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• W2052081639 abstract "Protein kinase Akt, an important downstream target of phosphatidylinositol 3-kinase, is one of the major survival factors in mammalian cells. It has been shown that phosphorylation of the C-terminal hydrophobic motif is required for Akt activation. The activated Akt then phosphorylates several pro-apoptotic proteins and prevents apoptosis mediated by caspases and the mitochondria. Interestingly, Akt has also been implicated to be a direct substrate of caspases in apoptotic cells induced by Fas (Widmann, C., Gibson, S., and Johnson, G. L. (1998) J. Biol. Chem. 273, 7141–7147) and anoikis (Bachelder, R. E., Wendt, M. A., Fujita, N., Tsuruo, T., and Mercurio, A. M. (2001) J. Biol. Chem. 276, 34702–34707). In this study we showed that cytokine withdrawal resulted in Akt degradation by caspases as well. Furthermore, we demonstrated residue Asp-462 of Akt1 which is just upstream of the hydrophobic motif to be the primary cleavage site. The Akt1 mutant (D462N) that prevented caspase cleavage was more stable during factor withdrawal and enhanced cell survival. The Akt truncation mutant mimicking the caspase cleavage product lost its kinase activity and functioned as a dominant negative to promote cell death. Our results suggest that the balance between Akt and caspase activity controls cell survival. In particular, caspases are able to render Akt inactive and dominantly inhibit the Akt pathway by cleaving off the C-terminal hydrophobic motif. Consequently, the survival signal is quickly down-regulated to allow apoptosis to occur. Protein kinase Akt, an important downstream target of phosphatidylinositol 3-kinase, is one of the major survival factors in mammalian cells. It has been shown that phosphorylation of the C-terminal hydrophobic motif is required for Akt activation. The activated Akt then phosphorylates several pro-apoptotic proteins and prevents apoptosis mediated by caspases and the mitochondria. Interestingly, Akt has also been implicated to be a direct substrate of caspases in apoptotic cells induced by Fas (Widmann, C., Gibson, S., and Johnson, G. L. (1998) J. Biol. Chem. 273, 7141–7147) and anoikis (Bachelder, R. E., Wendt, M. A., Fujita, N., Tsuruo, T., and Mercurio, A. M. (2001) J. Biol. Chem. 276, 34702–34707). In this study we showed that cytokine withdrawal resulted in Akt degradation by caspases as well. Furthermore, we demonstrated residue Asp-462 of Akt1 which is just upstream of the hydrophobic motif to be the primary cleavage site. The Akt1 mutant (D462N) that prevented caspase cleavage was more stable during factor withdrawal and enhanced cell survival. The Akt truncation mutant mimicking the caspase cleavage product lost its kinase activity and functioned as a dominant negative to promote cell death. Our results suggest that the balance between Akt and caspase activity controls cell survival. In particular, caspases are able to render Akt inactive and dominantly inhibit the Akt pathway by cleaving off the C-terminal hydrophobic motif. Consequently, the survival signal is quickly down-regulated to allow apoptosis to occur. Cell survival and apoptosis are tightly regulated processes in multicellular organisms, the disruption of which may lead to diseases such as cancer. The serine/threonine kinase Akt (also referred to as PKB or RAC-PK) has proven to be a critical player in cell survival pathways. Originally identified as the cellular homologue of the retroviral oncogene v-akt (3Bellicose A. Test J.R. Staal S.P. Tsichlis P.N. Science. 1991; 254: 274-277Crossref PubMed Scopus (786) Google Scholar), Akt is conserved throughout evolution. The activation of Akt occurs through a cascade of events and is thought to be directly downstream of phosphatidylinositol 3-kinase (4Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2493) Google Scholar, 5Franke T.F. Kaplan D.R. Cantley L.C. Cell. 1997; 88: 435-437Abstract Full Text Full Text PDF PubMed Scopus (1512) 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 (909) Google Scholar, 7Datta S.R. Brunet A. Greenberg M.E. Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3704) Google Scholar). A number of studies (8Franke 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 (1817) 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: 661-665Crossref PubMed Scopus (2212) Google Scholar, 10Songyang 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, 11Ahmed N.N. Grimes H.L. Bellacosa A. Chan T.O. Tsichlis P.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3627-3632Crossref PubMed Scopus (486) Google Scholar, 12Kulik G. Klippel A. Weber M.J. Mol. Cell. Biol. 1997; 17: 1595-1606Crossref PubMed Scopus (964) Google Scholar) have shown that many growth and survival factors, such as nerve growth factor, platelet-derived growth factor, insulin-like growth factor-1 and cytokines, can activate Aktin vivo. Akt contains an N-terminal pleckstrin homology domain (PH) 1The abbreviations used are: PH domain, pleckstrin homology domain; IL-3, interleukin-3; PDK1, 3-phosphoinositide-dependent protein kinase-1; PARP-1, poly(ADP-ribose) polymerase 1; FKHRs, forkhead related transcription factors; HA, hemagglutinin. 1The abbreviations used are: PH domain, pleckstrin homology domain; IL-3, interleukin-3; PDK1, 3-phosphoinositide-dependent protein kinase-1; PARP-1, poly(ADP-ribose) polymerase 1; FKHRs, forkhead related transcription factors; HA, hemagglutinin. and a C-terminal kinase domain. The PH domain binds to phosphatidylinositols (thereby recruiting Akt to the plasma membrane where PDK1 is located) (13Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1291) Google Scholar). Phosphorylation of both Thr-308 and Ser-473 is required for fully activating Akt (4Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2493) Google Scholar). Thr-308 is believed to be phosphorylated by PDK1 (14Alessi 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 (613) Google Scholar, 15Downward J. Curr. Opin. Cell Biol. 1998; 10: 262-267Crossref PubMed Scopus (1180) Google Scholar, 16Brazil D.P. Hemmings B.A. Trends Biochem. Sci. 2001; 26: 657-664Abstract Full Text Full Text PDF PubMed Scopus (1028) Google Scholar), although the kinase that phosphorylates the Ser-473 site still remains unclear (17Chan, T. O., and Tsichlis, P. N. (2001) stke.sciencemag.org/cgi/content/full/ oc_sigtrans;2001/66/pe/.Google Scholar). Interestingly, Ser-473 is within the hydrophobic motif (FXX(F/Y)(S/T)(F/Y)) in the C-terminal tail of Akt. This hydrophobic motif is also conserved among the AGC family of kinases. Basal phosphorylation of the Ser-473 site is important for the activation of Akt (4Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2493) Google Scholar), presumably necessary for maintaining the proper conformation of the kinase domain and docking to PDK1 (18Biondi R.M. Cheung P.C. Casamayor A. Deak M. Currie R.A. Alessi D.R. EMBO J. 2000; 19: 979-988Crossref PubMed Scopus (246) Google Scholar). A recent report (19Maira S.M. Galetic I. Brazil D.P. Kaech S. Ingley E. Thelen M. Hemmings B.A. Science. 2001; 294: 374-380Crossref PubMed Scopus (211) Google Scholar) has also identified a novel protein that is capable of binding to the C-terminal domain of Akt and negatively regulates its activity. These data point to the important regulatory role of the C-terminal region of Akt.Akt likely acts on multiple substrates in vivo, which may include Bad and FKHR family transcription factors (20Datta S.R. Dudek H. Tao X. Masters S., Fu, H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4912) Google Scholar, 21del Peso L. Gonzalez-Garcia M. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1978) Google Scholar, 22Tang E.D. Nunez G. Barr F.G. Guan K.L. J. Biol. Chem. 1999; 274: 16741-16746Abstract Full Text Full Text PDF PubMed Scopus (656) 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 (5361) Google Scholar). Phosphorylation of Bad and FKHRs leads to inactivation of its apoptotic activity (21del Peso L. Gonzalez-Garcia M. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1978) Google Scholar). It has also been found that Akt can phosphorylate and inactivate caspase-9 in vitro, suggesting that Akt may protect cells from apoptosis by inhibiting caspases (24Cardone M.H. Roy N. Stennicke H.R. Salvesen G.S. Franke T.F. Stanbridge E. Frisch S. Reed J.C. Science. 1998; 282: 1318-1321Crossref PubMed Scopus (2718) Google Scholar). Interestingly, Akt was reported to be degraded during apoptosis induced by Fas and anoikis, and inhibition of caspase activity prevented its degradation (1Widmann C. Gibson S. Johnson G.L. J. Biol. Chem. 1998; 273: 7141-7147Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, 2Bachelder R.E. Wendt M.A. Fujita N. Tsuruo T. Mercurio A.M. J. Biol. Chem. 2001; 276: 34702-34707Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 25Rokudai S. Fujita N. Hashimoto Y. Tsuruo T. J. Cell. Physiol. 2000; 182: 290-296Crossref PubMed Scopus (98) Google Scholar). These results suggest a possible negative feedback loop between Akt and caspases that may help to maintain the balance between cell survival and apoptosis.However, it is still unclear how Akt and caspases may interact with each other in cytokine-induced cell death. The exact site(s) where caspase cleavage may occur in vivo and how this cleavage may affect Akt survival activity remain to be elucidated. In this study, we examined the potential caspase cleavage sites in Akt in vitro and in vivo, and we identified a novel primary cleavage site located between the kinase domain and the hydrophobic motif. Mutation of this site (Asp-462) blocked caspase cleavage and significantly enhanced Akt survival activity against cytokine withdrawal. The Akt cleavage product that did not contain the C-terminal tail lost its kinase activity and could function as a dominant negative in IL-3-dependent 32D cells. Therefore, in addition to phosphorylation of Thr-308 and Ser-473, the activity of Akt is also regulated by caspases.DISCUSSIONAkt has been reported to be down-regulated during apoptosis induced by Fas ligand or anoikis (1Widmann C. Gibson S. Johnson G.L. J. Biol. Chem. 1998; 273: 7141-7147Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, 2Bachelder R.E. Wendt M.A. Fujita N. Tsuruo T. Mercurio A.M. J. Biol. Chem. 2001; 276: 34702-34707Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). In this report, we demonstrated that Akt could also be down-regulated during cytokine-induced cell death and it is likely mediated by caspases. In IL-3-dependent 32D cells, we found decreased levels of endogenous Akt at about 14–16 h after cytokine withdrawal. Such degradation is consistent with the time course of apoptosis in these cells, as most of the cells die between 14 and 20 h post-IL-3 depletion. Furthermore, addition of a caspase-3 inhibitor could block Akt degradation in 32D cells upon IL-3 withdrawal. We found that in vitro caspase-3 could cleave Akt at multiple sites, and a single point mutation of residue Asp-462 to Asn was able to block Akt cleavage and degradation both in vitro and in vivo. Interestingly, the D462N mutation enhanced Akt survival activity in vivo, possibly by stabilizing Akt without altering its kinase activity or subcellular localization.Other caspase cleavage sites (Asp-108 and Asp-119) on Akt have been reported recently (2Bachelder R.E. Wendt M.A. Fujita N. Tsuruo T. Mercurio A.M. J. Biol. Chem. 2001; 276: 34702-34707Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 25Rokudai S. Fujita N. Hashimoto Y. Tsuruo T. J. Cell. Physiol. 2000; 182: 290-296Crossref PubMed Scopus (98) Google Scholar), but the single mutation of any of these sites could not completely abolish in vitro caspase-3 cleavage. Taken together, these findings suggest that the Asp-462 site is likely the primary cleavage site. Subsequent cleavage might occur after this site has been cleaved. It is possible that in previous studies the Asp-462 site may have been overlooked because cleavage at Asp-462 results in only a slight change in the molecular weight of Akt and hence makes it difficult to distinguish from full-length Akt.The C-terminal tail of Akt downstream of the kinase domain contains a hydrophobic motif conserved in most of the AGC family kinases (32Alessi D.R. Cohen P. Curr. Opin. Genet. & Dev. 1998; 8: 55-62Crossref PubMed Scopus (674) Google Scholar). Phosphorylation of the Ser-473 residue inside this motif (by an as yet unidentified kinase) is thought to facilitate Akt interaction with PDK1 which then phosphorylates another regulatory site Thr-308 in the activation loop of Akt (4Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2493) Google Scholar, 32Alessi D.R. Cohen P. Curr. Opin. Genet. & Dev. 1998; 8: 55-62Crossref PubMed Scopus (674) Google Scholar). This hydrophobic motif therefore plays a central role in regulating Akt activity. Consistent with this notion, we found that this short tail is necessary for the kinase activity of Akt (Fig. 6). The D462* mutant that does not contain this tail retains an intact kinase domain. However, we found that this protein lost its kinase activity in vitro and could not protect cells from cytokine-induced apoptosis in vivo. It has been shown that the hydrophobic motifs of several AGC family protein kinases directly interact with PDK1 (18Biondi R.M. Cheung P.C. Casamayor A. Deak M. Currie R.A. Alessi D.R. EMBO J. 2000; 19: 979-988Crossref PubMed Scopus (246) Google Scholar, 33Biondi R.M. Kieloch A. Currie R.A. Deak M. Alessi D.R. EMBO J. 2001; 20: 4380-4390Crossref PubMed Scopus (303) Google Scholar). We hypothesize that the deletion of the hydrophobic motif by caspase cleavage at Asp-462 may have prevented PDK1 docking and subsequent phosphorylation of Thr-308. Alternatively, the loss of the tail may have disrupted potential intra-molecular interactions between the tail and other regions of Akt, thereby affecting its activity. The structures of cAMP-dependent protein kinase and Akt indicate that the non-catalytic C terminus including part of the hydrophobic motifs form a loop that interacts with the kinase domain (34Knighton D.R. Zheng J.H. Ten Eyck L.F. Xuong N.H. Taylor S.S. Sowadski J.M. Science. 1991; 253: 414-420Crossref PubMed Scopus (803) Google Scholar, 35Yang J. Cron P. Thompson V. Good V.M. Hess D. Hemmings B.A. Barford D. Mol. Cell. 2002; 9: 1227-1240Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar). In light of recent findings, it is also possible that such cleavage may affect the interaction of Akt with other regulatory proteins (e.g.CTMP) through the Akt tail (19Maira S.M. Galetic I. Brazil D.P. Kaech S. Ingley E. Thelen M. Hemmings B.A. Science. 2001; 294: 374-380Crossref PubMed Scopus (211) Google Scholar).Although our study has focused on Akt1, it is likely that similar regulation by caspases is common for other isoforms of Akt. A comparison of sequences of three Akt isoforms shows that several potential caspase sites are clustered at the equivalent position of Asp-462 on Akt2 and Akt3 (Fig. 2 A). Consistent with this prediction, the signal reactive with the C-20 antibody decreased upon IL-3 starvation. Given that the C-20 antibody recognizes all Akt isoforms, it suggests that other Akt isoforms are probably also cleaved at the C-terminal tail during apoptosis.The fate of a cell is determined by the balance between anti-apoptotic and pro-apoptotic signals. The observation that activated Akt inhibits caspases and caspases in turn degrade Akt indicates a feedback regulation mechanism in controlling cell survival and apoptosis. If cells maintain Akt activation, Akt can inhibit pro-apoptotic factors such as Bad, transcription factor FKHRs, and caspases, allowing the cells to survive. On the other hand, at the onset of apoptosis, caspase activity can be stimulated such that survival factors including Akt can be eliminated, thereby committing the cells to die. Consistent with this model, cleavage of Akt at Asp-462 was most apparent at about 16 h post-IL-3 withdrawal, which is also the time when most cells commit to apoptosis. It is possible that cleavage of Akt may have taken place during the early hours after IL-3 depletion. Because we could not detect the change in Akt levels at earlier time points using either the N- or C-terminal antibody, it is likely that new Akt protein synthesis counteracts Akt degradation. In support of this notion, the Akt protein level significantly increased in cells treated with a cell-permeable caspase-3 inhibitor. In addition, our results suggest that when cells undergo apoptosis, the primary cleavage of Akt by caspases at Asp-462 (which deletes the hydrophobic motif) is able to eliminate Akt survival activity before it is completely degraded.We have shown that the Akt cleavage product could function as a dominant negative. Interestingly, Akt cleavage products at two other caspase-3 cleavage sites have also been reported to function as dominant negative forms of Akt and accelerate the apoptotic process (2Bachelder R.E. Wendt M.A. Fujita N. Tsuruo T. Mercurio A.M. J. Biol. Chem. 2001; 276: 34702-34707Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar,25Rokudai S. Fujita N. Hashimoto Y. Tsuruo T. J. Cell. Physiol. 2000; 182: 290-296Crossref PubMed Scopus (98) Google Scholar). These sites are located N-terminal to the kinase domain, and cleavage is predicted to delete the PH domain. Therefore, cleavage of Akt results in the survival signal being quickly down-regulated thereby allowing the cells to commit suicide. Cell survival and apoptosis are tightly regulated processes in multicellular organisms, the disruption of which may lead to diseases such as cancer. The serine/threonine kinase Akt (also referred to as PKB or RAC-PK) has proven to be a critical player in cell survival pathways. Originally identified as the cellular homologue of the retroviral oncogene v-akt (3Bellicose A. Test J.R. Staal S.P. Tsichlis P.N. Science. 1991; 254: 274-277Crossref PubMed Scopus (786) Google Scholar), Akt is conserved throughout evolution. The activation of Akt occurs through a cascade of events and is thought to be directly downstream of phosphatidylinositol 3-kinase (4Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2493) Google Scholar, 5Franke T.F. Kaplan D.R. Cantley L.C. Cell. 1997; 88: 435-437Abstract Full Text Full Text PDF PubMed Scopus (1512) 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 (909) Google Scholar, 7Datta S.R. Brunet A. Greenberg M.E. Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3704) Google Scholar). A number of studies (8Franke 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 (1817) 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: 661-665Crossref PubMed Scopus (2212) Google Scholar, 10Songyang 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, 11Ahmed N.N. Grimes H.L. Bellacosa A. Chan T.O. Tsichlis P.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3627-3632Crossref PubMed Scopus (486) Google Scholar, 12Kulik G. Klippel A. Weber M.J. Mol. Cell. Biol. 1997; 17: 1595-1606Crossref PubMed Scopus (964) Google Scholar) have shown that many growth and survival factors, such as nerve growth factor, platelet-derived growth factor, insulin-like growth factor-1 and cytokines, can activate Aktin vivo. Akt contains an N-terminal pleckstrin homology domain (PH) 1The abbreviations used are: PH domain, pleckstrin homology domain; IL-3, interleukin-3; PDK1, 3-phosphoinositide-dependent protein kinase-1; PARP-1, poly(ADP-ribose) polymerase 1; FKHRs, forkhead related transcription factors; HA, hemagglutinin. 1The abbreviations used are: PH domain, pleckstrin homology domain; IL-3, interleukin-3; PDK1, 3-phosphoinositide-dependent protein kinase-1; PARP-1, poly(ADP-ribose) polymerase 1; FKHRs, forkhead related transcription factors; HA, hemagglutinin. and a C-terminal kinase domain. The PH domain binds to phosphatidylinositols (thereby recruiting Akt to the plasma membrane where PDK1 is located) (13Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1291) Google Scholar). Phosphorylation of both Thr-308 and Ser-473 is required for fully activating Akt (4Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2493) Google Scholar). Thr-308 is believed to be phosphorylated by PDK1 (14Alessi 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 (613) Google Scholar, 15Downward J. Curr. Opin. Cell Biol. 1998; 10: 262-267Crossref PubMed Scopus (1180) Google Scholar, 16Brazil D.P. Hemmings B.A. Trends Biochem. Sci. 2001; 26: 657-664Abstract Full Text Full Text PDF PubMed Scopus (1028) Google Scholar), although the kinase that phosphorylates the Ser-473 site still remains unclear (17Chan, T. O., and Tsichlis, P. N. (2001) stke.sciencemag.org/cgi/content/full/ oc_sigtrans;2001/66/pe/.Google Scholar). Interestingly, Ser-473 is within the hydrophobic motif (FXX(F/Y)(S/T)(F/Y)) in the C-terminal tail of Akt. This hydrophobic motif is also conserved among the AGC family of kinases. Basal phosphorylation of the Ser-473 site is important for the activation of Akt (4Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2493) Google Scholar), presumably necessary for maintaining the proper conformation of the kinase domain and docking to PDK1 (18Biondi R.M. Cheung P.C. Casamayor A. Deak M. Currie R.A. Alessi D.R. EMBO J. 2000; 19: 979-988Crossref PubMed Scopus (246) Google Scholar). A recent report (19Maira S.M. Galetic I. Brazil D.P. Kaech S. Ingley E. Thelen M. Hemmings B.A. Science. 2001; 294: 374-380Crossref PubMed Scopus (211) Google Scholar) has also identified a novel protein that is capable of binding to the C-terminal domain of Akt and negatively regulates its activity. These data point to the important regulatory role of the C-terminal region of Akt. Akt likely acts on multiple substrates in vivo, which may include Bad and FKHR family transcription factors (20Datta S.R. Dudek H. Tao X. Masters S., Fu, H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4912) Google Scholar, 21del Peso L. Gonzalez-Garcia M. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1978) Google Scholar, 22Tang E.D. Nunez G. Barr F.G. Guan K.L. J. Biol. Chem. 1999; 274: 16741-16746Abstract Full Text Full Text PDF PubMed Scopus (656) 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 (5361) Google Scholar). Phosphorylation of Bad and FKHRs leads to inactivation of its apoptotic activity (21del Peso L. Gonzalez-Garcia M. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1978) Google Scholar). It has also been found that Akt can phosphorylate and inactivate caspase-9 in vitro, suggesting that Akt may protect cells from apoptosis by inhibiting caspases (24Cardone M.H. Roy N. Stennicke H.R. Salvesen G.S. Franke T.F. Stanbridge E. Frisch S. Reed J.C. Science. 1998; 282: 1318-1321Crossref PubMed Scopus (2718) Google Scholar). Interestingly, Akt was reported to be degraded during apoptosis induced by Fas and anoikis, and inhibition of caspase activity prevented its degradation (1Widmann C. Gibson S. Johnson G.L. J. Biol. Chem. 1998; 273: 7141-7147Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, 2Bachelder R.E. Wendt M.A. Fujita N. Tsuruo T. Mercurio A.M. J. Biol. Chem. 2001; 276: 34702-34707Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 25Rokudai S. Fujita N. Hashimoto Y. Tsuruo T. J. Cell. Physiol. 2000; 182: 290-296Crossref PubMed Scopus (98) Google Scholar). These results suggest a possible negative feedback loop between Akt and caspases that may help to maintain the balance between cell survival and apoptosis. However, it is still unclear how Akt and caspases may interact with each other in cytokine-induced cell death. The exact site(s) where caspase cleavage may occur in vivo and how this cleavage may affect Akt survival activity remain to be elucidated. In this study, we examined the potential caspase cleavage sites in Akt in vitro and in vivo, and we identified a novel primary cleavage site located between the kinase domain and the hydrophobic motif. Mutation of this site (Asp-462) blocked caspase cleavage and significantly enhanced Akt survival activity against cytokine withdrawal. The Akt cleavage product that did not contain the C-terminal tail lost its kinase activity and could function as a dominant negative in IL-3-dependent 32D cells. Therefore, in addition to phosphorylation of Thr-308 and Ser-473, the activity of Akt is also regulated by caspases. DISCUSSIONAkt has been reported to be down-regulated during apoptosis induced by Fas ligand or anoikis (1Widmann C. Gibson S. Johnson G.L. J. Biol. Chem. 1998; 273: 7141-7147Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, 2Bachelder R.E. Wendt M.A. Fujita N. Tsuruo T. Mercurio A.M. J. Biol. Chem. 2001; 276: 34702-34707Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). In this report, we demonstrated that Akt could also be down-regulated during cytokine-induced cell death and it is likely mediated by caspases. In IL-3-dependent 32D cells, we found decreased levels of endogenous Akt at about 14–16 h after cytokine withdrawal. Such degradation is consistent with the time course of apoptosis in these cells, as most of the cells die between 14 and 20 h post-IL-3 depletion. Furthermore, addition of a caspase-3 inhibitor could block Akt degradation in 32D cells upon IL-3 withdrawal. We found that in vitro caspase-3 could cleave Akt at multiple sites, and a single point mutation of residue Asp-462 to Asn was able to block Akt cleavage and degradation both in vitro and in vivo. Interestingly, the D462N mutation enhanced Akt survival activity in vivo, possibly by stabilizing Akt without altering its kinase activity or subcellular localization.Other caspase cleavage sites (Asp-108 and Asp-119) on Akt have been reported recently (2Bachelder R.E. Wendt M.A. Fujita N. Tsuruo T. Mercurio A.M. J. Biol. Chem. 2001; 276: 34702-34707Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 25Rokudai S. Fujita N. Hashimoto Y. Tsuruo T. J. Cell. Physiol. 2000; 182: 290-296Crossref PubMed Scopus (98) Google Scholar), but the single mutation of any of these sites could not completely abolish in vitro caspase-3 cleavage. Taken together, these findings suggest that the Asp-462 site is likely the primary cleavage site. Subsequent cleavage might occur after this site has been cleaved. It is possible that in previous studies the Asp-462 site may have been overlooked because cleavage at Asp-462 results in only a slight change in the molecular weight of Akt and hence makes it difficult to distinguish from full-length Akt.The C-terminal tail of Akt downstream of the kinase domain contains a hydrophobic motif conserved in most of the AGC family kinases (32Alessi D.R. Cohen P. Curr. Opin. Genet. & Dev. 1998; 8: 55-62Crossref PubMed Scopus (674) Google Scholar). Phosphorylation of the Ser-473 residue inside this motif (by an as yet unidentified kinase) is thought to facilitate Akt interaction with PDK1 which then phosphorylates another regulatory site Thr-308 in the activation loop of Akt (4Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2493) Google Scholar, 32Alessi D.R. Cohen P. Curr. Opin. Genet. & Dev. 1998; 8: 55-62Crossref PubMed Scopus (674) Google Scholar). This hydrophobic motif therefore plays a central role in regulating Akt activity. Consistent with this notion, we found that this short tail is necessary for the kinase activity of Akt (Fig. 6). The D462* mutant that does not contain this tail retains an intact kinase domain. However, we found that this protein lost its kinase activity in vitro and could not protect cells from cytokine-induced apoptosis in vivo. It has been shown that the hydrophobic motifs of several AGC family protein kinases directly interact with PDK1 (18Biondi R.M. Cheung P.C. Casamayor A. Deak M. Currie R.A. Alessi D.R. EMBO J. 2000; 19: 979-988Crossref PubMed Scopus (246) Google Scholar, 33Biondi R.M. Kieloch A. Currie R.A. Deak M. Alessi D.R. EMBO J. 2001; 20: 4380-4390Crossref PubMed Scopus (303) Google Scholar). We hypothesize that the deletion of the hydrophobic motif by caspase cleavage at Asp-462 may have prevented PDK1 docking and subsequent phosphorylation of Thr-308. Alternatively, the loss of the tail may have disrupted potential intra-molecular interactions between the tail and other regions of Akt, thereby affecting its activity. The structures of cAMP-dependent protein kinase and Akt indicate that the non-catalytic C terminus including part of the hydrophobic motifs form a loop that interacts with the kinase domain (34Knighton D.R. Zheng J.H. Ten Eyck L.F. Xuong N.H. Taylor S.S. Sowadski J.M. Science. 1991; 253: 414-420Crossref PubMed Scopus (803) Google Scholar, 35Yang J. Cron P. Thompson V. Good V.M. Hess D. Hemmings B.A. Barford D. Mol. Cell. 2002; 9: 1227-1240Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar). In light of recent findings, it is also possible that such cleavage may affect the interaction of Akt with other regulatory proteins (e.g.CTMP) through the Akt tail (19Maira S.M. Galetic I. Brazil D.P. Kaech S. Ingley E. Thelen M. Hemmings B.A. Science. 2001; 294: 374-380Crossref PubMed Scopus (211) Google Scholar).Although our study has focused on Akt1, it is likely that similar regulation by caspases is common for other isoforms of Akt. A comparison of sequences of three Akt isoforms shows that several potential caspase sites are clustered at the equivalent position of Asp-462 on Akt2 and Akt3 (Fig. 2 A). Consistent with this prediction, the signal reactive with the C-20 antibody decreased upon IL-3 starvation. Given that the C-20 antibody recognizes all Akt isoforms, it suggests that other Akt isoforms are probably also cleaved at the C-terminal tail during apoptosis.The fate of a cell is determined by the balance between anti-apoptotic and pro-apoptotic signals. The observation that activated Akt inhibits caspases and caspases in turn degrade Akt indicates a feedback regulation mechanism in controlling cell survival and apoptosis. If cells maintain Akt activation, Akt can inhibit pro-apoptotic factors such as Bad, transcription factor FKHRs, and caspases, allowing the cells to survive. On the other hand, at the onset of apoptosis, caspase activity can be stimulated such that survival factors including Akt can be eliminated, thereby committing the cells to die. Consistent with this model, cleavage of Akt at Asp-462 was most apparent at about 16 h post-IL-3 withdrawal, which is also the time when most cells commit to apoptosis. It is possible that cleavage of Akt may have taken place during the early hours after IL-3 depletion. Because we could not detect the change in Akt levels at earlier time points using either the N- or C-terminal antibody, it is likely that new Akt protein synthesis counteracts Akt degradation. In support of this notion, the Akt protein level significantly increased in cells treated with a cell-permeable caspase-3 inhibitor. In addition, our results suggest that when cells undergo apoptosis, the primary cleavage of Akt by caspases at Asp-462 (which deletes the hydrophobic motif) is able to eliminate Akt survival activity before it is completely degraded.We have shown that the Akt cleavage product could function as a dominant negative. Interestingly, Akt cleavage products at two other caspase-3 cleavage sites have also been reported to function as dominant negative forms of Akt and accelerate the apoptotic process (2Bachelder R.E. Wendt M.A. Fujita N. Tsuruo T. Mercurio A.M. J. Biol. Chem. 2001; 276: 34702-34707Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar,25Rokudai S. Fujita N. Hashimoto Y. Tsuruo T. J. Cell. Physiol. 2000; 182: 290-296Crossref PubMed Scopus (98) Google Scholar). These sites are located N-terminal to the kinase domain, and cleavage is predicted to delete the PH domain. Therefore, cleavage of Akt results in the survival signal being quickly down-regulated thereby allowing the cells to commit suicide. Akt has been reported to be down-regulated during apoptosis induced by Fas ligand or anoikis (1Widmann C. Gibson S. Johnson G.L. J. Biol. Chem. 1998; 273: 7141-7147Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, 2Bachelder R.E. Wendt M.A. Fujita N. Tsuruo T. Mercurio A.M. J. Biol. Chem. 2001; 276: 34702-34707Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). In this report, we demonstrated that Akt could also be down-regulated during cytokine-induced cell death and it is likely mediated by caspases. In IL-3-dependent 32D cells, we found decreased levels of endogenous Akt at about 14–16 h after cytokine withdrawal. Such degradation is consistent with the time course of apoptosis in these cells, as most of the cells die between 14 and 20 h post-IL-3 depletion. Furthermore, addition of a caspase-3 inhibitor could block Akt degradation in 32D cells upon IL-3 withdrawal. We found that in vitro caspase-3 could cleave Akt at multiple sites, and a single point mutation of residue Asp-462 to Asn was able to block Akt cleavage and degradation both in vitro and in vivo. Interestingly, the D462N mutation enhanced Akt survival activity in vivo, possibly by stabilizing Akt without altering its kinase activity or subcellular localization. Other caspase cleavage sites (Asp-108 and Asp-119) on Akt have been reported recently (2Bachelder R.E. Wendt M.A. Fujita N. Tsuruo T. Mercurio A.M. J. Biol. Chem. 2001; 276: 34702-34707Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 25Rokudai S. Fujita N. Hashimoto Y. Tsuruo T. J. Cell. Physiol. 2000; 182: 290-296Crossref PubMed Scopus (98) Google Scholar), but the single mutation of any of these sites could not completely abolish in vitro caspase-3 cleavage. Taken together, these findings suggest that the Asp-462 site is likely the primary cleavage site. Subsequent cleavage might occur after this site has been cleaved. It is possible that in previous studies the Asp-462 site may have been overlooked because cleavage at Asp-462 results in only a slight change in the molecular weight of Akt and hence makes it difficult to distinguish from full-length Akt. The C-terminal tail of Akt downstream of the kinase domain contains a hydrophobic motif conserved in most of the AGC family kinases (32Alessi D.R. Cohen P. Curr. Opin. Genet. & Dev. 1998; 8: 55-62Crossref PubMed Scopus (674) Google Scholar). Phosphorylation of the Ser-473 residue inside this motif (by an as yet unidentified kinase) is thought to facilitate Akt interaction with PDK1 which then phosphorylates another regulatory site Thr-308 in the activation loop of Akt (4Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2493) Google Scholar, 32Alessi D.R. Cohen P. Curr. Opin. Genet. & Dev. 1998; 8: 55-62Crossref PubMed Scopus (674) Google Scholar). This hydrophobic motif therefore plays a central role in regulating Akt activity. Consistent with this notion, we found that this short tail is necessary for the kinase activity of Akt (Fig. 6). The D462* mutant that does not contain this tail retains an intact kinase domain. However, we found that this protein lost its kinase activity in vitro and could not protect cells from cytokine-induced apoptosis in vivo. It has been shown that the hydrophobic motifs of several AGC family protein kinases directly interact with PDK1 (18Biondi R.M. Cheung P.C. Casamayor A. Deak M. Currie R.A. Alessi D.R. EMBO J. 2000; 19: 979-988Crossref PubMed Scopus (246) Google Scholar, 33Biondi R.M. Kieloch A. Currie R.A. Deak M. Alessi D.R. EMBO J. 2001; 20: 4380-4390Crossref PubMed Scopus (303) Google Scholar). We hypothesize that the deletion of the hydrophobic motif by caspase cleavage at Asp-462 may have prevented PDK1 docking and subsequent phosphorylation of Thr-308. Alternatively, the loss of the tail may have disrupted potential intra-molecular interactions between the tail and other regions of Akt, thereby affecting its activity. The structures of cAMP-dependent protein kinase and Akt indicate that the non-catalytic C terminus including part of the hydrophobic motifs form a loop that interacts with the kinase domain (34Knighton D.R. Zheng J.H. Ten Eyck L.F. Xuong N.H. Taylor S.S. Sowadski J.M. Science. 1991; 253: 414-420Crossref PubMed Scopus (803) Google Scholar, 35Yang J. Cron P. Thompson V. Good V.M. Hess D. Hemmings B.A. Barford D. Mol. Cell. 2002; 9: 1227-1240Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar). In light of recent findings, it is also possible that such cleavage may affect the interaction of Akt with other regulatory proteins (e.g.CTMP) through the Akt tail (19Maira S.M. Galetic I. Brazil D.P. Kaech S. Ingley E. Thelen M. Hemmings B.A. Science. 2001; 294: 374-380Crossref PubMed Scopus (211) Google Scholar). Although our study has focused on Akt1, it is likely that similar regulation by caspases is common for other isoforms of Akt. A comparison of sequences of three Akt isoforms shows that several potential caspase sites are clustered at the equivalent position of Asp-462 on Akt2 and Akt3 (Fig. 2 A). Consistent with this prediction, the signal reactive with the C-20 antibody decreased upon IL-3 starvation. Given that the C-20 antibody recognizes all Akt isoforms, it suggests that other Akt isoforms are probably also cleaved at the C-terminal tail during apoptosis. The fate of a cell is determined by the balance between anti-apoptotic and pro-apoptotic signals. The observation that activated Akt inhibits caspases and caspases in turn degrade Akt indicates a feedback regulation mechanism in controlling cell survival and apoptosis. If cells maintain Akt activation, Akt can inhibit pro-apoptotic factors such as Bad, transcription factor FKHRs, and caspases, allowing the cells to survive. On the other hand, at the onset of apoptosis, caspase activity can be stimulated such that survival factors including Akt can be eliminated, thereby committing the cells to die. Consistent with this model, cleavage of Akt at Asp-462 was most apparent at about 16 h post-IL-3 withdrawal, which is also the time when most cells commit to apoptosis. It is possible that cleavage of Akt may have taken place during the early hours after IL-3 depletion. Because we could not detect the change in Akt levels at earlier time points using either the N- or C-terminal antibody, it is likely that new Akt protein synthesis counteracts Akt degradation. In support of this notion, the Akt protein level significantly increased in cells treated with a cell-permeable caspase-3 inhibitor. In addition, our results suggest that when cells undergo apoptosis, the primary cleavage of Akt by caspases at Asp-462 (which deletes the hydrophobic motif) is able to eliminate Akt survival activity before it is completely degraded. We have shown that the Akt cleavage product could function as a dominant negative. Interestingly, Akt cleavage products at two other caspase-3 cleavage sites have also been reported to function as dominant negative forms of Akt and accelerate the apoptotic process (2Bachelder R.E. Wendt M.A. Fujita N. Tsuruo T. Mercurio A.M. J. Biol. Chem. 2001; 276: 34702-34707Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar,25Rokudai S. Fujita N. Hashimoto Y. Tsuruo T. J. Cell. Physiol. 2000; 182: 290-296Crossref PubMed Scopus (98) Google Scholar). These sites are located N-terminal to the kinase domain, and cleavage is predicted to delete the PH domain. Therefore, cleavage of Akt results in the survival signal being quickly down-regulated thereby allowing the cells to commit suicide. We thank Amin Safari and Matthew O'Connor for technical support." @default.
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