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

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. 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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). 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In mammalian cells, the levels of PtdIns-3,5-P2 with and with In vitro, PtdIns-3,5-P2 can also be generated through phosphorylation of the lipid PtdIns-5-P by the Class 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 identified and direct target for this lipid has been PH proteins are likely for PtdIns-3,5-P2 studies of lipid binding specificity of PH domains not this mutations in the cause of the vacuole A. DeWald D.B. Anderson Emr S.D. D. Mol. Biol. Cell. 1995; PubMed Scopus Google Scholar), PtdIns-3,5-P2 be involved in vesicle trafficking (reviewed by Emr and colleagues Emr S.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google The majority of the in to extracellular most 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). 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J. 1996; PubMed Scopus Google Scholar). a pathway for PtdIns-3,4,5-P3 synthesis was PtdIns-P 5-kinases α and β have been shown to utilize PtdIns-3-P as a substrate to PtdIns-3,4,5-P3 by the 4- and the 5-positions of the inositol ring in a 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, X. Parker Chen J. Prestwich G.D. Anderson J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). The contribution of this new pathway to intracellular levels of PtdIns-3,4,5-P3 is unknown. PtdIns-3,4,5-P3 have now been special is the protein which can PtdIns-3,4,5-P3 at the 3-position T. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar), and which can the L. P. A.B. G. Proc. Natl. Acad. Sci. U. S. 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Chem. 1997; 272: PubMed Scopus Google Scholar). for in the PH domain of a mutation that causes in significantly the binding of PtdIns-3,4,5-P3 and inositol In vivo, of the Class PI 3-K enzyme constitutively form of PI or Class PI 3-K was shown to and family phosphorylation of A. Stephens L.R. Hawkins P.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: PubMed Scopus Google Scholar, D.A. S. Cantley L.C. D.J. EMBO J. 1998; 17: PubMed Scopus Google Scholar, in proof.Google Scholar). This was by wortmannin and required the PH domain of A. Stephens L.R. Hawkins P.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: PubMed Scopus Google Scholar, D.A. S. Cantley L.C. D.J. EMBO J. 1998; 17: PubMed Scopus Google Scholar). Phosphorylation of to its activation, as by its ability to phosphorylation of of and PI 3-K production of inositol in to cross-linking of in B cells. of to the receptor blocks B cell PtdIns-3,4,5-P3 elevation and these results that but not PtdIns-3,4-P2, is involved in activation of by family kinases through a mechanism that activation of Akt by The protein receptor was cloned based on its ability to bind PtdIns-3,4,5-P3 in vitro (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). of its acid a PH domain and a domain. The PH domain is for with other phosphoinositides J.K. Rameh L.E. Cantley L.C. J.M. Holik J.J. C. V. Corvera S. Czech M.P. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). The domain of was shown to function as a for the proteins and activity can be in vitro by that PtdIns-3,4,5-P3 regulates by recruiting it to membranes is J.K. Rameh L.E. Cantley L.C. J.M. Holik J.J. C. V. Corvera S. Czech M.P. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). The of PH domains in a of for proteins A. T. P. J. M. Trends Biochem. Sci. 1993; Full Text PDF PubMed Scopus Google Scholar) that phosphoinositide of these proteins be binding of to the protein depends on PI 3-K activation P.T. A. Stokoe D. Cooke F.T. R. S. L. T. M. Biol. 1995; Full Text