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The Role of Phosphoinositide 3-Kinase Lipid Products in Cell Function

1999; Elsevier BV; Volume: 274; Issue: 13 Linguagem: Inglês

10.1074/jbc.274.13.8347

ISSN

1083-351X

Autores

Lucia E. Rameh, Lewis C. Cantley,

Tópico(s)

Cellular transport and secretion

Resumo

Phosphoinositide 3-kinases (PI 3-Ks) 1The abbreviations used are:PI 3-K, phosphoinositide 3-kinase; PtdIns, phosphatidylinositol; PDGF, platelet-derived growth factor; fMLP, formyl-methionyl-leucyl-phenylalanine; PH, pleckstrin homology; PLC, phospholipase C; PKC, protein kinase C; MAPK, mitogen-activated protein kinase. are a subfamily of lipid kinases that catalyze the addition of a phosphate molecule specifically to the 3-position of the inositol ring of phosphoinositides. Phosphatidylinositol (PtdIns), the precursor of all phosphoinositides (PI), constitutes less than 10% of the total lipid in eukaryotic cell membranes (Fig.1). Approximately 5% of cellular PI is phosphorylated at the 4-position (PtdIns-4-P), and another 5% is phosphorylated at both the 4- and 5-positions (PtdIns-4,5-P2). However, less than 0.25% of the total inositol-containing lipids are phosphorylated at the 3-position, consistent with the idea that these lipids exert specific regulatory functions inside the cell, as opposed to a structural function. To date, nine members of the PI 3-K family have been isolated from mammalian cells. They are grouped, as suggested by Domin and Waterfield (1Domin J. Waterfield M.D. FEBS Lett. 1997; 410: 91-95Crossref PubMed Scopus (209) Google Scholar), into three classes according to the molecules that they preferentially utilize as substrates. Four different lipid products can be generated by the different PI 3-K members: the singly phosphorylated form PtdIns-3-P; the doubly phosphorylated forms PtdIns-3,4-P2 and PtdIns-3,5-P2; and finally the triply phosphorylated form PtdIns-3,4,5-P3 (Fig.1). PI 3-K was first described as a PI kinase activity associated with the viral oncoproteins, v-Src, v-Ros, and polyomavirus middle T. Mutational studies of these oncoproteins more than 10 years ago indicated a critical role for the associated PI kinase in cell transformation (reviewed by Ref. 2Cantley L.C. Auger K.R. Carpenter C. Duckworth B. Graziani A. Kapeller R. Soltoff S. Cell. 1991; 64: 281-302Abstract Full Text PDF PubMed Scopus (2186) Google Scholar). Recent advances in the field have been achieved by the development of new techniques to probe for the direct targets of PI 3-K lipid products. The chemical synthesis of short chain fatty acid versions of these lipids (3Wang D.-S. Chen C.-S. Org. Chem. 1996; 61: 5905-5910Crossref Scopus (56) Google Scholar, 4Falck J.R. Abdali A. Bioorg. Med. Chem. Lett. 1993; 3: 717-720Crossref Scopus (15) Google Scholar, 5Prestwich G.D. Acc. Chem. Res. 1996; 29: 503-513Crossref Scopus (107) Google Scholar) has been a crucial step in determining the specificity of lipid-binding proteins. Additionally, new cloning strategies have been developed to isolate new lipid-binding proteins (6Klarlund J.K. Guilherme A. Holik J.J. Virbasius J.V. Chawla A. Czech M.P. Science. 1997; 275: 1927-1930Crossref PubMed Scopus (371) Google Scholar). Here we will review the most recent advances in our understanding of the role of PI 3-K in cell function by dissecting the contribution of each of its lipid products. PtdIns-3-P is constitutively present in both mammalian and yeast cells (7Auger K.R. Serunian L.A. Soltoff S.P. Libby P. Cantley L.C. Cell. 1989; 57: 167-175Abstract Full Text PDF PubMed Scopus (683) Google Scholar, 8Auger K.R. Carpenter C.L. Cantley L.C. Varticovski L. J. Biol. Chem. 1989; 264: 20181-20184Abstract Full Text PDF PubMed Google Scholar). It can be produced in vitro via phosphorylation of PtdIns by Class I, II, or III PI 3-Ks (Fig. 1). However, the majority of PtdIns-3-P in mammalian cells is probably produced by Class III PI 3-K (9Vanhaesebroeck B. Leevers S.J. Panayotou G. Waterfield M.D. Trends Biochem. Sci. 1997; 22: 267-272Abstract Full Text PDF PubMed Scopus (833) Google Scholar). The mammalian Class III enzyme is highly related to the yeast Vps34 gene product (10Stack J.H. Horazdovsky B. Emr S.D. Annu. Rev. Cell Dev. Biol. 1995; 11: 1-33Crossref PubMed Scopus (172) Google Scholar) and, like the yeast enzyme, is specific for PtdIns and will not phosphorylate PtdIns-4-P or PtdIns-4,5-P2 (11Volinia S. Dhand R. Vanhaesebroeck B. MacDougall L.K. Stein R. Zvelebil M.J. Domin J. Panaretou C. Waterfield M.D. EMBO J. 1995; 14: 3339-3348Crossref PubMed Scopus (309) Google Scholar). PtdIns-3-P was recently shown to specifically interact with a 70-residue protein module called the FYVE finger domain. This domain is a special type of RING zinc finger that is characterized by two zinc-binding sites and a highly conserved stretch of basic residues surrounding the third zinc-coordinating cysteine. Liposomes containing PtdIns-3-P were shown to associate with several FYVE domains (15Patki V. Lawe D.C. Corvera S. Virbasius J.V. Chawla A. Nature. 1998; 394: 433-434Crossref PubMed Scopus (250) Google Scholar, 16Burd C.G. Emr S.D. Mol. Cell. 1998; 2: 157-162Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar, 17Gaullier J.M. Simonsen A. D'Arrigo A. Bremnes B. Stenmark H. Aasland R. Nature. 1998; 394: 432-433Crossref PubMed Scopus (444) Google Scholar). Other phosphoinositides bound poorly to the FYVE domains investigated, showing that this interaction is specific for PtdIns-3-P. Proper folding of the domain is important for its function because mutation in one of the zinc-coordinating cysteines or removal of zinc with EDTA or TPEN reduced PtdIns-3-P binding (16Burd C.G. Emr S.D. Mol. Cell. 1998; 2: 157-162Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar, 17Gaullier J.M. Simonsen A. D'Arrigo A. Bremnes B. Stenmark H. Aasland R. Nature. 1998; 394: 432-433Crossref PubMed Scopus (444) Google Scholar). Moreover, mutations in the basic motif also eliminated binding (16Burd C.G. Emr S.D. Mol. Cell. 1998; 2: 157-162Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar). The interaction between FYVE domains and PtdIns-3-P is presumed to occur in vivo, because localization of the FYVE-containing protein EEA1 on early endosomes depends on an intact FYVE domain (17Gaullier J.M. Simonsen A. D'Arrigo A. Bremnes B. Stenmark H. Aasland R. Nature. 1998; 394: 432-433Crossref PubMed Scopus (444) Google Scholar,18Stenmark H. Aasland R. Toh B.H. D'Arrigo A. J. Biol. Chem. 1996; 271: 24048-24054Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar) and on PI 3-K activity (based on wortmannin effects in mammalian cells) (19Patki V. Virbasius J. Lane W.S. Toh B.H. Shpetner H.S. Corvera S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7326-7330Crossref PubMed Scopus (202) Google Scholar). When overexpressed in cells, the FYVE domain of EEA1 is sufficient to determine subcellular localization (16Burd C.G. Emr S.D. Mol. Cell. 1998; 2: 157-162Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar). Simonsen and colleagues (20Simonsen A. Lippe R. Christoforidis S. Gaullier J.M. Brech A. Callaghan J. Toh B.H. Murphy C. Zerial M. Stenmark H. Nature. 1998; 394: 494-498Crossref PubMed Scopus (918) Google Scholar, 21.Deleted in proof.Google Scholar) have shown that, in addition to PtdIns-3-P, the EEA1 protein associates with GTP-bound Rab5 through separate domains, and interactions with both PtdIns-3-P and GTP-Rab5 are necessary for the stable association of EEA1 with membranes in vivo. Mutations in the yeast Class III PI 3-K, VPS34, cause missorting of vacuolar proteins, changes in vacuole morphology, and defects in the endocytic pathway (reviewed in Ref. 10Stack J.H. Horazdovsky B. Emr S.D. Annu. Rev. Cell Dev. Biol. 1995; 11: 1-33Crossref PubMed Scopus (172) Google Scholar). In mammalian cells, inhibition of PI 3-K by the drug wortmannin blocks transport of proteins from the Golgi to the lysosome, inhibits early endosome trafficking, and causes the accumulation of prelysosomal vesicles (22Brown W.J. DeWald D.B. Emr S.D. Plutner H. Balch W.E. J. Cell Biol. 1995; 130: 781-796Crossref PubMed Scopus (251) Google Scholar, 23Davidson H.W. J. Cell Biol. 1995; 130: 797-805Crossref PubMed Scopus (184) Google Scholar). Mutations in the PDGF receptor that disrupt its association with Class I PI 3-Ks interfere with trafficking of this receptor to the lysosome and its subsequent degradation (12Joly M. Kazlauskas A. Corvera S. J. Biol. Chem. 1995; 270: 13225-13230Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). With the identification of the FYVE-containing proteins as potential targets for PtdIns-3-P, the mechanism by which this lipid is involved in vesicle trafficking is now becoming clear. As discussed above, PtdIns-3-P is necessary for the subcellular localization of EEA1, a protein that regulates fusion of endocytic membranes (20Simonsen A. Lippe R. Christoforidis S. Gaullier J.M. Brech A. Callaghan J. Toh B.H. Murphy C. Zerial M. Stenmark H. Nature. 1998; 394: 494-498Crossref PubMed Scopus (918) Google Scholar, 24Mills I.G. Jones A.T. Clague M.J. Curr. Biol. 1998; 8: 881-884Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). The mammalian FYVE-containing protein Hrs-2 and the yeast proteins Fab1p, Vps27p, and Vac1p are involved in different vesicle trafficking events, such as secretion and vacuole targeting (reviewed by Ref. 14Wiedemann C. Cockcroft S. Nature. 1998; 394: 426-427Crossref PubMed Scopus (74) Google Scholar). PtdIns-3,4-P2 levels can be regulated by extracellular signals. PDGF stimulation of quiescent fibroblasts as well as fMLP peptide stimulation of neutrophils result in rapid PtdIns-3,4-P2 synthesis (7Auger K.R. Serunian L.A. Soltoff S.P. Libby P. Cantley L.C. Cell. 1989; 57: 167-175Abstract Full Text PDF PubMed Scopus (683) Google Scholar, 13Stephens L.R. Hughes K.T. Irvine R.F. Nature. 1991; 351: 33-39Crossref PubMed Scopus (388) Google Scholar, 25Hawkins P.T. Jackson T.R. Stephens L.R. Nature. 1992; 358: 157-159Crossref PubMed Scopus (199) Google Scholar). Stephens and collaborators (25Hawkins P.T. Jackson T.R. Stephens L.R. Nature. 1992; 358: 157-159Crossref PubMed Scopus (199) Google Scholar) have proposed that the elevation in PtdIns-3,4-P2 levels in these cells is caused by the dephosphorylation of PtdIns-3,4,5-P3 as opposed to the phosphorylation of PtdIns-4-P or PtdIns-3-P. Recent studies have indicated that, in platelets, PtdIns-3,4-P2 can also be synthesized by phosphorylation of the 4-position of PtdIns-3-P by an unidentified PtdIns-3-P 4-kinase (26Banfic H. Tang X. Batty I.H. Downes C.P. Chen C. Rittenhouse S.E. J. Biol. Chem. 1998; 273: 13-16Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 27Banfic H. Downes C.P. Rittenhouse S.E. J. Biol. Chem. 1998; 273: 11630-11637Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Although the PtdIns-5-P 4-kinase α (also called Type II PtdIns-P kinase) can catalyze this reaction in vitro, it is unlikely that this enzyme is responsible for the elevation of PtdIns-3,4-P2 levels in vivo because PtdIns-3-P is a poor substrate for this enzyme when compared with PtdIns-5-P (28Rameh L.E. Tolias K.F. Duckworth B.C. Cantley L.C. Nature. 1997; 390: 192-196Crossref PubMed Scopus (367) Google Scholar). The class II PI 3-Ks can phosphorylate PtdIns-4-P to generate PtdIns-3,4-P2, independent of PtdIns-3,4,5-P3synthesis (Fig. 1). The contribution of this pathway to the intracellular levels of PtdIns-3,4-P2 is unknown. In summary, it is clear that mammalian cells have evolved a variety of mechanisms for independently controlling the levels of PtdIns-3,4-P2 and PtdIns-3,4,5-P3. The serine/threonine protein kinase B (PKB), also known as Akt, is the most well characterized target of PtdIns-3,4-P2. The PH domain of Akt has been shown to bind phosphoinositides in vitro with the order of preference being PtdIns-3,4-P2 > PtdIns-3,4,5-P3 ≫ PtdIns-4,5-P2 (30Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1305) Google Scholar, 31Frech M. Andjelkovich 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 (363) Google Scholar). PtdIns-3,4-P2 binding to Akt causes a 3–5-fold stimulation of its activity in vitro (30Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1305) Google Scholar, 31Frech M. Andjelkovich 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 (363) Google Scholar, 32Klippel A. Kavanaugh W.M. Pot D. Williams L.T. Mol. Cell. Biol. 1997; 17: 338-344Crossref PubMed Scopus (447) Google Scholar). In thrombin-stimulated platelets, Akt activation correlates with PtdIns-3,4-P2 production rather than PtdIns-3,4,5-P3 production (30Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1305) Google Scholar). Consistent with this, integrin cross-linking causes PtdIns-3,4-P2 production without PtdIns-3,4,5-P3 production and results in Akt activation (27Banfic 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 vivo, full activation of Akt also depends on the phosphorylation of a threonine (Thr-308) and a serine (Ser-473) (33Alessi D.R. Andjelkovich M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2517) Google Scholar). Phosphorylation of these residues was shown to be dependent on PI 3-K. The Thr-308 kinase PDK1 (for phosphoinositide-dependent kinase; also called PKB kinase) was recently purified and cloned based on its ability to catalyze the phosphorylation of Thr-308 of Akt in a PtdIns-3,4,5-P3-dependent manner (34Alessi 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, 35Stokoe 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 (1048) Google Scholar, 36Alessi 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 (620) Google Scholar, 37Stephens 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 (914) Google Scholar). Like Akt, PDK1 contains a PH domain and binds with high affinity to PtdIns-3,4-P2 and PtdIns-3,4,5-P3 (37Stephens 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 (914) Google Scholar). To understand the mechanism by which these lipids activate Akt in vivo, a model was proposed in which the PI 3-K lipid products are involved in recruiting Akt and its upstream kinases to the membrane and also in promoting conformational changes in Akt that expose Thr-308 and Ser-473 to be phosphorylated by PDK1 and other kinases (reviewed in Refs. 29Franke T.F. Kaplan D.R. Cantley L.C. Cell. 1997; 88: 435-437Abstract Full Text Full Text PDF PubMed Scopus (1522) Google Scholar and 38Downward J. Science. 1998; 279: 673-674Crossref PubMed Scopus (181) Google Scholar). PDK1 can also phosphorylate the activation loop of p70S6-K(36Alessi 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 (620) Google Scholar, 39Pullen N. Dennis P.B. Andjelkovich M. Dufner A. Kozma S.C. Hemmings B.A. Thomas G. Science. 1998; 279: 707-710Crossref PubMed Scopus (727) Google Scholar) and PKC family members (40Chou M.M. Hou W. Johnson J. Graham L.K. Lee M.H. Chen C.-S. Newton A.C. Schaffhausen B.S. Toker A. Curr. Biol. 1998; 8: 1069-1077Abstract Full Text Full Text PDF PubMed Google Scholar, 41Le Good J.A. Ziegler W.H. Parekh D.B. Alessi D.R. Cohen P. Parker P.J. Science. 1998; 281: 2042-2045Crossref PubMed Scopus (973) Google Scholar) in vitro. In most cases, phosphorylation at the activation loop of these various kinases is necessary for activation, but phosphorylation of other residues is also required for full activity. PKCε expressed in baculovirus and PKCζ purified from brain are significantly activated by PtdIns-3,4-P2 and PtdIns-3,4,5-P3 (42Toker A. Meyer M. Reddy K.K. Falck J.R. Aneja R. Aneja S. Parra A. Burns D.J. Ballas L.M. Cantley L.C. J. Biol. Chem. 1994; 269: 32358-32367Abstract Full Text PDF PubMed Google Scholar, 43Nakanishi H. Brewer K.A. Exton J.H. J. Biol. Chem. 1993; 268: 13-16Abstract Full Text PDF PubMed Google Scholar). It is likely that the activity of these isoforms is affected by PDK1 phosphorylation (ε and ζ) as well as by a direct interaction with these phosphoinositides (ε). Consistent with these in vitro studies, inhibition of PI 3-K blocks PDGF and insulin-dependent activation of p70S6-K in vivo (44Chung J. Grammer T.C. Lemon K.P. Kazlauskas A. Blenis J. Nature. 1994; 370: 71-75Crossref PubMed Scopus (657) Google Scholar), insulin-dependent activation of PKCζ in vivo(45Standaert M.L. Galloway L. Karnam P. Bandyopadhyay G. Moscat J. Farese R.V. J. Biol. Chem. 1997; 272: 30075-30082Crossref PubMed Scopus (408) Google Scholar, 46Mendez R. Kollmorgen G. White M.F. Rhoads R.E. Mol. Cell. Biol. 1997; 17: 5184-5192Crossref PubMed Google Scholar), and PDGF-dependent membrane recruitment and activation of PKCε in vivo (47Moriya S. Kazlauskas A. Akimoto K. Hirai S. Mizuno K. Takenawa T. Fukui Y. Watanabe Y. Ozaki S. Ohno S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 151-155Crossref PubMed Scopus (167) Google Scholar). However, the enzymatic activity of PDK1 is not dependent on phosphoinositides (48Alessi D.R. Kozlowski M.T. Weng Q.P. Morrice N. Avruch J. Curr. Biol. 1998; 8: 69-81Abstract Full Text Full Text PDF PubMed Scopus (516) Google Scholar). Thus, the requirement of PI 3-K for PDK1-dependent phosphorylation of various enzymes probably reflects a role for PtdIns-3,4-P2and PtdIns-3,4,5-P3 in co-localizing PDK1 and its substrates at specific membranes. Activation of Akt mediates the transduction of cell survival signals. Activated Akt can phosphorylate and inactivate Bad, a protein involved in promoting cell death (reviewed by Ref. 50Franke T.F. Cantley L.C. Nature. 1997; 390: 116-117Crossref PubMed Scopus (170) Google Scholar). In addition to Bad, Akt must have other targets that mediate cell survival because it promotes survival of cells that lack Bad (49Datta 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 (4946) Google Scholar). Glycogen synthase kinase 3 and phosphofructokinase are also in vitro substrates for Akt, implicating PI 3-K lipid products in gluconeogenesis and glycolysis (51Cross D.A. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4376) Google Scholar, 52Deprez J. Vertommen D. Alessi D.R. Hue L. Rider M.H. J. Biol. Chem. 1997; 272: 17269-17275Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). In mouse fibroblasts, the major route for PtdIns-3,5-P2 synthesis is wortmannin-sensitive and involves the consecutive phosphorylation of PtdIns by a PI 3-K (presumably the Class III enzyme) and of PtdIns-3-P by a PtdIns-3-P 5-kinase (53Whiteford C.C. Brearley C.A. Ulug E.T. Biochem. J. 1997; 323: 597-601Crossref PubMed Scopus (128) Google Scholar). In vitro, this second reaction can be catalyzed by the PtdIns-P 5-kinases α and β (also known as type I PtdIns-P kinases) (54Tolias K.F. Rameh L.E. Ishihara H. Shibasaki Y. Chen J. Prestwich G.D. Cantley L.C. Carpenter C.L. J. Biol. Chem. 1998; 273: 18040-18046Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Likewise, PtdIns-3,5-P2 synthesis in yeast involves phosphorylation of PtdIns-3-P by a 5-kinase and requires Vps34p PI 3-K (55Dove S.K. Cooke F.T. Douglas M.R. Sayers L.G. Parker P.J. Michell R.H. Nature. 1997; 390: 187-192Crossref PubMed Scopus (392) Google Scholar). Fab1, a gene that is highly homologous to the mammalian PtdIns-P 5-kinases, was recently identified as the yeast PtdIns-3-P 5-kinase (56Gary J.D. Wurmser A.E. Bonangelino C.J. Weisman L.S. Emr S.D. J. Cell Biol. 1998; 143: 65-79Crossref PubMed Scopus (342) Google Scholar). Dramatic increases in PtdIns-3,5-P2 levels were observed in response to hyperosmotic shock of yeast cells. In mammalian cells, the levels of PtdIns-3,5-P2 decrease moderately with hyperosmotic shock and increase with hypo-osmotic shock. In vitro, PtdIns-3,5-P2 can also be generated through phosphorylation of the novel lipid PtdIns-5-P by the Class IA PI 3-K (28Rameh L.E. Tolias K.F. Duckworth B.C. Cantley L.C. Nature. 1997; 390: 192-196Crossref PubMed Scopus (367) Google Scholar). PtdIns-3,5-P2 is a newly identified molecule, and no direct target for this lipid has been found. PH domain-containing proteins are likely candidates for PtdIns-3,5-P2 downstream effectors. Previous studies of lipid binding specificity of PH domains did not investigate this lipid. Because mutations in the yeastFab1 cause enlargement of the vacuole (57Yamamoto A. DeWald D.B. Boronenkov I.V. Anderson R.A. Emr S.D. Koshland D. Mol. Biol. Cell. 1995; 6: 525-539Crossref PubMed Scopus (235) Google Scholar), PtdIns-3,5-P2 may be involved in vesicle trafficking (reviewed by Emr and colleagues (94Wurmser A.E. Gary J.D. Emr S.D. J. Biol. Chem. 1999; 274: 9129-9132Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar)). The majority of the PtdIns-3,4,5-P3synthesized in response to extracellular signals is, most likely, generated by phosphorylation of PtdIns-4,5-P2 at the 3-position of the inositol ring (25Hawkins P.T. Jackson T.R. Stephens L.R. Nature. 1992; 358: 157-159Crossref PubMed Scopus (199) Google Scholar). The Class I PI 3-Ks are the only enzymes that can use PtdIns-4,5-P2 as a substrate to synthesize PtdIns-3,4,5-P3 (Fig. 1). Activation of class IA PI 3-Ks by growth factor stimulation of cells is mediated in part by interaction of their SH2 domain with tyrosine-phosphorylated proteins and results in a rapid elevation of PtdIns-3,4,5-P3 levels (reviewed by Ref. 2Cantley L.C. Auger K.R. Carpenter C. Duckworth B. Graziani A. Kapeller R. Soltoff S. Cell. 1991; 64: 281-302Abstract Full Text PDF PubMed Scopus (2186) Google Scholar). Class IA PI 3-Ks can also be regulated by the GTP-bound form of the small G protein Ras (59Rodriguez V.P. Warne P.H. Dhand R. Vanhaesebroeck B. Gout I. Fry M.J. Waterfield M.D. Downward J. Nature. 1994; 370: 527-532Crossref PubMed Scopus (1727) Google Scholar). The Class IB PI 3-K can be directly activated by the βγ subunits of heterotrimeric G proteins (reviewed in Ref. 58Fruman D.A. Meyers R.E. Cantley L.C. Annu. Rev. Biochem. 1998; 67: 481-507Crossref PubMed Scopus (1319) Google Scholar). In addition, one of the Class IAenzymes (p110β) can be activated synergistically by phosphotyrosine peptides plus βγ subunits (60Okada T. Hazeki O. Ui M. Katada T. Biochem. J. 1996; 317: 475-480Crossref PubMed Scopus (49) Google Scholar). Recently, a PtdIns-4,5-P2-independent pathway for PtdIns-3,4,5-P3 synthesis was described. PtdIns-P 5-kinases α and β have been shown to utilize PtdIns-3-P as a substrate to produce PtdIns-3,4,5-P3 by phosphorylating the 4- and the 5-positions of the inositol ring in a concerted reaction (54Tolias K.F. Rameh L.E. Ishihara H. Shibasaki Y. Chen J. Prestwich G.D. Cantley L.C. Carpenter C.L. J. Biol. Chem. 1998; 273: 18040-18046Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 61Zhang X. Loijens J.C. Boronenkov I.V. Parker G.J. Norris F.A. Chen J. Thum O. Prestwich G.D. Majerus P.W. Anderson R.A. J. Biol. Chem. 1997; 272: 17756-17761Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). The contribution of this new pathway to intracellular levels of PtdIns-3,4,5-P3 is still unknown. Several PtdIns-3,4,5-P3 phosphatases have now been isolated. Of special interest is the PTEN tumor suppressor protein (see below), which can dephosphorylate PtdIns-3,4,5-P3 at the 3-position (62Maehama T. Dixon J.E. J. Biol. Chem. 1998; 273: 13375-13378Abstract Full Text Full Text PDF PubMed Scopus (2601) Google Scholar), and SHIP (discussed above), which can dephosphorylate the 5-position (63Damen 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 (566) Google Scholar). Little is known about the in vivometabolism of this lipid. However, it is resistant to hydrolysis by phospholipase C, types β, γ, and δ (64Serunian L.A. Haber M.T. Fukui T. Kim J.W. Rhee S.G. Lowenstein J.M. Cantley L.C. J. Biol. Chem. 1989; 264: 17809-17815Abstract Full Text PDF PubMed Google Scholar). In addition to Akt, PDK1, and PKCε (discussed above), many targets for PtdIns-3,4,5-P3 have now been described. Several of these proteins have PH domains that mediate binding (for a review on PH domains see Ref. 65Hemmings B.A. Science. 1997; 275: 1899Crossref PubMed Scopus (67) Google Scholar). Many PtdIns-3,4,5-P3-binding PH domains can also bind to PtdIns-4,5-P2, and only those that have at least a 10-fold higher affinity for PtdIns-3,4,5-P3 than for PtdIns-4,5-P2 will be considered here. The PH domain of the Bruton's tyrosine kinase (Btk) was shown to interact with PtdIns-3,4,5-P3 and its head group, inositol 1,3,4,5-P4, with high affinity (66Fukuda M. Kojima T. Kabayama H. Mikoshiba K. J. Biol. Chem. 1996; 271: 30303-30306Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 67Salim K. Bottomley M.J. Querfurth E. Zvelebil M.J. Gout I. Scaife R. Margolis R.L. Gigg R. Smith C.I.E. Driscoll P.C. Waterfield M.D. Panayotou G. EMBO J. 1996; 15: 6241-6250Crossref PubMed Scopus (494) Google Scholar, 68Rameh L.E. Arvidsson A. Carraway III, K.L. Couvillon A.D. Rathbun G. Crompton A. VanRenterghem B. Czech M.P. Ravichandran K.S. Burakoff S.J. Wang D.-S. Chen C.-S. Cantley L.C. J. Biol. Chem. 1997; 272: 22059-22066Crossref PubMed Scopus (425) Google Scholar). Substituting cysteine for arginine 28 (R28C) in the PH domain of Btk, a natural mutation that causes X-linked immunodeficiency in mice, significantly affects the binding of PtdIns-3,4,5-P3 and inositol 1,3,4,5-P4. In vivo, overexpression of the Class IA PI 3-K enzyme p110* (a constitutively active form of PI 3-K) or Class IB PI 3-K γ was shown to enhance Btk autophosphorylation and Src family kinase-mediated tyrosine phosphorylation of Btk (69Li Z. Wahl M.I. Eguinoa A. Stephens L.R. Hawkins P.T. Witte O.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13820-13825Crossref PubMed Scopus (187) Google Scholar, 70Scharenberg 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, 71.Deleted in proof.Google Scholar). This effect was inhibitable by wortmannin and required the PH domain of Btk (69Li Z. Wahl M.I. Eguinoa A. Stephens L.R. Hawkins P.T. Witte O.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13820-13825Crossref PubMed Scopus (187) Google Scholar, 70Scharenberg 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). Phosphorylation of Btk leads to its activation, as measured by its ability to regulate tyrosine phosphorylation of PLCγ2. Overexpression of Btk and PI 3-K enhanced production of inositol 1,4,5-P3 in response to cross-linking of surface immunoglobulin in B cells. Engagement of SHIP to the inhibitory receptor FcγRIIB1 blocks B cell receptor-induced PtdIns-3,4,5-P3 elevation and Btk activation. Altogether, these results indicate that PtdIns-3,4,5-P3, but not PtdIns-3,4-P2, is involved in activation of Btk by Src family kinases through a mechanism that resembles activation of Akt by PDK1. The protein Grp1 (general receptor forphosphoinositides) was cloned based on its ability to bind PtdIns-3,4,5-P3 in vitro (6Klarlund J.K. 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