The Inositol Phosphatase SHIP Inhibits Akt/PKB Activation in B Cells
1998; Elsevier BV; Volume: 273; Issue: 51 Linguagem: Inglês
10.1074/jbc.273.51.33922
ISSN1083-351X
AutoresM. Javad Aman, Thomas D. Lamkin, Hidetaka Okada, Tomohiro Kurosaki, Kodi S. Ravichandran,
Tópico(s)Chronic Lymphocytic Leukemia Research
ResumoThe serine-threonine kinase Akt/PKB is activated downstream of phosphatidylinositol 3-kinase in response to several growth factor stimuli and has been implicated in the promotion of cell survival. Although both phosphatidylinositol 3,4,5-trisphosphate (PIP3) and phosphatidylinositol 3,4-bisphosphate (PI 3,4-P2) have been implicated in the regulation of Akt activity in vitro, the relative roles of these two phospholipids in vivo are not well understood. Co-ligation of the B cell receptor (BCR) and the inhibitory FcγRIIB1 on B cells results in the recruitment of the 5′-inositol phosphatase SHIP to the signaling complex. Since SHIP is known to cleave PIP3 to generate PI 3,4-P2 both in vivo and in vitro, and Akt activity has been reported to be regulated by either PIP3 or PI 3,4-P2, we hypothesized that recruitment of SHIP through FcγRIIB1 co-cross-linking to the BCR in B cells might regulate Akt activity. The nature of this regulation, positive or negative, might also reveal the relative contribution of PIP3 and PI 3,4-P2 to Akt activation in vivo. Here we report that Akt is activated by stimulation through the BCR in a phosphatidylinositol 3-kinase-dependent manner and that this activation is inhibited by co-cross-linking of the BCR to FcγRIIB1. Using mutants of FcγRIIB1 and SHIP-deficient B cells, we demonstrate that inhibition of Akt activity is mediated by the immune cell tyrosine-based inhibitory motif within FcγRIIB1 as well as SHIP. The SHIP-dependent inhibition of Akt activation also suggests that PIP3 plays a greater role in Akt activation than PI 3,4-P2 in vivo. The serine-threonine kinase Akt/PKB is activated downstream of phosphatidylinositol 3-kinase in response to several growth factor stimuli and has been implicated in the promotion of cell survival. Although both phosphatidylinositol 3,4,5-trisphosphate (PIP3) and phosphatidylinositol 3,4-bisphosphate (PI 3,4-P2) have been implicated in the regulation of Akt activity in vitro, the relative roles of these two phospholipids in vivo are not well understood. Co-ligation of the B cell receptor (BCR) and the inhibitory FcγRIIB1 on B cells results in the recruitment of the 5′-inositol phosphatase SHIP to the signaling complex. Since SHIP is known to cleave PIP3 to generate PI 3,4-P2 both in vivo and in vitro, and Akt activity has been reported to be regulated by either PIP3 or PI 3,4-P2, we hypothesized that recruitment of SHIP through FcγRIIB1 co-cross-linking to the BCR in B cells might regulate Akt activity. The nature of this regulation, positive or negative, might also reveal the relative contribution of PIP3 and PI 3,4-P2 to Akt activation in vivo. Here we report that Akt is activated by stimulation through the BCR in a phosphatidylinositol 3-kinase-dependent manner and that this activation is inhibited by co-cross-linking of the BCR to FcγRIIB1. Using mutants of FcγRIIB1 and SHIP-deficient B cells, we demonstrate that inhibition of Akt activity is mediated by the immune cell tyrosine-based inhibitory motif within FcγRIIB1 as well as SHIP. The SHIP-dependent inhibition of Akt activation also suggests that PIP3 plays a greater role in Akt activation than PI 3,4-P2 in vivo. pleckstrin homology phosphatidylinositol 3-kinase phosphatidylinositol 3,4,5-trisphosphate 4-P2, phosphatidylinositol 3,4-bisphosphate B cell receptor Src homology 2 immune cell tyrosine-based inhibitory motif glycogen sythase kinase-3. Akt (also called protein kinase B) is a serine/threonine kinase that is activated upon ligation of several cell surface receptors, including the receptors for insulin and platelet-derived growth factor (1Franke 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 (1820) Google Scholar, 2Burgering B.M. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1872) Google Scholar, 3Kohn A.D. Takeuchi F. Roth R.A. J. Biol. Chem. 1996; 271: 21920-21926Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar, 4Cross D.A. Watt P.W. Shaw M. van der Kaay J. Downes C.P. Holder J.C. Cohen P. FEBS Lett. 1997; 406: 211-215Crossref PubMed Scopus (191) Google Scholar). The biological significance of Akt has been demonstrated by its ability to protect a variety of cell types from apoptosis (5Dudek 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 (2213) Google Scholar, 6Kauffmann-Zeh A. Rodriguez-Viciana P. Ulrich E. Gilbert C. Coffer P. Downward J. Evan G. 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Nature. 1997; 387: 673-676Crossref PubMed Scopus (1218) Google Scholar), since the specific PI3K inhibitor wortmannin can block Akt activation, and platelet-derived growth factor receptor mutants that fail to activate PI3K also fail to activate Akt (1Franke 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 (1820) Google Scholar, 2Burgering B.M. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1872) Google Scholar, 3Kohn A.D. Takeuchi F. Roth R.A. J. Biol. Chem. 1996; 271: 21920-21926Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar).Recent studies have established the importance of both 3′-phosphorylated inositol phosphates (PI 3,4-P2 and PIP3) and Akt phosphorylation in activation of Akt. PI 3,4-P2 has been shown to directly activate Akt in vitro via interaction with the Akt PH domain, while PIP3 was inhibitory in this experimental system (11Klippel A. Kavanaugh W.M. Pot D. Williams L.T. Mol. Cell. Biol. 1997; 17: 338-344Crossref PubMed Scopus (446) Google Scholar, 12Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1294) Google Scholar, 13Frech M. Andjelkovic M. Ingley E. Reddy K.K. Falck J.R. Hemmings B.A. J. Biol. Chem. 1997; 272: 8474-8481Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar). Additionally, Akt enzymatic activity was shown to be dependent on phosphorylation of Akt on a specific serine (Ser473) and a specific threonine (Thr308) (14Andjelkovic M. Alessi D.R. Meier R. Fernandez A. Lamb N.J. Frech M. Cron P. Cohen P. Lucocq J.M. Hemmings B.A. J. Biol. Chem. 1997; 272: 31515-31524Abstract Full Text Full Text PDF PubMed Scopus (895) Google Scholar, 15Alessi 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). Akt is phosphorylated by at least two serine-threonine kinases only in the presence of 3′-phosphorylated phospholipids (16Alessi D.R. Cohen P. Curr. Opin. Genet. Dev. 1998; 8: 55-62Crossref PubMed Scopus (674) Google Scholar, 17Downward J. Science. 1998; 279: 673-674Crossref PubMed Scopus (181) Google Scholar). One of the kinases that phosphorylates Akt, PDK1, has recently been cloned (18Alessi 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 (616) Google Scholar, 19Stephens 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, 20Pullen N. Dennis P.B. Andjelkovic M. Dufner A. Kozma S.C. Hemmings B.A. Thomas G. Science. 1998; 279: 707-710Crossref PubMed Scopus (723) Google Scholar). The studies mentioned above demonstrating direct activation of Akt by PI 3,4-P2 and its inhibition by PIP3 were performed with immunoprecipitated Akt in the absence of PDK1 (11Klippel A. Kavanaugh W.M. Pot D. Williams L.T. Mol. Cell. Biol. 1997; 17: 338-344Crossref PubMed Scopus (446) Google Scholar, 12Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1294) Google Scholar, 13Frech M. Andjelkovic M. Ingley E. Reddy K.K. Falck J.R. Hemmings B.A. J. Biol. Chem. 1997; 272: 8474-8481Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar). In contrast, when purified PDK1 was present, activation of Akt was dependent on the presence of 3′-phosphorylated inositol lipids, with PIP3 being at least 2–3-fold more effective than PI 3,4-P2 in allowing Akt activation (18Alessi 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 (616) Google Scholar, 19Stephens 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, 21Alessi 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, 22Stokoe 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). Studies utilizing PH mutants of Akt and PDK1 revealed that the regulatory action of PIP3 in the PDK-mediated activation of Akt is primarily directed toward Akt rather than PDK1 (17Downward J. Science. 1998; 279: 673-674Crossref PubMed Scopus (181) Google Scholar, 18Alessi 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 (616) Google Scholar, 20Pullen N. Dennis P.B. Andjelkovic M. Dufner A. Kozma S.C. Hemmings B.A. Thomas G. Science. 1998; 279: 707-710Crossref PubMed Scopus (723) Google Scholar, 22Stokoe 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). Based on these data, the current model of Akt activation is that Akt is recruited to the membrane by its PH domain binding to 3-OH-phosphorylated phosphatidylinositol phosphates, which causes a conformational change in Akt, which allows phosphorylation and activation by PDK1 and at least one other kinase. Although there is some indication that PIP3 may be more permissive in allowing PDK1-mediated activation of Akt than PI 3,4-P2 in vitro (18Alessi 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 (616) Google Scholar, 19Stephens 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), the relative roles of PIP3and PI 3,4-P2 in Akt activation in vivo remain to be determined.Antigen-mediated activation of B cells through the B cell receptor (BCR) initiates a cascade of intracellular biochemical events including activation of tyrosine kinases, activation of PI3K and PLCγ, subsequent generation of phospholipid and inositol phosphate second messengers, and calcium flux (23DeFranco A.L. Curr. Opin. Immunol. 1997; 9: 296-308Crossref PubMed Scopus (282) Google Scholar). The significance of PI3K activation in BCR signaling has been demonstrated by the ability of the PI3K inhibitor wortmannin to inhibit BCR-induced calcium flux (24Kiener P.A. Lioubin M.N. Rohrschneider L.R. Ledbetter J.A. Nadler S.G. Diegel M.L. J. Biol. Chem. 1997; 272: 3838-3844Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) and anti-Ig-induced proliferation of the human B cell line RL (25Beckwith M. Fenton R.G. 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Chem. 1997; 272: 3838-3844Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 29Ono M. Bolland S. Tempst P. Ravetch J.V. Nature. 1996; 383: 263-266Crossref PubMed Scopus (644) Google Scholar, 30Tridandapani S. Kelley T. Pradhan M. Cooney D. Justement L.B. Coggeshall K.M. Mol. Cell. Biol. 1997; 17: 4305-4311Crossref PubMed Google Scholar, 31D'Ambrosio D. Fong D.C. Cambier J.C. Immunol. Lett. 1996; 54: 77-82Crossref PubMed Scopus (85) Google Scholar). The enzymatic activity of SHIP has also been shown to be critical for this inhibitory effect (32Ono M. Okada H. Bolland S. Yanagi S. Kurosaki T. Ravetch J.V. Cell. 1997; 90: 293-301Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar). SHIP has been shown in vitro (33Damen J.E. Liu L. Rosten P. Humphries R.K. Jefferson A.B. Majerus P.W. Krystal G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1689-1693Crossref PubMed Scopus (561) Google Scholar, 34Lioubin M.N. Algate P.A. Tsai S. Carlberg K. Aebersold A. Rohrschneider L.R. Genes Dev. 1996; 10: 1084-1095Crossref PubMed Scopus (378) Google Scholar) and in vivo (35Scharenberg A.M. El-Hillal O. Fruman D.A. Beitz L.O. Li Z. Lin S. Gout I. Cantley L.C. Rawlings D.J. Kinet J.P. EMBO J. 1998; 17: 1961-1972Crossref PubMed Scopus (386) Google Scholar) to have 5′-phosphatase activity toward PIP3, resulting in dephosphorylation of PIP3 and production of PI 3,4-P2. Consistent with the requirement for enzymatically active SHIP for FcγRIIB1-mediated inhibition, FcγRIIB1 co-cross-linking to the BCR diminishes the BCR-induced levels of PIP3 (35Scharenberg A.M. El-Hillal O. Fruman D.A. Beitz L.O. Li Z. Lin S. Gout I. Cantley L.C. Rawlings D.J. Kinet J.P. EMBO J. 1998; 17: 1961-1972Crossref PubMed Scopus (386) Google Scholar) in vivo. The failure of recruitment by PIP3 of the kinase Btk (via its PH domain) under FcγRIIB1 cross-linking conditions has been shown to be responsible for the FcγRIIB1–mediated inhibition of BCR-mediated calcium entry (35Scharenberg A.M. El-Hillal O. Fruman D.A. Beitz L.O. Li Z. Lin S. Gout I. Cantley L.C. Rawlings D.J. Kinet J.P. EMBO J. 1998; 17: 1961-1972Crossref PubMed Scopus (386) Google Scholar, 36Fluckiger A.C. Li Z. Kato R.M. Wahl M.I. Ochs H.D. Longnecker R. Kinet J.P. Witte O.N. Scharenberg A.M. Rawlings D.J. EMBO J. 1998; 17: 1973-1985Crossref PubMed Scopus (357) Google Scholar, 37Bolland S. Pearse R.N. Kurosaki T. Ravetch J.V. Immunity. 1998; 8: 509-516Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). A similar mechanism could be involved in regulation of other downstream effectors whose activity is dependent on phospholipids.Although BCR stimulation results in PI3K activation and Akt is activated downstream of PI3K in several cell types, BCR regulation of Akt has not been reported. In this report, we show that Akt is activated by BCR cross-linking in a PI3K-dependent manner. Since there is conflicting evidence on the relative importance of PIP3 and PI 3,4-P2 in Akt activation (11Klippel A. Kavanaugh W.M. Pot D. Williams L.T. Mol. Cell. Biol. 1997; 17: 338-344Crossref PubMed Scopus (446) Google Scholar, 12Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1294) Google Scholar, 13Frech M. Andjelkovic M. Ingley E. Reddy K.K. Falck J.R. Hemmings B.A. J. Biol. Chem. 1997; 272: 8474-8481Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar,16Alessi D.R. Cohen P. Curr. Opin. Genet. Dev. 1998; 8: 55-62Crossref PubMed Scopus (674) Google Scholar, 18Alessi 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 (616) Google Scholar, 19Stephens 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, 21Alessi 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, 22Stokoe 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) and FcγRIIB1-associated SHIP is known to dephosphorylate PIP3 to generate PI 3,4-P2(33Damen J.E. Liu L. Rosten P. Humphries R.K. Jefferson A.B. Majerus P.W. Krystal G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1689-1693Crossref PubMed Scopus (561) Google Scholar, 34Lioubin M.N. Algate P.A. Tsai S. Carlberg K. Aebersold A. Rohrschneider L.R. Genes Dev. 1996; 10: 1084-1095Crossref PubMed Scopus (378) Google Scholar), we hypothesized that recruitment of SHIP through FcγRIIB1 co-cross-linking to the BCR in B cells would regulate Akt activity. The nature of this regulation, positive or negative, might also reveal the relative contribution of PIP3 and PI 3,4-P2 to Akt activation in vivo. We demonstrate here that Akt activity is down-regulated by co-cross-linking FcγRIIB1 to the BCR, as compared with BCR cross-linking alone, and that this Akt down-regulation is dependent upon the ITIM motif of FcγRIIB1. Finally, we show that FcγRIIB1-mediated regulation of Akt is dependent on the 5′-inositol phosphatase SHIP.DISCUSSIONCo-ligation of FcγRIIB1 and the BCR results in a potent inhibitory signal, which leads to a selective attenuation of BCR-mediated signals (26Ravetch J.V. Curr. Opin. Immunol. 1997; 9: 121-125Crossref PubMed Scopus (213) Google Scholar). This phenomenon represents a negative feedback mechanism triggered by immune complex or anti-idiotypic antibodies to suppress excessive B cell immune response (23DeFranco A.L. Curr. Opin. Immunol. 1997; 9: 296-308Crossref PubMed Scopus (282) Google Scholar, 26Ravetch J.V. Curr. Opin. Immunol. 1997; 9: 121-125Crossref PubMed Scopus (213) Google Scholar). The negative regulation by FcγRIIB1 requires the recruitment and the enzymatic activity of the inositol phosphatase SHIP (32Ono M. Okada H. Bolland S. Yanagi S. Kurosaki T. Ravetch J.V. Cell. 1997; 90: 293-301Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar). The mechanism of this FcγRIIB1/SHIP-mediated inhibitory action is not fully understood. Recently, it has been suggested that SHIP-mediated dephosphorylation of PIP3 and consequently the inhibition of Btk membrane localization and activation plays a central role in inhibition of calcium flux by FcγRIIB1 (35Scharenberg A.M. El-Hillal O. Fruman D.A. Beitz L.O. Li Z. Lin S. Gout I. Cantley L.C. Rawlings D.J. Kinet J.P. EMBO J. 1998; 17: 1961-1972Crossref PubMed Scopus (386) Google Scholar, 36Fluckiger A.C. Li Z. Kato R.M. Wahl M.I. Ochs H.D. Longnecker R. Kinet J.P. Witte O.N. Scharenberg A.M. Rawlings D.J. EMBO J. 1998; 17: 1973-1985Crossref PubMed Scopus (357) Google Scholar, 37Bolland S. Pearse R.N. Kurosaki T. Ravetch J.V. Immunity. 1998; 8: 509-516Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar).It has been shown that co-ligation of FcγRIIB1 and BCR induces apoptosis in mouse splenocytes (38Ashman R.F. Peckham D. Stunz L.L. J. Immunol. 1996; 157: 5-11PubMed Google Scholar, 39Yamashita Y. Miyake K. Miura Y. Kaneko Y. Yagita H. Suda T. Nagata S. Nomura J. Sakaguchi N. Kimoto M. J. Exp. Med. 1996; 184: 113-120Crossref PubMed Scopus (48) Google Scholar). The serine threonine kinase Akt, an enzyme activated downstream of PI3K, is a major signaling protein involved in protection from apoptosis (5Dudek 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 (2213) Google Scholar, 6Kauffmann-Zeh A. Rodriguez-Viciana P. Ulrich E. Gilbert C. Coffer P. Downward J. Evan G. Nature. 1997; 385: 544-548Crossref PubMed Scopus (1069) Google Scholar, 7Kulik G. Klippel A. Weber M.J. Mol. Cell. Biol. 1997; 17: 1595-1606Crossref PubMed Scopus (965) Google Scholar, 8Ahmed 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, 40Franke T.F. Kaplan D.R. Cantley L.C. Cell. 1997; 88: 435-437Abstract Full Text Full Text PDF PubMed Scopus (1514) Google Scholar). Although BCR stimulation activates PI3K, as demonstrated by the ability of wortmannin to inhibit BCR signaling (23DeFranco A.L. Curr. Opin. Immunol. 1997; 9: 296-308Crossref PubMed Scopus (282) Google Scholar, 24Kiener P.A. Lioubin M.N. Rohrschneider L.R. Ledbetter J.A. Nadler S.G. Diegel M.L. J. Biol. Chem. 1997; 272: 3838-3844Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), activation of Akt by BCR engagement has not been reported previously. Here, we show the activation of Akt by BCR stimulation in a PI3K-dependent manner. Furthermore, our data demonstrate that co-ligation of FcγRIIB1 with BCR results in inhibition of Akt kinase activity in murine A20 cells as well as in chicken DT40 cells transfected with murine FcγRIIB1. Using an FcγRIIB1-deficient subclone of A20 (IIA1.6) reconstituted with either wild type FcγRIIB1 or a carboxyl-terminally truncated FcγRIIB1 (lacking ITIM), we demonstrate that the inhibition of Akt activity is dependent on the ITIM motif. Additionally, in SHIP-deficient DT40 cells stably transfected with murine FcγRIIB1, co-ligation of BCR and FcγRIIB1 could not significantly inhibit Akt activation. These data indicate that the inhibition of Akt kinase activity is mediated by the inositol phosphatase SHIP. However, we consistently observed a slight inhibition of Akt by BCR plus FcγRIIB1 stimulation in SHIP-deficient cells. This may reflect minor roles played by other molecules such as SHP-1, known to interact with FcγRIIB1 (41Scharenberg A.M. Kinet J.-P. Cell. 1996; 87: 961-964Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Alternatively, there could be some redundancy for SHIP, since a second SH2-containing inositol phosphatase (SHIP2) has been recently described (42Habib T. Hejna J. Moses R. Decker S. J. Biol. Chem. 1998; 273: 18605-18609Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 43Pesesse X. Deleu S. De Smedt F. Drayer L. Erneux C. Biochem. Biophys. Res. Commun. 1997; 239: 697-700Crossref PubMed Scopus (199) Google Scholar).There have been conflicting data in the literature on the role of PIP3 versus PI 3,4-P2 in the activation of Akt. In the absence of PDK1, one of the kinases that phosphorylates Akt, the addition of PI 3,4-P2 containing micelles to immunoprecipitated Akt increased its enzymatic activity, whereas PIP3 either had no effect or was inhibitory (11Klippel A. Kavanaugh W.M. Pot D. Williams L.T. Mol. Cell. Biol. 1997; 17: 338-344Crossref PubMed Scopus (446) Google Scholar, 12Franke T.F. Kaplan D.R. Cantley L.C. Toker A. 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It has been proposed that SHIP functions upstream of Akt and that the generation of PI 3,4-P2 positively regulates Akt activation (10Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1218) Google Scholar, 11Klippel A. Kavanaugh W.M. Pot D. Williams L.T. Mol. Cell. Biol. 1997; 17: 338-344Crossref PubMed Scopus (446) Google Scholar, 12Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1294) Google Scholar, 13Frech M. Andjelkovic M. Ingley E. Reddy K.K. Falck J.R. Hemmings B.A. J. Biol. Chem. 1997; 272: 8474-8481Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar). Inhibition of BCR-induced signals by FcγRIIB1 involves recruitment of SHIP to the BCR signaling complex (26Ravetch J.V. Curr. Opin. Immunol. 1997; 9: 121-125Crossref PubMed Scopus (213) Google Scholar). Recently, Scharenberg et al. (35Scharenberg A.M. El-Hillal O. Fruman D.A. Beitz L.O. Li Z. Lin S. Gout I. Cantley L.C. Rawlings D.J. Kinet J.P. EMBO J. 1998; 17: 1961-1972Crossref PubMed Scopus (386) Google Scholar) have demonstrated in A20 cells that FcγRIIB1 co-cross-linking to the BCR decreases cellular PIP3 levels generated by BCR stimulation. Therefore, this system provides a useful tool to indirectly address the role of PIP3 versus PI 3,4-P2 in in vivo activation of Akt. Our data strongly suggest that, in vivo, dephosphorylation of PIP3 inhibits Akt activation and that SHIP-mediated generation of PI 3,4-P2does not positively regulate Akt. Therefore, in this system, PIP3 appears to be a more potent activator of Akt than PI 3,4-P2. Additionally, in several experiments, we observed a greater level of BCR-mediated activation of Akt in SHIP-deficient DT40 cells compared with SHIP-expressing cells, most likely due to the absence of PIP3 dephosphorylation. This suggests that SHIP also directly regulates the BCR-mediated rise in PIP3levels. This is consistent with the reported increase in Btk activation and calcium flux (another PIP3-dependent event) in SHIP-deficient cells as compared with SHIP-expressing cells (37Bolland S. Pearse R.N. Kurosaki T. Ravetch J.V. Immunity. 1998; 8: 509-516Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). Thus, SHIP, through its effects on PIP3, could set the threshold for the magnitude of Akt activation and the downstream consequences.Others have suggested a biphasic pattern of Akt activation, with PIP3 being responsible for the early phase and PI 3,4-P2 mediating the late activ
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