DMEM with 10% fetal bovine serum. 3T3-vSrc cells were kindly provided by Dr. W. Tao at the University of Minnesota. The SYF cells are a generous gift of Dr. P. Soriano of the Fred Hutchinson Cancer Research Center. Purified c-Src and PTPβ were purchased from Upstate Biotechnology Inc. Akt in vitrokinase kit, anti-active caspase-3, and anti-phospho-MAPK were purchased from Cell Signaling Technology, Inc. Polyclonal Akt antibody was described previously (15Franke T.F. Yang S.I. Chan T.O. Datta K. Kazlauskas A. Morrison D.K. Kaplan D.R. Tsichlis P.N. Cell. 1995; 81: 727-736Abstract Full Text PDF PubMed Scopus (1810) Google Scholar). Anti-HA antibody was from Babco. pCMV6-HA-Akt, HA-Akt-KM, and myr-Akt were kindly provided by Dr. Philip Tsichlis. Site mutagenesis was carried out by using the polymerase chain reaction-based approach described previously (16Qiu Y. Robinson D. Pretlow T.G. Kung H.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3644-3649Crossref PubMed Scopus (213) Google Scholar). Mutations were all confirmed by sequencing. Transfections were performed by using Fugene 6 (Roche Molecular Biochemicals) or LipofactAMINE 2000 (Life Technologies, Inc.) according to the manufacturer's instructions. At 24 h posttransfection, the cells were serum-starved for 24 h followed by treatment as indicated in the legends of Figs. 1and 2.Figure 2Requirement of tyrosine phosphorylation for activation of Akt. a, constitutively active Src induces tyrosine phosphorylation of Akt. HA-tagged Akt or kinase-inactive Akt (Akt-KM) were cotransfected with a constitutively active Src (Src527F) or kinase-inactive Src (Src-KD) into COS1 cells. The cells were serum-starved for 24 h after transfection. One dish of the cells (lane 3) was treated with 10 µm LY294002 for 2 h before the cells were lysed. Cell lysates were subjected to immunoprecipitation and IVK as in Fig. 1 b. Tyrosine phosphorylation and phosphorylation of Thr308 or Ser473 of Akt were determined by immunoblotting with anti-phosphotyrosine (αpY), anti-phospho-Thr308 (αpT308), or anti-phospho-Ser473 (αpS473) antibodies.b, increase of the kinase activity of a membrane-bound Akt by Src527F. myr-HA-Akt was cotransfected with vector, p110CAAX, or Src527F into SYF cells. At 24 h posttransfection, the cells were serum-starved for 6 h. Akt activities were then assayed as in Fig.1 b. c, alignment of the subdomains VII and VIII of the kinase domains of Akt family kinases. The conserved tyrosine residues and Thr308 were highlighted and numbered according to mouse Akt1 sequence. d, the effect of mutation of the conserved tyrosine residues on tyrosine phosphorylation and kinase activity of Akt induced by Src527F. HA-tagged Aktwt or its derivatives carrying substitution of tyrosine residue(s) at the indicated position with phenylalanine(s) were cotransfected with Src527F. The cells were treated as in a and e. The effect of tyrosine residue mutation on EGF-induced Akt activity and tyrosine phosphorylation. HA-tagged Aktwt or its mutants was transfected into COS1 cells. After 24 h of serum starvation, the cells were treated with 100 ng/ml EGF for 10 min. Immunoprecipitation and IVK were performed as in Fig. 1 b. Tyrosine phosphorylation and phosphorylation of Thr308 of Akt were determined as ina. f, the effect of PTPβ treatment on Akt activity. HA-Akt was transfected with or without Src527F into COS1 cells. At 24 h posttransfection, the cells were serum-starved for 24 h. The Akt immunoprecipitates were pretreated with purified recombinant PTPβ (Upstate Biotechnology Inc.) at the indicated concentrations for 1 h. Akt activity was determined by IVK, and immunobloting with anti-phospho-Thr308, anti-Tyr(P), or anti-HA was performed as in a. g, Src phosphorylates Akt in vitro. IVK assays were performed as described under “Experimental Procedures.” Purified c-Src(3U) (Upstate Biotechnology Inc.) and 500 ng of purified GST-Akt-KM or GST-Akt-KM/325F/326F were incubated in the kinase buffer containing [γ-32P]ATP. Phosphorylation of both c-Src and its substrates was detected by autoradiography (top panel). PP2 was added in one sample to further corroborate that the detected phosphorylation resulted from Src kinase activity. The amount of the substrate in each reaction was monitored by Coomassie Blue staining shown in the bottom panel.View Large Image Figure ViewerDownload (PPT) The transfected cells were lysed in the buffer (20 mm Tris/HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton-X-100, 2.5 mm sodium pyrophosphate, 1 mm β-glycerol phosphate, 1 mm Na3VO4, 1 µg/ml aprotinin, 1 µg/ml leupetin, and 1 mmphenylmethylsulfonly fluoride). Insoluble material was removed by centrifugation, and antibodies were added to lysates for 1 h at 4 °C. Antibodies were collected with protein A- and protein G (1:1)-Sepharose beads, and protein complexes were washed three times at 4 °C with the lysis buffer. Immunoprecipitates were divided equally into two aliquots: one for kinase assay and the other for Western blot. The Akt immunoprecipitates were resuspended in nonreducing SDS sample buffer, which will minimize the interference of IgG, and resolved by SDS-polyacrylamide gel electrophoresis. Immunoblotting was performed as described previously (17Chen R. Kim O. Li M. Xiong X. Guan J. Kung H. Chen H. Shimizu Y. Qiu Y. Nat. Cell Biol. 2001; 3: 439-444Crossref PubMed Scopus (132) Google Scholar). Akt in vitro kinase assays were performed as reported by Franke et al. (15Franke T.F. Yang S.I. Chan T.O. Datta K. Kazlauskas A. Morrison D.K. Kaplan D.R. Tsichlis P.N. Cell. 1995; 81: 727-736Abstract Full Text PDF PubMed Scopus (1810) Google Scholar). Briefly, anti-HA immunoprecipitates were washed twice with kinase buffer (25 mm Tris, pH 7.5, 5 mm β-glycerol phosphate, 2 mm dithiothreitol, 0.1 mmNa3VO4, 10 mm MgCl2). The immune complex was then incubated at room temperature for 15 min in 30 µl of the kinase buffer with 0.1 µg/ml histone 2B, 2 µm ATP, and 10 µci of [γ-32P]ATP. The reaction was separated by 12% SDS-polyacrylamide gel electrophoresis. After the gels were dried, the phosphorylation of histone-H2B was visualized by autoradiography. For measurement of endogenous Akt activity, equivalent amounts of protein (determined by Bradford assay) were immunoprecipitated with anti-PKB/Akt antibody prebound to protein A-Sepharose beads, and kinase assays were carried out according to the instruction manual of the Akt Kinase Assay Kit (Cell Signaling Technology, Inc.). GSK-3 fusion protein was used as a substrate for PKB/Akt. The phosphorylated GSK-3 was detected by Western blot using phospho-GSK-3 (Ser 21/9) antibody. Src in vitro kinase assays were performed according to manufacturer's instructions (Upstate Biotechnology Inc.). 293 cells grown in a 12-well plate were transfected with 3XIRS (a luciferase reporter under the control of triple insulin-responsive sequence), empty vector, or FKHR expression plasmid and HA-tagged Akt or its mutants. Luciferase assays were performed as reported previously (18Tang E.D. Nunez G. Barr F.G. Guan K.L. J. Biol. Chem. 1999; 274: 16741-16746Abstract Full Text Full Text PDF PubMed Scopus (653) Google Scholar). At 24 h posttransfection, MDCK cells were detached by using trypsin/EDTA, washed, and resuspended with DMEM supplemented with 5% fetal bovine serum. Cells were maintained in suspension in poly-(2-hydroxyethyl methacrylate) (poly-HEMA)-coated 100-mm Petri dished. Coating was carried out with two rounds of 0.24 mg/ml poly-HEMA in ethanol and subsequent washing with DMEM. MDCK cells were kept in suspension for 12 h (19Rytomaa M. Lehmann K. Downward J. Oncogene. 2000; 19: 4461-4468Crossref PubMed Scopus (125) Google Scholar). The cells were then stained by Annexin V-PE (PharMingen) according to the manufacturer's procedure and the number of Annexin V-positive and GFP-positive cells was determined by flow cytometry. The number of the apoptotic cells was normalized with the transfection efficiency for each sample. Treatment of COS1 and prostate cancer cell line PC3M with EGF induces tyrosine phosphorylation of Akt, which is correlated with increased Akt kinase activity (Fig.1 a). The effect of EGF is inhibited by PP2, a selective inhibitor for Src family tyrosine kinases. Similar observations are obtained in COS1 cells transiently expressing HA-tagged Akt (Fig. 1 b), while MAPK activity induced by EGF is not affected by PP2. We also examined the endogenous Akt activity in v-Src-transformed NIH3T3 cells. As shown in Fig.1 c, accompanied by its tyrosine phosphorylation, Akt kinase activity is dramatically increased in v-Src-transformed NIH3T3 cells in comparison with the parental cell line. These results prompted us to examine Akt activation in response to growth factors in SYF, a cell line lacking Src, Yes, and Fyn (20Klinghoffer R.A. Sachsenmaier C. Cooper J.A. Soriano P. EMBO J. 1999; 18: 2459-2471Crossref PubMed Scopus (644) Google Scholar). In Fig. 1 d, treatment of SYF cells with 5 ng/ml platelet-derived growth factor or 50 ng/ml EGF failed to stimulate endogenous Akt activity. Akt activity is restored by introducing wild-type c-Src but not kinase-inactive Src (SrcKD), back to these cells. Taken together, these data suggest that Src family tyrosine kinases may enhance Akt activation by a tyrosine phosphorylation-dependent mechanism. To further demonstrate that Src kinases may directly regulate Akt activity, we tested whether Src is able to induce tyrosine phosphorylation of Akt in a cotransfection experiment. As shown in Fig.2 a, HA-tagged Akt is highly tyrosine-phosphorylated in the presence of a constitutively active Src (Src527F) but not kinase-inactive Src (Src-KD). Tyrosine phosphorylation of Akt is correlated with its increased kinase activity, evidenced by in vitro kinase assays using histone 2B as a substrate. Akt activity induced by Src is inhibited by the PI 3-kinase inhibitor LY294002, although the tyrosine phosphorylation of Akt remains unchanged. This is in agreement with previous observations that PI 3-kinase activity is required for Akt activation and that Src can up-regulate PI 3-kinase activity (11Datta K. Bellacosa A. Chan T.O. Tsichlis P.N. J. Biol. Chem. 1996; 271: 30835-30839Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 15Franke T.F. Yang S.I. Chan T.O. Datta K. Kazlauskas A. Morrison D.K. Kaplan D.R. Tsichlis P.N. Cell. 1995; 81: 727-736Abstract Full Text PDF PubMed Scopus (1810) Google Scholar,21Lee A.W. States D.J. Mol. Cell. Biol. 2000; 20: 6779-6798Crossref PubMed Scopus (77) Google Scholar). However, in SYF cells, Src527F can still significantly enhance the kinase activity of a membrane-bound myristoylated Akt (myr-Akt) without affecting the phosphorylation of Thr308 (Fig.2 b), while a constitutively active PI 3-kinase p110CAAX has little effect on the kinase activity of myr-Akt, which is consistent with the previous observation that the activity of myr-Akt is independent of PI 3-kinase (22Andjelkovic 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 (893) Google Scholar). These data raise the possibility that Src527F may regulate Akt activity through additional mechanism(s) in which PI 3-kinase is not involved and most likely by directly phosphorylating Akt. To define the potential tyrosine phosphorylation sites on Akt induced by Src, we were particularly interested in looking for those highly conserved tyrosine residues near the activation loop or C-terminal regulatory region. These tyrosine residues are often subjected to regulation by phosphorylation and more likely play a critical role in regulation of this highly conserved kinase family. Alignment of the subdomains VII and VIII of the kinase domains of Akt family from several organisms revealed three evolutionarily conserved tyrosine residues Tyr315, Tyr326, and Tyr340, which are close to the activation loop of Akt kinases (Fig. 2 c). To test whether these tyrosine residues are important for regulation of Akt activity, we substituted these residues with phenylalanines. As shown in Fig. 2 d, while mutation of Tyr340 has little effect on either tyrosine phosphorylation or kinase activity of Akt induced by Src527F, substitution of Tyr315 or Tyr326 with a phenylalanine, respectively, dramatically reduces both the tyrosine phosphorylation and kinase activity of Akt. The combination of these two mutations abolishes Src-induced tyrosine phosphorylation of Akt as well as its kinase activity. Similar results are obtained when we examine the effect of EGF on these mutants in COS1 cells (Fig.2 e). Interestingly, EGF-induced phosphorylation of Thr308 of all these YF mutants is largely unaffected, suggesting that the phenylalanine substitution has not perturbed the local conformation of the kinase domain. The lack of Akt kinase activity in the double mutant is more likely due to loss of tyrosine phosphorylation, which may be required for kinase activation. This is further corroborated by our data from the following in vitrokinase assays. As shown in Fig. 2 f, treatment of tyrosine-phosphorylated Akt with a phosphotyrosine-specific phosphatase PTPβ reduced both the tyrosine phosphorylation and kinase activity of Akt, while phosphorylation of Thr308 remains unchanged. Furthermore, purified c-Src can induce phosphorylation of purified kinase-inactive GST-Akt-KM, and this phosphorylation is inhibited by PP2 (Fig. 2 g). However, the mutant Akt carrying 315F/326F mutations (GST-Akt-KM/315F/326F) can no longer be phosphorylated. Taken together, these data indicate that Akt is a direct substrate of Src both in vivo and in vitro and that tyrosine phosphorylation of Akt is essential for its full activation by growth factors. To address whether tyrosine phosphorylation of Akt is required for its biological function, we first examined the effects of the Akt mutants carrying mutations at these tyrosine residues on the activity of Forkhead transcription factor, which is phosphorylated and negatively regulated by Akt in many cell types (18Tang E.D. Nunez G. Barr F.G. Guan K.L. J. Biol. Chem. 1999; 274: 16741-16746Abstract Full Text Full Text PDF PubMed Scopus (653) Google Scholar, 23Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5333) Google Scholar, 24Biggs W.H. Meisenhelder J. Hunter T. Cavenee W.K. Arden K.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7421-7426Crossref PubMed Scopus (936) Google Scholar). As shown in Fig.3a, cotransfection of wild-type Akt with FKHR into 293 cells inhibits FKHR transcription activity measured by the luciferase activity under the control of a promoter containing the FKHR recognition motif IRS. AktY326F has little effect on FKHR transcription activity, which is correlated with its poor kinase activity. The mutant AktY315F/Y326F and kinase-inactive mutant Akt-KM show strong dominant-negative effects on endogenous Akt and enhance FKHR transcription activity, consistent with complete loss of kinase activity in these mutants. The stronger inhibition by the double mutant over Akt-KM is possibly due to a slightly higher expression level of AktY315F/Y326F than Akt-KM. These data suggest that tyrosine phosphorylation of Akt is required for inhibition of FKHR transcription activity. Since one of biological consequences of inhibiting FKHR is to promote cell survival, we next examined whether tyrosine phosphorylation of Akt is required for its anti-apoptotic effect. Fig. 3 b shows that about 42% MDCK cells undergo apoptosis upon detachment from matrix as evidenced by increased Annexin V staining and activation of caspase-3. Overexpression of a constitutively active myristoylated Akt (myr-Akt) significantly reduces cell death to 26%, in agreement with previous studies (19Rytomaa M. Lehmann K. Downward J. Oncogene. 2000; 19: 4461-4468Crossref PubMed Scopus (125) Google Scholar). However, myr-AktY315F/Y326F fails to block the cell death and renders more than 50% of the cells susceptible to detachment induced apoptosis. Therefore, the integrity of these two tyrosine residues seems to be important for Akt's anti-apoptotic function. As another functional assay for Akt, we examined the effect of myr-Akt and its derivative on the growth of MDCK cells. Overexpression of myr-Akt results in enhanced proliferation of MDCK cells, while myr-AktY315F/Y326F has a dominant-negative effect on cell growth (data not shown). Our data suggest that tyrosine phosphorylation of Akt may be critical for its biological functions. In general, phosphorylation plays an essential role in regulation of the function of virtually all kinases. With no exception, Akt is also regulated by several phosphorylation events. In this report, we demonstrated that, in addition to phosphorylation of Thr308and Ser473, tyrosine phosphorylation may also play an important role in regulation of Akt activity. This notion is supported by our observation that EGF-induced tyrosine phosphorylation and kinase activity of Akt are both blocked by a Src family tyrosine kinase inhibitor PP2 in several cell lines tested as well as in SYF cells, and activation of Akt in response to EGF (50 ng/ml) or platelet-derived growth factor (5 ng/ml) is severely impaired and can be restored by cotransfection with c-Src but not kinase-inactive Src. These data suggest that Src family kinases may be required for Akt activation in response to growth factors in these cells. This is corroborated by our observation that Src527F can further enhance the kinase activity of myr-Akt, which is shown to be independent of PI 3-kinase. We also identified two tyrosine residues Tyr315 and Tyr326, which can be phosphorylated by Src both in vivo and in vitro. The substitution of these two tyrosine residues with phenylalanines renders Akt's loss of both tyrosine phosphorylation and kinase activity in response to growth factors. As a consequence, these Akt mutants can no longer inhibit their downstream target FKHR transcription activity and fail to promote cell survival. In this study, we demonstrated that the tyrosine kinase Src is able to phosphorylate Akt both in vivo and in vitro; therefore, Src family kinases may directly regulate Akt activity through a tyrosine phosphorylation-dependent manner rather than solely depends on activation of PI 3-kinase as suggested previously. Tyr315 lies in the activation loop, and phosphorylation of this residue may have a similar impact on the conformation of the kinase domain as that of Thr308 to open up the activation loop, hence resembling the mode of MAPK activation, which requires phosphorylation of both Thr202and Thr204 (25Payne D.M. Rossomando A.J. Martino P. Erickson A.K. Her J.H. Shabanowitz J. Hunt D.F. Weber M.J. Sturgill T.W. EMBO J. 1991; 10: 885-892Crossref PubMed Scopus (835) Google Scholar). Tyr326 is closed to subdomain VIII and more likely involved in stabilizing the substrate. Based on the fact that several residues preceding to Tyr326 are predominantly negative charge amino acids, and the preferred substrates for Akt all carry positively charged RXRXXS motifs, it is possible that phosphorylation of Tyr326 will allow the kinase form a more stable complex with its substrate(s) and enhance its catalytic activity. It is likely that other tyrosine kinases such as Syk or Btk family kinases may also be involved in Akt phosphorylation in different cell contexts or responding to different stimuli, since lack of B cell antigen receptor-mediated activation of Akt in Syk−/− or Btk−/− B lymphocytes has also been reported (7Li H.L. Davis W.W. Whiteman E.L. Birnbaum M.J. Pure E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6890-6895Crossref PubMed Scopus (56) Google Scholar, 8Craxton A. Jiang A. Kurosaki T. Clark E.A. J. Biol. Chem. 1999; 274: 30644-30650Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Given that these two tyrosine residues are highly conserved among all Akt isoforms, we anticipate that such regulation may also be applicable to other Akt isoforms. It is noteworthy that these two tyrosine residues are conserved in about 50% of Ser/Thr kinases, and phosphorylation of the corresponding residues Tyr512 and Tyr523 in PKCδ has been shown to be critical for PKCδ activation in response to H2O2 (14Konishi H. Tanaka M. Takemura Y. Matsuzaki H. Ono Y. Kikkawa U. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11233-11237Crossref PubMed Scopus (531) Google Scholar), suggesting that tyrosine phosphorylation of these two tyrosine residues may be a general mechanism by which these kinases are regulated. The level of detectable tyrosine phosphorylation of these kinases may depend on cell contexts and likely be affected by the content of tyrosine phosphotase(s). Our preliminary data show that phosphotyrosine is detectable after alkali treatment by two-dimentional electrophoretic analysis ofin vivo 32Pi-labeled Akt purified from EGF-treated COS1 cells (data not shown). Our current model for activation of Akt in response to growth factors is that multiple upstream events are required for Akt to achieve fully activated and biologically functional including: 1) translocation to the plasma membrane by binding to PI 3-kinase generated phospholipids, 2) phosphorylation of Thr308 and Ser473, and 3) phosphorylation of Tyr315 and Tyr326. However, the tyrosine phosphorylation and threonine/serine phosphorylation appear to be independent events, since AktY315F/Y326F is still phosphorylated at Thr308 and AktT308A/S473A is also tyrosine-phosphorylated (data not shown). Our data suggest that, in addition to phosphorylation of Thr308 and Ser473, tyrosine phosphorylation of Akt may be required for its full activation and biological functions. The mechanisms by which extracellular stimuli regulate Akt activity are actually far more complex than we previously thought and appear to be controlled by more than the PI 3-kinase-dependent pathway(s). We anticipate that more factors involved in regulation of Akt will be discovered in the future. We thank Tung Chan and Philip Tsichlis for providing many essential reagents, Philippe Soriano for providing SYF cells, Wufan Tao for providing NIH-v-Src cells, and Kun-Liang Guan for providing FKHR construct. We are also grateful to Hsing-Jien Kung, Tony Hunter, Jun-Lin Guan, Wenqun Li, and Keyong Du for helpful discussion." @default.
• W2039604857 created "2016-06-24" @default.
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• W2039604857 creator A5026598031 @default.
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• W2039604857 date "2001-08-01" @default.
• W2039604857 modified "2023-09-30" @default.
• W2039604857 title "Regulation of Akt/PKB Activation by Tyrosine Phosphorylation" @default.
• W2039604857 cites W1479869774 @default.
• W2039604857 cites W1532882622 @default.
• W2039604857 cites W1726025741 @default.
• W2039604857 cites W1743742701 @default.
• W2039604857 cites W1972239225 @default.
• W2039604857 cites W1973837499 @default.
• W2039604857 cites W1979147887 @default.
• W2039604857 cites W1980935526 @default.
• W2039604857 cites W2000586934 @default.
• W2039604857 cites W2004078164 @default.
• W2039604857 cites W2023245305 @default.
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