PI3K: Downstream AKTion Blocks Apoptosis

Extracellular stimuli are transmitted intracellularly by signaling cascades that involve the interaction of macromolecules and/or the generation of second messenger molecules that transduce signaling events over a distance between the origin and target of a signal. One class of such second messenger molecules is generated via phosphorylation of phosphoinositides on the D-3 position by phosphoinositide 3-kinase (PI3K). These products of PI3K can act on multiple downstream effectors that include Src homology-2 (SH2) and Pleckstrin homology (PH) domains of serine/threonine and tyrosine kinases and various cytoskeletal proteins (reviewed by4Carpenter C.L. Cantley L.C. Curr. Opin. Cell Biol. 1996; 8: 153-158Crossref PubMed Scopus (572) Google Scholar). The role of PI3K in intracellular signaling has been underscored by its implication in a plethora of biological responses. Although it is unlikely that these multiple responses will be explained by the action of a single downstream target, recent research from several laboratories indicates that a signaling pathway from PI3K to the serine/threonine protein kinase Akt/PKB may mediate some cellular responses of PI3K (3Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1846) Google Scholar, 5Cross D.A.E. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4167) Google Scholar, 9Franke 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 (1784) Google Scholar, 15Kohn A.D. Kovacina K.S. Roth R.A. EMBO J. 1995; 14: 4288-4295Crossref PubMed Scopus (313) Google Scholar), including protection from apoptosis (7Dudek 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 (2140) Google Scholar, 11Kauffmann-Zeh A. Rodriguez-Viciana P. Ulrich E. Gilbert C. Coffer P. Downward J. Evan G. Nature, in press. 1997; Google Scholar, 12Khwaja A. Rodriguez-Viciana P. Wennström S. Warne P.H. Downward J. EMBO J., in press. 1997; Google Scholar, 17Kulik G. Klippel A. Weber M.J. Mol. Cell. Biol., in press. 1997; Google Scholar). Multiple forms of PI3K with distinct mechanisms of regulation and different substrate specificities exist in mammalian cells (reviewed by4Carpenter C.L. Cantley L.C. Curr. Opin. Cell Biol. 1996; 8: 153-158Crossref PubMed Scopus (572) Google Scholar, 20Zvelebil M.J. MacDougall L. Leevers S. Volinia S. Vanhaesebroeck B. Gout I. Panayotou G. Domin J. Stein R. Pages F. et al.Phil. Trans. R. Soc. Lond. 1996; 351: 217-223Crossref PubMed Scopus (88) Google Scholar). The various forms of PI3K are able to phosphorylate the D-3 position of phosphatidylinositol (PtdIns), phosphatidylinositol-4-phosphate (PtdIns-4-P), and phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P2)to produce phosphatidylinositol-3-phosphate (PtdIns-3-P), phosphatidylinositol-3,4-bisphosphate (PtdIns- 3,4-P2), and phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P3), respectively. PtdIns-3-P is constitutively produced in the absence of growth factor stimulation and is implicated in vesicle trafficking. PtdIns-3,4-P2 and PtdIns-3,4,5-P3 are nominally absent in quiescent cells, but they appear acutely within seconds to minutes of addition of extracellular stimuli and are ultimately degraded by phosphatases (Figure 1). In most cells that have been investigated, PtdIns-3,4-P2 appears with a lag after the peak in PtdIns-3,4,5-P3, suggesting that it results from hydrolysis of the D-5 phosphate from PtdIns-3,4,5-P3. There is evidence that PtdIns-3,4-P2 can also be synthesized by pathways that do not involve production of PtdIns-3,4,5-P3. In vitro, this lipid can be produced by the p170/mCpk-type PI3K that phosphorylates PtdIns-4-P at the D-3 position (Figure 1) or by a kinase that phosphorylates PtdIns-3-P at the D-4 position (not shown). Thus, the relative levels of PtdIns-3,4-P2 and PtdIns-3,4,5-P3 are independently controlled by a complex set of kinases and phosphatases that have not yet been fully characterized. The importance of this regulation is emphasized by recent evidence that certain SH2 and PH domains interact with PtdIns-3,4,5-P3, but not with PtdIns-3,4-P2 (reviewed by4Carpenter C.L. Cantley L.C. Curr. Opin. Cell Biol. 1996; 8: 153-158Crossref PubMed Scopus (572) Google Scholar), while the serine/threonine protein kinase Akt/PKB is activated by PtdIns-3,4-P2 but not by PtdIns-3,4,5-P3 (8Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1266) Google Scholar, 13Klippel A. Kavanaugh W.M. Pot D. Williams L.T. Mol. Cell. Biol. 1997; 17: 338-344Crossref PubMed Scopus (440) Google Scholar). Akt/PKB is homologous to the PKA and PKC families of protein kinases (hence named PKB or elated to and protein kinase: RAC-PK). It is also the cellular homolog of the retroviral oncogene v-akt (see references in3Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1846) Google Scholar, 5Cross D.A.E. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4167) Google Scholar, 9Franke 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 (1784) Google Scholar, 15Kohn A.D. Kovacina K.S. Roth R.A. EMBO J. 1995; 14: 4288-4295Crossref PubMed Scopus (313) Google Scholar). In vivo, the activity of Akt/PKB is regulated by serum and growth factors that activate PI3K (3Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1846) Google Scholar, 9Franke 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 (1784) Google Scholar, 8Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1266) Google Scholar, 15Kohn A.D. Kovacina K.S. Roth R.A. EMBO J. 1995; 14: 4288-4295Crossref PubMed Scopus (313) Google Scholar, 1Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2419) Google Scholar, 2Andjelkovic M. Jakubowicz T. Cron P. Ming X.-F. Han J.-W. Hemmings B.A. Proc. Natl. Acad. Sci. USA. 1996; 93: 5699-5704Crossref PubMed Scopus (419) Google Scholar). Three types of experiments have suggested that PI3K is necessary and sufficient for growth factor-dependent activation of Akt. First, PDGF receptor mutants that are deficient in activating PI3K fail to mediate activation of Akt/PKB (3Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1846) Google Scholar, 9Franke 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 (1784) Google Scholar). Second, dominant-inhibitory alleles of PI3K prevent activation of Akt (3Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1846) Google Scholar), and constitutively activated PI3K increases Akt/PKB activity independent of growth factor stimulation (14Klippel A. Reinhard C. Kavanaugh W.M. Apell G. Escobedo M.-A. Williams L.T. Mol. Cell. Biol. 1996; 16: 4117-4127Crossref PubMed Scopus (409) Google Scholar, 19Marte B.M. Rodriguez-Viciana P. Wennström S. Warne P.H. Downward J. Curr. Biol. 1996; 7: 63-70Abstract Full Text Full Text PDF Google Scholar, 8Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1266) Google Scholar). Third, the PI3K inhibitor wortmannin blocks activation of Akt/PKB by growth factors (3Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1846) Google Scholar, 9Franke 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 (1784) Google Scholar, 8Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1266) Google Scholar, 15Kohn A.D. Kovacina K.S. Roth R.A. EMBO J. 1995; 14: 4288-4295Crossref PubMed Scopus (313) Google Scholar, 2Andjelkovic M. Jakubowicz T. Cron P. Ming X.-F. Han J.-W. Hemmings B.A. Proc. Natl. Acad. Sci. USA. 1996; 93: 5699-5704Crossref PubMed Scopus (419) Google Scholar). Akt/PKB activation is partially dependent upon the activity of Ras (9Franke 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 (1784) Google Scholar), and activated mutants of R-Ras and Ras may stimulate Akt/PKB by activating PI3K (14Klippel A. Reinhard C. Kavanaugh W.M. Apell G. Escobedo M.-A. Williams L.T. Mol. Cell. Biol. 1996; 16: 4117-4127Crossref PubMed Scopus (409) Google Scholar, 19Marte B.M. Rodriguez-Viciana P. Wennström S. Warne P.H. Downward J. Curr. Biol. 1996; 7: 63-70Abstract Full Text Full Text PDF Google Scholar). Although these studies indicate that PI3K is necessary to activate this enzyme in response to certain extracellular stimuli, an additional pathway for activation has been suggested that is independent of PI3K and could involve p38/HOG (1Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2419) Google Scholar, 16Konishi H. Matsuzaki H. Tanaka M. Ono Y. Tokunaga C. Kuroda S.i. Kikkawa U. Proc. Natl. Acad. Sci. USA. 1996; 93: 7639-7643Crossref PubMed Scopus (186) Google Scholar). The diversity of factors leading to the activation of Akt/PKB may explain the existence of conflicting models of Akt/PKB regulation. Akt/PKB molecules are able to dimerize and to interact with other proteins through an NH2-terminal region that includes a PH domain (16Konishi H. Matsuzaki H. Tanaka M. Ono Y. Tokunaga C. Kuroda S.i. Kikkawa U. Proc. Natl. Acad. Sci. USA. 1996; 93: 7639-7643Crossref PubMed Scopus (186) Google Scholar, 8Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1266) Google Scholarreferences therein). Homo-oligomerization of Akt/PKB is induced by interaction with PtdIns-3,4-P2 and increases Akt/PKB activity (8Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1266) Google Scholar); homo-oligomerization is also involved in the regulation of the serine/threonine kinase Raf and could possibly be a general mechanism of regulation of serine/threonine kinases that will have to be examined further (reviewed by18Marshall C.J. Nature. 1996; 383: 127-128Crossref PubMed Scopus (96) Google Scholar). The integrity of the NH2-terminal PH domain is required for in vivo activation of Akt/PKB by several growth factors, by constitutively active PI3K, and also by certain pathways independent of PI3K (9Franke 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 (1784) Google Scholar, 8Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1266) Google Scholar, 2Andjelkovic M. Jakubowicz T. Cron P. Ming X.-F. Han J.-W. Hemmings B.A. Proc. Natl. Acad. Sci. USA. 1996; 93: 5699-5704Crossref PubMed Scopus (419) Google Scholar, 14Klippel A. Reinhard C. Kavanaugh W.M. Apell G. Escobedo M.-A. Williams L.T. Mol. Cell. Biol. 1996; 16: 4117-4127Crossref PubMed Scopus (409) Google Scholar, 16Konishi H. Matsuzaki H. Tanaka M. Ono Y. Tokunaga C. Kuroda S.i. Kikkawa U. Proc. Natl. Acad. Sci. USA. 1996; 93: 7639-7643Crossref PubMed Scopus (186) Google Scholar). In contrast, the Akt/PKB PH domain is not required for Akt/PKB activation by okadaic acid or insulin in cells overexpressing the insulin receptor (15Kohn A.D. Kovacina K.S. Roth R.A. EMBO J. 1995; 14: 4288-4295Crossref PubMed Scopus (313) Google Scholar; 2Andjelkovic M. Jakubowicz T. Cron P. Ming X.-F. Han J.-W. Hemmings B.A. Proc. Natl. Acad. Sci. USA. 1996; 93: 5699-5704Crossref PubMed Scopus (419) Google Scholar). These results indicate that the relative importance of the Akt/PKB PH domain for activation depends on the cell type or the stimulus used. Treatment of cells with inhibitors of serine/threonine phosphatases and direct treatment of purified Akt/PKB with phosphatases has shown that Akt/PKB phosphorylation is critical for its activity. The major phosphorylation sites required for activation have been identified as threonine 308 and serine 473, and MAPKAP kinase 2 is able to phosphorylate one of these sites (serine 473) to partially activate Akt/PKB in vitro (1Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2419) Google Scholar). Since MAPKAP kinase 2 is activated by p38/HOG under conditions of cellular stress that do not activate PI3K, it may contribute to PI3K-independent activation of Akt/PKB in vivo (1Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2419) Google Scholar, 16Konishi H. Matsuzaki H. Tanaka M. Ono Y. Tokunaga C. Kuroda S.i. Kikkawa U. Proc. Natl. Acad. Sci. USA. 1996; 93: 7639-7643Crossref PubMed Scopus (186) Google Scholar). The kinase that phosphorylates Akt/PKB in its catalytic loop on threonine 308 and activates Akt/PKB in the growth factor- and PI3K-dependent pathway is not known. A direct mechanism of Akt/PKB activation by PI3K that involves the binding of PtdIns-3,4-P2 to the Akt/PKB PH domain has been described recently (8Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1266) Google Scholar, 13Klippel A. Kavanaugh W.M. Pot D. Williams L.T. Mol. Cell. Biol. 1997; 17: 338-344Crossref PubMed Scopus (440) Google Scholar). PtdIns-3,4,5-P3 does not activate Akt/PKB and may cause some inhibition of the basal activity. The failure of the structural isomer PtdIns-4,5-P2 to activate Akt/PKB supports the view that the activation of Akt/PKB by PtdIns-3,4-P2 is physiologically relevant. PtdIns-3,4-P2 causes dimerization of Akt/PKB, which could be the mechanism of activation (8Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1266) Google Scholar). These results suggest the model presented in Figure 2. Activation of PI3K by growth factors results in the production of PtdIns-3,4-P2 at the membrane. Akt/PKB binds to this lipid, dimerizes, and is stabilized in a partially active state. The location at the membrane and/or the dimerization then enhances the ability of Akt/PKB to be phosphorylated. Although Ras is involved in activation of Akt/PKB (9Franke 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 (1784) Google Scholar; 14Klippel A. Reinhard C. Kavanaugh W.M. Apell G. Escobedo M.-A. Williams L.T. Mol. Cell. Biol. 1996; 16: 4117-4127Crossref PubMed Scopus (409) Google Scholar, 19Marte B.M. Rodriguez-Viciana P. Wennström S. Warne P.H. Downward J. Curr. Biol. 1996; 7: 63-70Abstract Full Text Full Text PDF Google Scholar), Akt/PKB does not appear to be in the pathway leading to the activation of MAPK (9Franke 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 (1784) Google Scholar). This is consistent with the fact that R-Ras activates Akt/PKB but does not activate MAPK (19Marte B.M. Rodriguez-Viciana P. Wennström S. Warne P.H. Downward J. Curr. Biol. 1996; 7: 63-70Abstract Full Text Full Text PDF Google Scholar). These results indicate the existence of a Ras signaling cascade parallel to the Raf/MEK/MAPK pathway: Ras binds and activates PI3K, causing activation of Akt/PKB. The only known protein target of Akt/PKB is glycogen synthase kinase-3 (GSK3): Akt/PKB phosphorylates N-terminal sequences in GSK3 in vitro that are important for GSK3 inhibition by insulin stimulation in vivo, thereby linking PI3K and Akt/PKB to insulin-dependent glycogen synthesis (5Cross D.A.E. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4167) Google Scholar). There is also evidence that p70S6kinase is downstream of constitutively activated Akt/PKB (3Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1846) Google Scholar). Exciting insights into the function of Akt/PKB have been revealed by studies investigating its function in PI3K-dependent pathways that are involved in the regulation of cell survival. Previous studies have demonstrated that PI3K is involved in serum-dependent survival of PC12 cells (reviewed by4Carpenter C.L. Cantley L.C. Curr. Opin. Cell Biol. 1996; 8: 153-158Crossref PubMed Scopus (572) Google Scholar). Recent results indicate that PI3K also mediates type 1 insulin-like growth factor (IGF-1)-dependent survival of Rat-1 and COS-7 cells (17Kulik G. Klippel A. Weber M.J. Mol. Cell. Biol., in press. 1997; Google Scholar) and granule neurons (6D'Mello S.R. Borodezt K. Soltoff S. J. Neurosci., in press. 1997; Google Scholar). A role for Akt/PKB in IGF-1-mediated cell survival has been indicated (7Dudek 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 (2140) Google Scholar): overexpression of Akt/PKB prevents apoptosis in primary cultures of cerebellar neurons that are induced by survival factor withdrawal or inhibition of PI3K. The expression of dominant-negative forms of Akt/PKB interferes with growth factor-mediated survival in these cells, indicating that Akt/PKB is necessary and sufficient for neuronal survival. Studies from Weber and colleagues have shown that overexpression of constitutively activated Akt/PKB also blocks UV-induced apoptosis in Rat-1 and COS-7 cells (17Kulik G. Klippel A. Weber M.J. Mol. Cell. Biol., in press. 1997; Google Scholar). A different approach taken by Downward and colleagues indicated that activated Akt/PKB prevents apoptosis that is induced by detachment of MDCK cells from their extracellular matrix (anoikis; 12Khwaja A. Rodriguez-Viciana P. Wennström S. Warne P.H. Downward J. EMBO J., in press. 1997; Google Scholar). Finally, Evan and colleagues demonstrated that mutants of V12 Ras that selectively stimulate PI3K and Akt/PKB but not the Raf/MEK/MAPK pathway are able to prevent c-myc-induced cell death in Rat-1 cells (11Kauffmann-Zeh A. Rodriguez-Viciana P. Ulrich E. Gilbert C. Coffer P. Downward J. Evan G. Nature, in press. 1997; Google Scholar). In this system, activated forms of PI3K and Akt/PKB are sufficient to prevent apoptosis that is induced by c-myc. p70S6kinase activity was not necessary for the prevention of apoptosis caused by deregulated c-myc (11Kauffmann-Zeh A. Rodriguez-Viciana P. Ulrich E. Gilbert C. Coffer P. Downward J. Evan G. Nature, in press. 1997; Google Scholar), nor was it necessary for Akt/PKB-dependent neuronal survival in primary cerebellar neurons (7Dudek 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 (2140) Google Scholar). Therefore, IGF-1 and certain other growth factors stimulate a cell survival pathway that involves Ras-dependent stimulation of PI3K, leading to activation of Akt/PKB. This pathway appears to be independent of MAPK and p70S6kinase and to prevent apoptosis induced by a variety of cellular challenges. Its importance in tissue culture systems as well as in primary neuronal cells suggests that this pathway may be of general significance. Further studies are needed to determine if Akt/PKB can be a suitable target for drug therapy directed at neurodegenerative and other degenerative human diseases. Many groups are currently extending these findings and contributing to the further understanding of Akt/PKB in survival in other cell systems. GSK3 has not been implicated in the regulation of survival (see references in11Kauffmann-Zeh A. Rodriguez-Viciana P. Ulrich E. Gilbert C. Coffer P. Downward J. Evan G. Nature, in press. 1997; Google Scholar), suggesting that Akt/PKB has more widespread roles in cell regulation and employs distinct subsets of substrates in different signaling systems. Additional direct downstream targets of Akt/PKB other than GSK3 must exist that have not been identified. Recent studies have provided some insight into the mechanism by which cell survival factors cause modifications to members of the Bcl-2 family of proteins (reviewed by10Gajewski T.F. Thompson C.B. Cell. 1996; 87: 589-592Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). The exact mechanisms of prevention of apoptosis by Akt/PKB are undetermined; future studies will address whether Akt/PKB mediates survival by phosphorylation and inhibition of proteins that are involved in programmed cell death. The observation that PI3K and Akt/PKB are involved in cell survival certainly could explain why so many oncoproteins, growth factors, and survival factors have evolved mechanisms for activation of PI3K.