Cyclic AMP Inhibits Akt Activity by Blocking the Membrane Localization of PDK1
2001; Elsevier BV; Volume: 276; Issue: 16 Linguagem: Inglês
10.1074/jbc.m001492200
ISSN1083-351X
AutoresSunhong Kim, Kwangho Jee, Dohoon Kim, Hyongjong Koh, Jongkyeong Chung,
Tópico(s)Cancer Mechanisms and Therapy
ResumoAkt is a protein serine/threonine kinase that plays an important role in the mitogenic responses of cells to variable stimuli. Akt contains a pleckstrin homology (PH) domain and is activated by phosphorylation at threonine 308 and serine 473. Binding of 3′-OH phosphorylated phosphoinositides to the PH domain results in the translocation of Akt to the plasma membrane where it is activated by upstream kinases such as (phosphoinositide-dependent kinase-1 (PDK1). Over-expression of constitutively active forms of Akt promotes cell proliferation and survival, and also stimulates p70 S6 kinase (p70S6K). In many cells, an increase in levels of intracellular cyclic AMP (cAMP) diminishes cell growth and promotes differentiation, and in certain conditions cAMP is even antagonistic to the effect of growth factors. Here, we show that cAMP has inhibitory effects on the phosphatidylinositol 3-kinase/PDK/Akt signaling pathway. cAMP potently inhibits phosphorylation at threonine 308 and serine 473 of Akt, which is required for the protein kinase activities of Akt. cAMP also negatively regulates PDK1 by inhibiting its translocation to the plasma membrane, despite not affecting its protein kinase activities. Furthermore, when we co-expressed myristoylated Akt and PDK1 mutants which constitutively co-localize in the plasma membrane, Akt activity was no longer sensitive to raised intracellular cAMP concentrations. Finally, cAMP was also found to inhibit the lipid kinase activity of PI3K and to decrease the levels of phosphatidylinositol 3,4,5-triphosphate in vivo, which are required for the membrane localization of PDK1. Collectively, these data strongly support the theory that the cAMP-dependent signaling pathway inhibits Akt activity by blocking the coupling between Akt and its upstream regulators, PDK, in the plasma membrane. Akt is a protein serine/threonine kinase that plays an important role in the mitogenic responses of cells to variable stimuli. Akt contains a pleckstrin homology (PH) domain and is activated by phosphorylation at threonine 308 and serine 473. Binding of 3′-OH phosphorylated phosphoinositides to the PH domain results in the translocation of Akt to the plasma membrane where it is activated by upstream kinases such as (phosphoinositide-dependent kinase-1 (PDK1). Over-expression of constitutively active forms of Akt promotes cell proliferation and survival, and also stimulates p70 S6 kinase (p70S6K). In many cells, an increase in levels of intracellular cyclic AMP (cAMP) diminishes cell growth and promotes differentiation, and in certain conditions cAMP is even antagonistic to the effect of growth factors. Here, we show that cAMP has inhibitory effects on the phosphatidylinositol 3-kinase/PDK/Akt signaling pathway. cAMP potently inhibits phosphorylation at threonine 308 and serine 473 of Akt, which is required for the protein kinase activities of Akt. cAMP also negatively regulates PDK1 by inhibiting its translocation to the plasma membrane, despite not affecting its protein kinase activities. Furthermore, when we co-expressed myristoylated Akt and PDK1 mutants which constitutively co-localize in the plasma membrane, Akt activity was no longer sensitive to raised intracellular cAMP concentrations. Finally, cAMP was also found to inhibit the lipid kinase activity of PI3K and to decrease the levels of phosphatidylinositol 3,4,5-triphosphate in vivo, which are required for the membrane localization of PDK1. Collectively, these data strongly support the theory that the cAMP-dependent signaling pathway inhibits Akt activity by blocking the coupling between Akt and its upstream regulators, PDK, in the plasma membrane. The phosphatidylinositol 3-kinase (PI3K)1-dependent cell signaling pathway has emerged as a key regulatory pathway involved in a number of cellular events (1Rameh L.E. Cantley L.C. J. Biol. Chem. 1999; 274: 8347-8350Abstract Full Text Full Text PDF PubMed Scopus (848) Google Scholar). Upon activation of growth factor tyrosine kinase receptors, the p85 regulatory subunit of PI3K recruits the p110 catalytic subunit to the plasma membrane (2Vanhaesebroeck B. Leevers S.J. Panayotou G. Waterfield M.D. Trends Biochem. Sci. 1997; 22: 267-272Abstract Full Text PDF PubMed Scopus (827) Google Scholar). The p110 catalytic subunit increases the level of PtdIns-3,4,5-P3and phosphatidylinositol 3,4-bisphosphate (PtdIns-3,4-P2), which induce the membrane translocation of PDK1 and Akt (also called PKB or RAC-PK) by binding to the pleckstrin homology domain (3Anderson K.E. Coadwell J. Stephens L.R. Hawkins P.T. Curr. Biol. 1998; 8: 684-691Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). In the membrane, PDK1 phosphorylates and activates Akt in a PtdIns-3,4,5-P3- or PtdIns-3,4-P2-dependent manner (4Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar, 5Stephens 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 (910) Google Scholar). By a mechanism that involves phospholipase C (6Ferguson K.M. Lemmon M.A. Sigler P.B. Schlessinger J. Nature Struct. Biol. 1995; 2: 715-718Crossref PubMed Scopus (59) Google Scholar), activated Akt is released from the membrane and phosphorylates various targets.This complex and unique signaling pathway has been implicated in a variety of cellular events such as cell proliferation and survival (1Rameh L.E. Cantley L.C. J. Biol. Chem. 1999; 274: 8347-8350Abstract Full Text Full Text PDF PubMed Scopus (848) Google Scholar,7Datta S.R. Brunet A. Greenberg M.E. Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3707) Google Scholar). Previously, it has been shown that various survival factors, such as nerve growth factor, require the activation of PI3K to prevent various cell types from undergoing apoptosis (8Yao R.J. Cooper G.M. Science. 1995; 267: 2003-2006Crossref PubMed Scopus (1288) Google Scholar, 9Kulik G. Klippel A. Weber M.J. Mol. Cell. Biol. 1997; 17: 1595-1606Crossref PubMed Scopus (964) Google Scholar). The mechanism by which the PI3K pathway protects cells from programmed cell death has been studied intensively. Recently, it was shown that Akt can phosphorylate serine 136 of BAD, a member of the pro-apoptotic Bcl-2 family, forming a binding site for 14-3-3 (10Del Peso L. Gonzalez-Garcia M. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1978) Google Scholar, 11Datta 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 (4914) Google Scholar). As BAD binds 14-3-3, it can no longer bind to Bcl-2 and Bcl-XL to inhibit their pro-survival activity. Akt also phosphorylates other important cellular factors involved in apoptosis, such as caspase-9 (12Cardone 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 (2719) Google Scholar) and fork-head transcription factors (13Brunet 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 (5366) Google Scholar), which results in the inhibition of apoptosis.Besides blocking apoptosis, the PI3K signaling pathway is involved in glycogen synthesis (14Cross D.A.E. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4324) Google Scholar, 15Frevert E.U. Kahn B.B. Mol. Cell. Biol. 1997; 17: 190-198Crossref PubMed Scopus (156) Google Scholar), glucose transport (16Cheatham B. Vlahos C.J. Cheatham L. Wang L. Blenis J. Kahn C.R. Mol. Cell. Biol. 1994; 14: 4902-4911Crossref PubMed Scopus (998) Google Scholar), and protein synthesis (17Thomas G. Hall M.N. Curr. Opin. Cell Biol. 1997; 9: 782-787Crossref PubMed Scopus (411) Google Scholar). The activities of Akt especially are closely correlated with these important biological activities. For example, GSK-3β (glycogen synthase kinase-3β) is phosphorylated at serine 9, and its activities are down-modulated by Akt (14Cross D.A.E. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4324) Google Scholar). Meanwhile, Tor (target of rapamycin) and p70S6K, which phosphorylate a translation initiation factor, 4E-BP (eIF4E-binding protein), and a ribosomal protein, S6, respectively, are positioned as downstream targets of Akt (18Scott P.H. Brunn G.J. Kohn A.D. Roth R.A. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7772-7777Crossref PubMed Scopus (408) Google Scholar, 19Burgering B.M. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1871) Google Scholar, 20Kohn A.D. Summers S.A. Birnbaum M.J. Roth R.A. J. Biol. Chem. 1996; 271: 31372-31378Abstract Full Text Full Text PDF PubMed Scopus (1085) Google Scholar, 21Koh H. Jee K. Lee B. Kim J. Kim D. Yun Y.H. Kim J.W. Choi H.S. Chung J. Oncogene. 1999; 18: 5115-5119Crossref PubMed Scopus (67) Google Scholar).cAMP and cAMP-dependent protein kinase (PKA) are regulators of development in many organisms and are involved in many other cellular processes. For example, cAMP is a key regulator ofDrosophila development by a mechanism in which PKA inhibits the Hedgehog-dependent activation of the Cubitus interruptus transcription factor (22Ohlmeyer J.T. Kalderon D. Genes Dev. 1997; 11: 2250-2258Crossref PubMed Scopus (74) Google Scholar). In Dictyostelium amoebae, extracellular cAMP regulates G-protein-dependent and -independent pathways to control aggregation as well as the activity of GSK-3 and the transcription factors GBF (G-box binding factor) and STAT during multicellular development (23Wang B. Kuspa A. Science. 1997; 277: 251-254Crossref PubMed Scopus (88) Google Scholar, 24Thomason P.A. Traynor D. Cavet G. Chang W. Harwood A.J. Kay R.R. EMBO J. 1998; 17: 2838-2845Crossref PubMed Scopus (122) Google Scholar).More importantly, cAMP and its effector, PKA, are implicated in a variety of cross-talks between intracellular signaling pathways. It was reported that the exit from mitosis in Xenopus egg extracts required the MPF (maturation-promoting factor)-dependent activation of the cAMP/PKA pathway (25Grieco D. Porcellini A. Avvedimento E.V. Gottesman M.E. Science. 1996; 271: 1718-1723Crossref PubMed Scopus (124) Google Scholar). There is also evidence that cross-talks between the Ca2+- and cAMP-dependent pathways exist in the cytoplasm (26DeBernadi M.A. Brooker G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4577-4582Crossref PubMed Scopus (40) Google Scholar) and the nucleus (27Rogue P.J. Humbert J.P. Meyer A. Freyermuth S. Krady M.M. Malviya A.N. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9178-9183Crossref PubMed Scopus (31) Google Scholar). Also, it was demonstrated that cAMP inhibits the Jak/STAT pathway (28David M. Petricoin 3rd, E. Larner A.C. J. Biol. Chem. 1996; 271: 4585-4588Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), which is important in cytokine signaling. cAMP-dependent protein kinase inhibits Gβγ-activated PLCβ2activity by phosphorylating PLCβ2 at a serine residuein vivo (29Liu M. Simon M.I. Nature. 1996; 382: 83-87Crossref PubMed Scopus (189) Google Scholar). In addition, the Ras-mediated mitogen-activated protein kinase pathway is strongly inhibited by cAMP-dependent signaling in various mechanisms (30Wu J. Dent P. Jelinek T. Wolfman A. Weber M.J. Sturgill T.W. Science. 1993; 262: 1065-1069Crossref PubMed Scopus (817) Google Scholar, 31Cook S.J. McCormick F. Science. 1993; 262: 1069-1072Crossref PubMed Scopus (860) Google Scholar, 32Burgering B.M. Pronk G.J. Van Weesen P. Chardin P. Bos J.L. EMBO J. 1993; 12: 4211-4220Crossref PubMed Scopus (313) Google Scholar, 33Graves L.M. Bornfeldt K.E. Raines E.W. Potts B.C. Macdonald S.G. Ross R. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10300-10304Crossref PubMed Scopus (400) Google Scholar).Recent findings suggest that there is also cross-talk between the PI3K pathway and the cAMP-dependent pathway. For example, an increased level of cAMP inhibits the interleukin-2-dependent activation of p70S6K (34Monfar M. Lemon K.P. Grammer T.C. Cheatham L. Chung J. Vlahos C.J. Blenis J. Mol. Cell. Biol. 1995; 15: 326-337Crossref PubMed Scopus (157) Google Scholar). In addition, CREB (cAMP response element-binding protein), in which transcriptional activities are induced by phosphorylation at serine 133, is phosphorylated at the same serine residue by both PKA and Aktin vivo (35Du K. Montminy M. J. Biol. Chem. 1998; 273: 32377-32379Abstract Full Text Full Text PDF PubMed Scopus (812) Google Scholar).Here, we demonstrate that an increase in the level of intracellular cAMP inhibits the activities of Akt, PI3K, and their downstream target, p70S6K. Interestingly, PDK1 activity was not affected by cAMP treatments, but its plasma membrane localization was dramatically reduced. Taken together, these results support that the cAMP-dependent signaling pathway inhibits Akt through inhibition of PI3K lipid kinase activity and the subsequent inhibition of PDK1 localization at the plasma membrane.DISCUSSIONWe have demonstrated that cAMP down-modulates Akt activity by interfering with the membrane localization of PDK1 and inhibiting the lipid kinase activity of PI3K. When we observed that the kinase activity of myristoylated Akt was strongly inhibited by forskolin, we first suspected that PKA might directly phosphorylate Akt and consequently inhibit the phosphotransferase activity of Akt, as Akt has a putative PKA phosphorylation site in its catalytic domain. However, the catalytic subunit of PKA was unable to phosphorylate Akt in vitro, and an Akt mutant that lacks the putative PKA phosphorylation site was still strongly inhibited by forskolin in vivo (data not shown). Furthermore, the phosphorylations required for the activity of Akt were greatly diminished by forskolin treatmentin vivo (Figs. 4 B and 5). These results led us to study the effects of cAMP on PDK1 and PI3K, two known upstream regulators of Akt.Previous studies have suggested that PDK1 is constitutively active and that its activity is regulated mainly by membrane localization via binding to PtdIns-3,4,5-P3 (3Anderson K.E. Coadwell J. Stephens L.R. Hawkins P.T. Curr. Biol. 1998; 8: 684-691Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 4Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar, 40Banfic H. Downes C.P. Rittenhouse S.E. J. Biol. Chem. 1998; 273: 11630-11637Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). In support of this hypothesis, a PDK1 mutant containing an N-terminal myristoylation signal constitutively activates co-expressed Akt proteinin vivo (3Anderson K.E. Coadwell J. Stephens L.R. Hawkins P.T. Curr. Biol. 1998; 8: 684-691Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). These results suggest that the pleckstrin homology domain-mediated localization of PDK1 and Akt in the plasma membrane is critical for the functional coupling between the two protein kinases. Interestingly, our results strongly support the theory that cAMP signaling interferes with this process by inhibiting the lipid kinase activity of PI3K and the in vivoproduction of PtdIns-3,4,5-P3 (Fig. 9, A andB, respectively). However, we do not currently understand how cAMP inhibits PI3K in the cell. Our preliminary data suggest at least that a direct phosphorylation of the p110 and p85 subunits of PI3K by PKA is not a mechanism behind the inhibition of the PI3K activity. 2J. Chung and S. Kim, unpublished results. We also examined the changes in tyrosine phosphorylation status of the p85 subunit of PI3K by cAMP but found that basal and EGF-stimulated tyrosine phosphorylation of the p85 protein was not significantly changed by cAMP under our experimental conditions.2 In addition, we found that cAMP does not affect the expression of PTEN.2 Thus, although we have excluded several possibilities for the mechanism of PI3K inhibition by cAMP, further studies are needed to elucidate this matter fully.Although we consistently observed an inhibitory role of cAMP for Akt in various cultured cells (Fig. 5), Van Obberghen and colleagues (44Stokoe D. Stephens L.R. Copeland T. Gaffney P.R. Reese C.B. Painter G.F. Holmes A.B. McCormick F. Hawkins P.T. Mol. Cell Biol. 1997; 19: 4989-5000Google Scholar) reported on the activation of Akt by PKA through a PI3K-independent pathway. However, not only was the activation of Akt by cAMP and PKA only minor, cAMP rather inhibited phosphorylation at serine 473 of Akt (44Stokoe D. Stephens L.R. Copeland T. Gaffney P.R. Reese C.B. Painter G.F. Holmes A.B. McCormick F. Hawkins P.T. Mol. Cell Biol. 1997; 19: 4989-5000Google Scholar). This finding does not agree with studies by other groups demonstrating that phosphorylation at serine 473 of Akt is necessary for its activity (4Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar, 42Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2495) Google Scholar, 43Stokoe 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 (1045) Google Scholar). On the other hand, we demonstrated that the EGF-stimulated phosphorylation of endogenous Akt at both threonine 308 and serine 473 was inhibited to basal levels by forskolin treatments (Fig. 5). We also demonstrated the inhibition of PI3K activity (Fig. 9 A), the in vivo production of PtdIns-3,4,5-P3 (Fig. 9 B), translocation of PDK1 to the plasma membrane (Fig. 7), and p70S6K activity (Fig.2 B) by cAMP. We believe these findings reflect the general nature of the inhibitory effect of cAMP on the PI3K pathway, as it would be illogical for cAMP to activate Akt while inhibiting its upstream regulators. Furthermore, our findings were consistent in a variety of cell lines. While we were revising this manuscript, others also revealed, in agreement with our data, that cAMP cannot induce the phosphorylations of Akt at threonine 308 and serine 473 and cannot activate Akt activity (45Fang X. Lu Y. Bast Jr., R.C. Woodgett J.R. Mills G.B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11960-11965Crossref PubMed Scopus (626) Google Scholar, 46Li M. Wang X. Meintzer M.K. Laessig T. Birnbaum M.J. Heidenreich K.A. Mol. Cell. Biol. 2000; 20: 9356-9363Crossref PubMed Scopus (334) Google Scholar). They also mentioned that cAMP decreases the activity of Akt (45Fang X. Lu Y. Bast Jr., R.C. Woodgett J.R. Mills G.B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11960-11965Crossref PubMed Scopus (626) Google Scholar). Therefore, we believe that we have demonstrated without a doubt the inhibition of Akt and other components of the PI3K pathway by cAMP under more relevant and natural experimental conditions. This conclusion is further supported by other groups' results that cAMP indirectly inhibits p70S6K, a downstream target of Akt and PI3K, in vivo (34Monfar M. Lemon K.P. Grammer T.C. Cheatham L. Chung J. Vlahos C.J. Blenis J. Mol. Cell. Biol. 1995; 15: 326-337Crossref PubMed Scopus (157) Google Scholar) and that cell-permeable cAMP fails to activate Akt in vivo (47Moule S.K. Welsh G.I. Edgell N.J. Foulstone E.J. Proud C.G. Denton R.M. J. Biol. Chem. 1997; 272: 7713-7719Crossref PubMed Scopus (226) Google Scholar).Akt plays important roles in protecting cells from various apoptotic pressures. Recent studies have shown that Akt exerts its anti-apoptotic activities by phosphorylating important regulators for apoptosis such as BAD (10Del Peso L. Gonzalez-Garcia M. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1978) Google Scholar, 11Datta 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 (4914) Google Scholar), caspase-9 (12Cardone 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 (2719) Google Scholar), Forkhead transcription factors (13Brunet 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 (5366) Google Scholar), etc. However, activation of the cAMP-dependent pathway can promote apoptosis. Lanotte and colleagues (48Ruchaud S. Seite P. Foulkes N.S. Sassone-Corsi P. Lanotte M. Oncogene. 1997; 15: 827-836Crossref PubMed Scopus (40) Google Scholar) have shown that activation of the cAMP pathway induces apoptosis within 4–6 h via CREM (cAMP-responsive element modulator) activation in a leukemia cell. In the v-Abl-transformed cell, cAMP induces programmed cell death via Raf-1 inhibition (49Weissinger E.M. Eissner G. Grammer C. Fackler S. Haefner B. Yoon L.S. Lu K.S. Bazarov A. Sedivy J.M. Mischak H. Kolch W. Mol. Cell. Biol. 1997; 17: 3229-3241Crossref PubMed Scopus (59) Google Scholar). In addition, retinoic acid and cAMP act in a synergistic fashion to induce apoptosis via caspase-3 activation (50Srivastava R.K. Srivastava A.R. Cho-Chung Y.S. Longo D.L. Oncogene. 1999; 18: 1755-1763Crossref PubMed Scopus (28) Google Scholar). However, we believe that these findings have yet to link the cAMP-dependent pathway directly to the known molecular mechanisms for regulating apoptosis. With our present findings, we now propose that inhibition of the Akt activity may be one of the major mechanisms behind the induction of apoptosis.In the present study, we have shown that cAMP inhibits the in vivo production of PtdIns-3,4,5-P3 and the activities of PI3K, PDK1, Akt and p70S6K, which suggests that other downstream targets of the PI3K pathway are also regulated by cAMP. As the PI3K- and cAMP-dependent pathways are deeply involved in various cellular events including cell growth, life span, metabolism, mobility and development, our findings may provide important clues to understanding the cAMP-PI3K cross-talk network and its role in the cell. The phosphatidylinositol 3-kinase (PI3K)1-dependent cell signaling pathway has emerged as a key regulatory pathway involved in a number of cellular events (1Rameh L.E. Cantley L.C. J. Biol. Chem. 1999; 274: 8347-8350Abstract Full Text Full Text PDF PubMed Scopus (848) Google Scholar). Upon activation of growth factor tyrosine kinase receptors, the p85 regulatory subunit of PI3K recruits the p110 catalytic subunit to the plasma membrane (2Vanhaesebroeck B. Leevers S.J. Panayotou G. Waterfield M.D. Trends Biochem. Sci. 1997; 22: 267-272Abstract Full Text PDF PubMed Scopus (827) Google Scholar). The p110 catalytic subunit increases the level of PtdIns-3,4,5-P3and phosphatidylinositol 3,4-bisphosphate (PtdIns-3,4-P2), which induce the membrane translocation of PDK1 and Akt (also called PKB or RAC-PK) by binding to the pleckstrin homology domain (3Anderson K.E. Coadwell J. Stephens L.R. Hawkins P.T. Curr. Biol. 1998; 8: 684-691Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). In the membrane, PDK1 phosphorylates and activates Akt in a PtdIns-3,4,5-P3- or PtdIns-3,4-P2-dependent manner (4Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar, 5Stephens 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 (910) Google Scholar). By a mechanism that involves phospholipase C (6Ferguson K.M. Lemmon M.A. Sigler P.B. Schlessinger J. Nature Struct. Biol. 1995; 2: 715-718Crossref PubMed Scopus (59) Google Scholar), activated Akt is released from the membrane and phosphorylates various targets. This complex and unique signaling pathway has been implicated in a variety of cellular events such as cell proliferation and survival (1Rameh L.E. Cantley L.C. J. Biol. Chem. 1999; 274: 8347-8350Abstract Full Text Full Text PDF PubMed Scopus (848) Google Scholar,7Datta S.R. Brunet A. Greenberg M.E. Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3707) Google Scholar). Previously, it has been shown that various survival factors, such as nerve growth factor, require the activation of PI3K to prevent various cell types from undergoing apoptosis (8Yao R.J. Cooper G.M. Science. 1995; 267: 2003-2006Crossref PubMed Scopus (1288) Google Scholar, 9Kulik G. Klippel A. Weber M.J. Mol. Cell. Biol. 1997; 17: 1595-1606Crossref PubMed Scopus (964) Google Scholar). The mechanism by which the PI3K pathway protects cells from programmed cell death has been studied intensively. Recently, it was shown that Akt can phosphorylate serine 136 of BAD, a member of the pro-apoptotic Bcl-2 family, forming a binding site for 14-3-3 (10Del Peso L. Gonzalez-Garcia M. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1978) Google Scholar, 11Datta 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 (4914) Google Scholar). As BAD binds 14-3-3, it can no longer bind to Bcl-2 and Bcl-XL to inhibit their pro-survival activity. Akt also phosphorylates other important cellular factors involved in apoptosis, such as caspase-9 (12Cardone 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 (2719) Google Scholar) and fork-head transcription factors (13Brunet 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 (5366) Google Scholar), which results in the inhibition of apoptosis. Besides blocking apoptosis, the PI3K signaling pathway is involved in glycogen synthesis (14Cross D.A.E. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4324) Google Scholar, 15Frevert E.U. Kahn B.B. Mol. Cell. Biol. 1997; 17: 190-198Crossref PubMed Scopus (156) Google Scholar), glucose transport (16Cheatham B. Vlahos C.J. Cheatham L. Wang L. Blenis J. Kahn C.R. Mol. Cell. Biol. 1994; 14: 4902-4911Crossref PubMed Scopus (998) Google Scholar), and protein synthesis (17Thomas G. Hall M.N. Curr. Opin. Cell Biol. 1997; 9: 782-787Crossref PubMed Scopus (411) Google Scholar). The activities of Akt especially are closely correlated with these important biological activities. For example, GSK-3β (glycogen synthase kinase-3β) is phosphorylated at serine 9, and its activities are down-modulated by Akt (14Cross D.A.E. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4324) Google Scholar). Meanwhile, Tor (target of rapamycin) and p70S6K, which phosphorylate a translation initiation factor, 4E-BP (eIF4E-binding protein), and a ribosomal protein, S6, respectively, are positioned as downstream targets of Akt (18Scott P.H. Brunn G.J. Kohn A.D. Roth R.A. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7772-7777Crossref PubMed Scopus (408) Google Scholar, 19Burgering B.M. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1871) Google Scholar, 20Kohn A.D. Summers S.A. Birnbaum M.J. Roth R.A. J. Biol. Chem. 1996; 271: 31372-31378Abstract Full Text Full Text PDF PubMed Scopus (1085) Google Scholar, 21Koh H. Jee K. Lee B. Kim J. Kim D. Yun Y.H. Kim J.W. Choi H.S. Chung J. Oncogene. 1999; 18: 5115-5119Crossref PubMed Scopus (67) Google Scholar). cAMP and cAMP-dependent protein kinase (PKA) are regulators of development in many organisms and are involved in many other cellular processes. For example, cAMP is a key regulator ofDrosophila development by a mechanism in which PKA inhibits the Hedgehog-dependent activation of the Cubitus interruptus transcription factor (22Ohlmeyer J.T. Kalderon D. Genes Dev. 1997; 11: 2250-2258Crossref PubMed Scopus (74) Google Scholar). In Dictyostelium amoebae, extracellular cAMP regulates G-protein-dependent and -independent pathways to control aggregation as well as the activity of GSK-3 and the transcription factors GBF (G-box binding factor) and STAT during multicellular development (23Wang B. Kuspa A. Science. 1997; 277: 251-254Crossref PubMed Scopus (88) Google Scholar, 24Thomason P.A. Traynor D. Cavet G. Chang W. Harwood A.J. Kay R.R. EMBO J. 1998; 17: 2838-2845Crossref PubMed Scopus (122) Google Scholar). More importantly, cAMP and its effector, PKA, are implicated in a variety of cross-talks between intracellular signaling pathways. It was reported that the exit from mitosis in Xenopus egg extracts required the MPF (maturation-promoting factor)-dependent activation of the cAMP/PKA pathway (25Grieco D. Porcellini A. Avvedimento E.V. Gottesman M.E. Science. 1996; 271: 1718-1723Crossref PubMed Scopus (124) Google Scholar). There is also evidence that cross-talks between the Ca2+- and cAMP-dependent pathways exist in the cytoplasm (26DeBernadi M.A. Brooker G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4577-4582Crossref PubMed Scopus (40) Google Scholar) and the nucleus (27Rogue P.J. Humbert J.P. Meyer A. Freyermuth S. Krady M.M. Malviya A.N. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9178-9183Crossref PubMed Scopus (31) Google Scholar). Also, it was demonstrated that cAMP inhibits the Jak/STAT pathway (28David M. Petricoin 3rd, E. Larner A.C. J. Biol. Chem. 1996; 271: 4585-4588Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), which is important in cytokine signaling. cAMP-dependent protein kinase inhibits Gβγ-activated PLCβ2activity by phosphorylating PLCβ2 at a serine residuein vivo (29Liu M. Simon M.I. Nature. 1996; 382: 83-87Crossref PubMed Scopus (189) Google Scholar). In addition, the Ras-mediated mitogen-activated protein kinase pathway is strongly inhibited by cAMP-dependent signaling in various mechanisms (30Wu J. Dent P. Jelinek T. Wolfman A. Weber M.J. Sturgill T.W. Science. 1993; 262: 1065-1069Crossref PubMed Scopus (817) Google Scholar, 31Cook S.J. McCormick F. Science. 1993; 262: 1069-1072Crossref PubMed Scopus (860) Google Scholar, 32Burgering B.M. Pronk G.J. Van Weesen P. Chardin P. Bos J.L. EMBO J. 1993; 12: 4211-4220Crossref PubMed Scopus (313) Google Scholar, 33Graves L.M. Bornfeldt K.E. Raines E.W. Potts B.C. Macdonald S.G. Ross R. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10300-10304Crossref PubMed Scopus (400) Google Scholar). Recent findings suggest that there is also cross-talk between the PI3K pathway and the cAMP-dependent pathway. For example, an increased level of cAMP inhibits the interleukin-2-dependent activation of p70S6K (34Monfar M. Lemon K.P. Grammer T.C. Cheatham L. Chung J. Vlahos C.J. Blenis J. Mol. Cell. Biol. 1995; 15: 326-337Crossref PubMed Scopus (157) Google Scholar). In addition, CREB (cAMP response element-binding protein), in which transcriptional activities are induced by phosphorylation at serine 133, is phosphorylated at the same serine residue by both PKA and Aktin vivo (35Du K. Montminy M. J. Biol. Chem. 1998; 273: 32377-32379Abstract Full Text Full Text PDF PubMed Scopus (812) Google Scholar). Here, we demonstrate that an increase in the level of intracellular cAMP inhibits the activities of Akt, PI3K, and their downstream target, p70S6K. Interestingly, PDK1 activity was not affected by cAMP treatments, but its plasma membrane localization was dramatically reduced. Taken together, these results support that the cAMP-dependent signaling pathway inhibits Akt through inhibition of PI3K lipid kinase activity and the subsequent inhibition of PDK1 localization at the plasma membrane. DISCUSSIONWe have demonstrated that cAMP down-modulates Akt activity by interfering with the membrane localization of PDK1 and inhibiting the lipid kinase activity of PI3K. When we observed that the kinase activity of myristoylated Akt was strongly inhibited by forskolin, we first suspected that PKA might directly phosphorylate Akt and consequently inhibit the phosphotransferase activity of Akt, as Akt has a putative PKA phosphorylation site in its catalytic domain. However, the catalytic subunit of PKA was unable to phosphorylate Akt in vitro, and an Akt mutant that lacks the putative PKA phosphorylation site was still strongly inhibited by forskolin in vivo (data not shown). Furthermore, the phosphorylations required for the activity of Akt were greatly diminished by forskolin treatmentin vivo (Figs. 4 B and 5). These results led us to study the effects of cAMP on PDK1 and PI3K, two known upstream regulators of Akt.Previous studies have suggested that PDK1 is constitutively active and that its activity is regulated mainly by membrane localization via binding to PtdIns-3,4,5-P3 (3Anderson K.E. Coadwell J. Stephens L.R. Hawkins P.T. Curr. Biol. 1998; 8: 684-691Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 4Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar, 40Banfic H. Downes C.P. Rittenhouse S.E. J. Biol. Chem. 1998; 273: 11630-11637Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). In support of this hypothesis, a PDK1 mutant containing an N-terminal myristoylation signal constitutively activates co-expressed Akt proteinin vivo (3Anderson K.E. Coadwell J. Stephens L.R. Hawkins P.T. Curr. Biol. 1998; 8: 684-691Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). These results suggest that the pleckstrin homology domain-mediated localization of PDK1 and Akt in the plasma membrane is critical for the functional coupling between the two protein kinases. Interestingly, our results strongly support the theory that cAMP signaling interferes with this process by inhibiting the lipid kinase activity of PI3K and the in vivoproduction of PtdIns-3,4,5-P3 (Fig. 9, A andB, respectively). However, we do not currently understand how cAMP inhibits PI3K in the cell. Our preliminary data suggest at least that a direct phosphorylation of the p110 and p85 subunits of PI3K by PKA is not a mechanism behind the inhibition of the PI3K activity. 2J. Chung and S. Kim, unpublished results. We also examined the changes in tyrosine phosphorylation status of the p85 subunit of PI3K by cAMP but found that basal and EGF-stimulated tyrosine phosphorylation of the p85 protein was not significantly changed by cAMP under our experimental conditions.2 In addition, we found that cAMP does not affect the expression of PTEN.2 Thus, although we have excluded several possibilities for the mechanism of PI3K inhibition by cAMP, further studies are needed to elucidate this matter fully.Although we consistently observed an inhibitory role of cAMP for Akt in various cultured cells (Fig. 5), Van Obberghen and colleagues (44Stokoe D. Stephens L.R. Copeland T. Gaffney P.R. Reese C.B. Painter G.F. Holmes A.B. McCormick F. Hawkins P.T. Mol. Cell Biol. 1997; 19: 4989-5000Google Scholar) reported on the activation of Akt by PKA through a PI3K-independent pathway. However, not only was the activation of Akt by cAMP and PKA only minor, cAMP rather inhibited phosphorylation at serine 473 of Akt (44Stokoe D. Stephens L.R. Copeland T. Gaffney P.R. Reese C.B. Painter G.F. Holmes A.B. McCormick F. Hawkins P.T. Mol. Cell Biol. 1997; 19: 4989-5000Google Scholar). This finding does not agree with studies by other groups demonstrating that phosphorylation at serine 473 of Akt is necessary for its activity (4Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar, 42Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2495) Google Scholar, 43Stokoe 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 (1045) Google Scholar). On the other hand, we demonstrated that the EGF-stimulated phosphorylation of endogenous Akt at both threonine 308 and serine 473 was inhibited to basal levels by forskolin treatments (Fig. 5). We also demonstrated the inhibition of PI3K activity (Fig. 9 A), the in vivo production of PtdIns-3,4,5-P3 (Fig. 9 B), translocation of PDK1 to the plasma membrane (Fig. 7), and p70S6K activity (Fig.2 B) by cAMP. We believe these findings reflect the general nature of the inhibitory effect of cAMP on the PI3K pathway, as it would be illogical for cAMP to activate Akt while inhibiting its upstream regulators. Furthermore, our findings were consistent in a variety of cell lines. While we were revising this manuscript, others also revealed, in agreement with our data, that cAMP cannot induce the phosphorylations of Akt at threonine 308 and serine 473 and cannot activate Akt activity (45Fang X. Lu Y. Bast Jr., R.C. Woodgett J.R. Mills G.B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11960-11965Crossref PubMed Scopus (626) Google Scholar, 46Li M. Wang X. Meintzer M.K. Laessig T. Birnbaum M.J. Heidenreich K.A. Mol. Cell. Biol. 2000; 20: 9356-9363Crossref PubMed Scopus (334) Google Scholar). They also mentioned that cAMP decreases the activity of Akt (45Fang X. Lu Y. Bast Jr., R.C. Woodgett J.R. Mills G.B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11960-11965Crossref PubMed Scopus (626) Google Scholar). Therefore, we believe that we have demonstrated without a doubt the inhibition of Akt and other components of the PI3K pathway by cAMP under more relevant and natural experimental conditions. This conclusion is further supported by other groups' results that cAMP indirectly inhibits p70S6K, a downstream target of Akt and PI3K, in vivo (34Monfar M. Lemon K.P. Grammer T.C. Cheatham L. Chung J. Vlahos C.J. Blenis J. Mol. Cell. Biol. 1995; 15: 326-337Crossref PubMed Scopus (157) Google Scholar) and that cell-permeable cAMP fails to activate Akt in vivo (47Moule S.K. Welsh G.I. Edgell N.J. Foulstone E.J. Proud C.G. Denton R.M. J. Biol. Chem. 1997; 272: 7713-7719Crossref PubMed Scopus (226) Google Scholar).Akt plays important roles in protecting cells from various apoptotic pressures. Recent studies have shown that Akt exerts its anti-apoptotic activities by phosphorylating important regulators for apoptosis such as BAD (10Del Peso L. Gonzalez-Garcia M. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1978) Google Scholar, 11Datta 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 (4914) Google Scholar), caspase-9 (12Cardone 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 (2719) Google Scholar), Forkhead transcription factors (13Brunet 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 (5366) Google Scholar), etc. However, activation of the cAMP-dependent pathway can promote apoptosis. Lanotte and colleagues (48Ruchaud S. Seite P. Foulkes N.S. Sassone-Corsi P. Lanotte M. Oncogene. 1997; 15: 827-836Crossref PubMed Scopus (40) Google Scholar) have shown that activation of the cAMP pathway induces apoptosis within 4–6 h via CREM (cAMP-responsive element modulator) activation in a leukemia cell. In the v-Abl-transformed cell, cAMP induces programmed cell death via Raf-1 inhibition (49Weissinger E.M. Eissner G. Grammer C. Fackler S. Haefner B. Yoon L.S. Lu K.S. Bazarov A. Sedivy J.M. Mischak H. Kolch W. Mol. Cell. Biol. 1997; 17: 3229-3241Crossref PubMed Scopus (59) Google Scholar). In addition, retinoic acid and cAMP act in a synergistic fashion to induce apoptosis via caspase-3 activation (50Srivastava R.K. Srivastava A.R. Cho-Chung Y.S. Longo D.L. Oncogene. 1999; 18: 1755-1763Crossref PubMed Scopus (28) Google Scholar). However, we believe that these findings have yet to link the cAMP-dependent pathway directly to the known molecular mechanisms for regulating apoptosis. With our present findings, we now propose that inhibition of the Akt activity may be one of the major mechanisms behind the induction of apoptosis.In the present study, we have shown that cAMP inhibits the in vivo production of PtdIns-3,4,5-P3 and the activities of PI3K, PDK1, Akt and p70S6K, which suggests that other downstream targets of the PI3K pathway are also regulated by cAMP. As the PI3K- and cAMP-dependent pathways are deeply involved in various cellular events including cell growth, life span, metabolism, mobility and development, our findings may provide important clues to understanding the cAMP-PI3K cross-talk network and its role in the cell. We have demonstrated that cAMP down-modulates Akt activity by interfering with the membrane localization of PDK1 and inhibiting the lipid kinase activity of PI3K. When we observed that the kinase activity of myristoylated Akt was strongly inhibited by forskolin, we first suspected that PKA might directly phosphorylate Akt and consequently inhibit the phosphotransferase activity of Akt, as Akt has a putative PKA phosphorylation site in its catalytic domain. However, the catalytic subunit of PKA was unable to phosphorylate Akt in vitro, and an Akt mutant that lacks the putative PKA phosphorylation site was still strongly inhibited by forskolin in vivo (data not shown). Furthermore, the phosphorylations required for the activity of Akt were greatly diminished by forskolin treatmentin vivo (Figs. 4 B and 5). These results led us to study the effects of cAMP on PDK1 and PI3K, two known upstream regulators of Akt. Previous studies have suggested that PDK1 is constitutively active and that its activity is regulated mainly by membrane localization via binding to PtdIns-3,4,5-P3 (3Anderson K.E. Coadwell J. Stephens L.R. Hawkins P.T. Curr. Biol. 1998; 8: 684-691Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 4Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar, 40Banfic H. Downes C.P. Rittenhouse S.E. J. Biol. Chem. 1998; 273: 11630-11637Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). In support of this hypothesis, a PDK1 mutant containing an N-terminal myristoylation signal constitutively activates co-expressed Akt proteinin vivo (3Anderson K.E. Coadwell J. Stephens L.R. Hawkins P.T. Curr. Biol. 1998; 8: 684-691Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). These results suggest that the pleckstrin homology domain-mediated localization of PDK1 and Akt in the plasma membrane is critical for the functional coupling between the two protein kinases. Interestingly, our results strongly support the theory that cAMP signaling interferes with this process by inhibiting the lipid kinase activity of PI3K and the in vivoproduction of PtdIns-3,4,5-P3 (Fig. 9, A andB, respectively). However, we do not currently understand how cAMP inhibits PI3K in the cell. Our preliminary data suggest at least that a direct phosphorylation of the p110 and p85 subunits of PI3K by PKA is not a mechanism behind the inhibition of the PI3K activity. 2J. Chung and S. Kim, unpublished results. We also examined the changes in tyrosine phosphorylation status of the p85 subunit of PI3K by cAMP but found that basal and EGF-stimulated tyrosine phosphorylation of the p85 protein was not significantly changed by cAMP under our experimental conditions.2 In addition, we found that cAMP does not affect the expression of PTEN.2 Thus, although we have excluded several possibilities for the mechanism of PI3K inhibition by cAMP, further studies are needed to elucidate this matter fully. Although we consistently observed an inhibitory role of cAMP for Akt in various cultured cells (Fig. 5), Van Obberghen and colleagues (44Stokoe D. Stephens L.R. Copeland T. Gaffney P.R. Reese C.B. Painter G.F. Holmes A.B. McCormick F. Hawkins P.T. Mol. Cell Biol. 1997; 19: 4989-5000Google Scholar) reported on the activation of Akt by PKA through a PI3K-independent pathway. However, not only was the activation of Akt by cAMP and PKA only minor, cAMP rather inhibited phosphorylation at serine 473 of Akt (44Stokoe D. Stephens L.R. Copeland T. Gaffney P.R. Reese C.B. Painter G.F. Holmes A.B. McCormick F. Hawkins P.T. Mol. Cell Biol. 1997; 19: 4989-5000Google Scholar). This finding does not agree with studies by other groups demonstrating that phosphorylation at serine 473 of Akt is necessary for its activity (4Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar, 42Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2495) Google Scholar, 43Stokoe 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 (1045) Google Scholar). On the other hand, we demonstrated that the EGF-stimulated phosphorylation of endogenous Akt at both threonine 308 and serine 473 was inhibited to basal levels by forskolin treatments (Fig. 5). We also demonstrated the inhibition of PI3K activity (Fig. 9 A), the in vivo production of PtdIns-3,4,5-P3 (Fig. 9 B), translocation of PDK1 to the plasma membrane (Fig. 7), and p70S6K activity (Fig.2 B) by cAMP. We believe these findings reflect the general nature of the inhibitory effect of cAMP on the PI3K pathway, as it would be illogical for cAMP to activate Akt while inhibiting its upstream regulators. Furthermore, our findings were consistent in a variety of cell lines. While we were revising this manuscript, others also revealed, in agreement with our data, that cAMP cannot induce the phosphorylations of Akt at threonine 308 and serine 473 and cannot activate Akt activity (45Fang X. Lu Y. Bast Jr., R.C. Woodgett J.R. Mills G.B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11960-11965Crossref PubMed Scopus (626) Google Scholar, 46Li M. Wang X. Meintzer M.K. Laessig T. Birnbaum M.J. Heidenreich K.A. Mol. Cell. Biol. 2000; 20: 9356-9363Crossref PubMed Scopus (334) Google Scholar). They also mentioned that cAMP decreases the activity of Akt (45Fang X. Lu Y. Bast Jr., R.C. Woodgett J.R. Mills G.B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11960-11965Crossref PubMed Scopus (626) Google Scholar). Therefore, we believe that we have demonstrated without a doubt the inhibition of Akt and other components of the PI3K pathway by cAMP under more relevant and natural experimental conditions. This conclusion is further supported by other groups' results that cAMP indirectly inhibits p70S6K, a downstream target of Akt and PI3K, in vivo (34Monfar M. Lemon K.P. Grammer T.C. Cheatham L. Chung J. Vlahos C.J. Blenis J. Mol. Cell. Biol. 1995; 15: 326-337Crossref PubMed Scopus (157) Google Scholar) and that cell-permeable cAMP fails to activate Akt in vivo (47Moule S.K. Welsh G.I. Edgell N.J. Foulstone E.J. Proud C.G. Denton R.M. J. Biol. Chem. 1997; 272: 7713-7719Crossref PubMed Scopus (226) Google Scholar). Akt plays important roles in protecting cells from various apoptotic pressures. Recent studies have shown that Akt exerts its anti-apoptotic activities by phosphorylating important regulators for apoptosis such as BAD (10Del Peso L. Gonzalez-Garcia M. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1978) Google Scholar, 11Datta 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 (4914) Google Scholar), caspase-9 (12Cardone 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 (2719) Google Scholar), Forkhead transcription factors (13Brunet 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 (5366) Google Scholar), etc. However, activation of the cAMP-dependent pathway can promote apoptosis. Lanotte and colleagues (48Ruchaud S. Seite P. Foulkes N.S. Sassone-Corsi P. Lanotte M. Oncogene. 1997; 15: 827-836Crossref PubMed Scopus (40) Google Scholar) have shown that activation of the cAMP pathway induces apoptosis within 4–6 h via CREM (cAMP-responsive element modulator) activation in a leukemia cell. In the v-Abl-transformed cell, cAMP induces programmed cell death via Raf-1 inhibition (49Weissinger E.M. Eissner G. Grammer C. Fackler S. Haefner B. Yoon L.S. Lu K.S. Bazarov A. Sedivy J.M. Mischak H. Kolch W. Mol. Cell. Biol. 1997; 17: 3229-3241Crossref PubMed Scopus (59) Google Scholar). In addition, retinoic acid and cAMP act in a synergistic fashion to induce apoptosis via caspase-3 activation (50Srivastava R.K. Srivastava A.R. Cho-Chung Y.S. Longo D.L. Oncogene. 1999; 18: 1755-1763Crossref PubMed Scopus (28) Google Scholar). However, we believe that these findings have yet to link the cAMP-dependent pathway directly to the known molecular mechanisms for regulating apoptosis. With our present findings, we now propose that inhibition of the Akt activity may be one of the major mechanisms behind the induction of apoptosis. In the present study, we have shown that cAMP inhibits the in vivo production of PtdIns-3,4,5-P3 and the activities of PI3K, PDK1, Akt and p70S6K, which suggests that other downstream targets of the PI3K pathway are also regulated by cAMP. As the PI3K- and cAMP-dependent pathways are deeply involved in various cellular events including cell growth, life span, metabolism, mobility and development, our findings may provide important clues to understanding the cAMP-PI3K cross-talk network and its role in the cell. We are grateful to Drs. D. R. Alessi, J. Blenis, J. Downward, and R. A. Roth for providing reagents. phosphatidylinositol 3-kinase epidermal growth factor phosphoinositide-dependent kinase cyclic AMP bromo cAMP-dependent protein kinase myristoylated Akt p70 S6 kinase 4,5-P3, phosphatidylinositol 3,4,5-trisphosphate 4-P2, phosphatidylinositol 3,4-bisphosphate hemagglutinin Janus kinase/signal transducers and activators of transcription
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