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W2124186695 abstract "In cells expressing the oncogenic Bcr-Abl tyrosine kinase, the regulatory p85 subunit of phosphatidylinositol 3-kinase is phosphorylated on tyrosine residues. We report that this phosphorylation event is readily catalyzed by the Abl and Lck protein-tyrosine kinases in vitro, by Bcr-Abl or a catalytically activated Lck-Y505F in co-transfected COS cells, and by endogenous kinases in transfected Jurkat T cells upon triggering of their T cell antigen receptor. Using these systems, we have mapped a major phosphorylation site to Tyr-688 in the C-terminal SH2 domain of p85. Tyrosine phosphorylation of p85 in vitro or in vivo was not associated with detectable change in the enzymatic activity of the phosphatidylinositol 3-kinase heterodimer, but correlated with a strong reduction in the binding of some, but not all, phosphoproteins to the SH2 domains of p85. This provides an additional candidate to the list of SH2 domains regulated by tyrosine phosphorylation and may explain why association of phosphatidylinositol 3-kinase with some cellular ligands is transient or of lower stoichiometry than anticipated. In cells expressing the oncogenic Bcr-Abl tyrosine kinase, the regulatory p85 subunit of phosphatidylinositol 3-kinase is phosphorylated on tyrosine residues. We report that this phosphorylation event is readily catalyzed by the Abl and Lck protein-tyrosine kinases in vitro, by Bcr-Abl or a catalytically activated Lck-Y505F in co-transfected COS cells, and by endogenous kinases in transfected Jurkat T cells upon triggering of their T cell antigen receptor. Using these systems, we have mapped a major phosphorylation site to Tyr-688 in the C-terminal SH2 domain of p85. Tyrosine phosphorylation of p85 in vitro or in vivo was not associated with detectable change in the enzymatic activity of the phosphatidylinositol 3-kinase heterodimer, but correlated with a strong reduction in the binding of some, but not all, phosphoproteins to the SH2 domains of p85. This provides an additional candidate to the list of SH2 domains regulated by tyrosine phosphorylation and may explain why association of phosphatidylinositol 3-kinase with some cellular ligands is transient or of lower stoichiometry than anticipated. Phosphatidylinositol 3-kinases (PI3Ks) 1The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; HA, hemagglutinin; Tyr(P), phosphotyrosine; SH2, Src homology 2 region; SH3, Src homology 3 region; mAb, monoclonal antibody; TPCK,l-1-tosylamido-2-phenylethyl chloromethyl ketone; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis.1The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; HA, hemagglutinin; Tyr(P), phosphotyrosine; SH2, Src homology 2 region; SH3, Src homology 3 region; mAb, monoclonal antibody; TPCK,l-1-tosylamido-2-phenylethyl chloromethyl ketone; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis. are a family of enzymes involved in a multiplicity of cellular functions, including cell proliferation and transformation (1Auger K.R. Cantley L.C. Cancer Cells. 1991; 3: 263-275PubMed Google Scholar, 2Coughlin S.R. Escobedo J.A. Williams L.T. Science. 1989; 243: 1191-1194Crossref PubMed Scopus (287) Google Scholar, 3Kaplan D.R. Whitman M. Schaffhausen B. Pallas D.C. White M. Cantley L.C. Roberts T.M. Cell. 1987; 50: 1021-1029Abstract Full Text PDF PubMed Scopus (407) Google Scholar), lymphocyte activation (4Gold M.R. Chan V.W.-F. Turck C. DeFranco A.L. J. Immunol. 1992; 148: 2012-2022PubMed Google Scholar, 5Jascur T. Gilman J. Mustelin T. J. Biol. Chem. 1997; 272: 14483-14488Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 6von Willebrand M. Jascur T. Bonnefoy-Bérard N. Yano H. Altman A. Matsuda Y. Mustelin T. Eur. J. Biochem. 1996; 235: 828-835Crossref PubMed Scopus (64) Google Scholar, 7Ward S.G. Ley S.C. MacPhee C. Cantrell D.A. Eur. J. Immunol. 1992; 22: 45-49Crossref PubMed Scopus (96) Google Scholar), G protein signaling (8Stoyanov B. Volinia S. Hanck T. Rubio I. Loubtchenkov M. Malek D. Stoyanova S. Vanhaesebroeck B. Dhand R. Nürnberg B. Gierschik P. Seedorf K. Hsuan J.J. Waterfield M.D. Wetzker R. Science. 1995; 269: 690-693Crossref PubMed Scopus (638) Google Scholar), DNA repair (9Savitsky K. Bar-Shira A. Gilad S. Rotman G. Ziv Y. Vanagaite L. Tagle D.A. Smith S. Uziel T. Sfez S. Ashkenazi M. Pecker I. Frydman M. Harnik R. Patanjali S.R. Simmons A. Clines G.A. Sartiel A. Gatti R.A. Chessa L. Sanal O. Lavin M.F. Jaspers N.G.J. Taylor A.M.R. Arlett C.F. Miki T. Weissman S.M. Lovett M. Collins F.S. Shiloh Y. Science. 1995; 268: 1749-1753Crossref PubMed Scopus (2355) Google Scholar), intracellular vesicle trafficking (10Schu P.V. Takegawa K. Fry M.J. Stack J.H. Waterfield M.D. Emr S.D. Science. 1993; 260: 88-91Crossref PubMed Scopus (802) Google Scholar, 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 (306) Google Scholar), and inhibition of programmed cell death (12Minshall C. Arkins S. Freund G.G. Kelley K.W. J. Immunol. 1996; 156: 939-947PubMed Google Scholar, 13Yao R. Cooper G.M. Science. 1995; 267: 2003-2006Crossref PubMed Scopus (1288) Google Scholar). The currently best characterized type of PI3K is the heterodimeric enzymes that consist of a 110-kDa catalytic subunit (p110α or p110β; Refs. 14Hiles I. Otsu M. Volinia S. Fry M.J. Gout I. Dhand R. Panayotou G. Ruiz-Larrea F. Thompson A. Totty N. Hsuan J. Courtneidge S.A. Parker P.J. Waterfield M.D. Cell. 1992; 70: 419-425Abstract Full Text PDF PubMed Scopus (539) Google Scholar and 15Hu P. Mondino A. Skolnik E.Y. Schlessinger J. Mol. Cell. Biol. 1993; 13: 7677-7688Crossref PubMed Scopus (234) Google Scholar) and a 85-kDa regulatory subunit (p85α or p85β; Refs. 16Escobedo J.A. Navankasattusas S. Kavanaugh W.M. Milfay D. Fried V.A. Williams L.T. Cell. 1991; 65: 75-82Abstract Full Text PDF PubMed Scopus (373) Google Scholar, 17Otsu M. Hiles I. Gout I. Fry M.J. Ruiz-Larrea F. Panayotou G. Thompson A. Dhand R. Hsuan J. Totty N. Smith A.D. Morgan S.J. Courtneidge S.A. Parker P.J. Waterfield M.D. Cell. 1991; 65: 91-97Abstract Full Text PDF PubMed Scopus (539) Google Scholar, 18Skolnik E.Y. Margolis B. Mohammadi M. Lowenstein E. Fischer R. Drepps A. Ullrich A. Schlessinger J. Cell. 1991; 65: 83-90Abstract Full Text PDF PubMed Scopus (436) Google Scholar), and that are utilized for signaling by activated growth factor, cytokine, and antigen receptors. In these heterodimeric PI3Ks, the p85 subunit also functions as an adaptor protein that mediates protein-protein interactions through its two Src homology 2 (SH2) domains, one SH3 domain, two proline-rich sequences, and a region with similarity to the breakpoint cluster region gene (16Escobedo J.A. Navankasattusas S. Kavanaugh W.M. Milfay D. Fried V.A. Williams L.T. Cell. 1991; 65: 75-82Abstract Full Text PDF PubMed Scopus (373) Google Scholar, 17Otsu M. Hiles I. Gout I. Fry M.J. Ruiz-Larrea F. Panayotou G. Thompson A. Dhand R. Hsuan J. Totty N. Smith A.D. Morgan S.J. Courtneidge S.A. Parker P.J. Waterfield M.D. Cell. 1991; 65: 91-97Abstract Full Text PDF PubMed Scopus (539) Google Scholar, 18Skolnik E.Y. Margolis B. Mohammadi M. Lowenstein E. Fischer R. Drepps A. Ullrich A. Schlessinger J. Cell. 1991; 65: 83-90Abstract Full Text PDF PubMed Scopus (436) Google Scholar). The two SH2 domains of p85 are involved in recruitment of PI3K to activated growth factor receptors (3Kaplan D.R. Whitman M. Schaffhausen B. Pallas D.C. White M. Cantley L.C. Roberts T.M. Cell. 1987; 50: 1021-1029Abstract Full Text PDF PubMed Scopus (407) Google Scholar, 16Escobedo J.A. Navankasattusas S. Kavanaugh W.M. Milfay D. Fried V.A. Williams L.T. Cell. 1991; 65: 75-82Abstract Full Text PDF PubMed Scopus (373) Google Scholar) or other proteins having the general motif phosphotyrosine Tyr(P)-X-X-methionine (19Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Leichleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2373) Google Scholar), or, in some cases, Tyr(P)-X-X-leucine (20Exley M. Varticovski L. Peter M. Sancho J. Terhorst C. J. Biol. Chem. 1994; 269: 15140-15146Abstract Full Text PDF PubMed Google Scholar, 21Zenner G. Vorherr T. Mustelin T. Burn P. J. Cell. Biochem. 1996; 63: 94-103Crossref PubMed Scopus (38) Google Scholar). In T cells, the physiologically relevant ligands for p85 include tyrosine-phosphorylated CD28 (22Pages F. Ragueneau M. Rottapel R. Truneh A. Nunes J. Imbert J. Olive D. Nature. 1994; 369: 327-329Crossref PubMed Scopus (345) Google Scholar), subunits of the T cell antigen receptor (20Exley M. Varticovski L. Peter M. Sancho J. Terhorst C. J. Biol. Chem. 1994; 269: 15140-15146Abstract Full Text PDF PubMed Google Scholar, 21Zenner G. Vorherr T. Mustelin T. Burn P. J. Cell. Biochem. 1996; 63: 94-103Crossref PubMed Scopus (38) Google Scholar, 23Carrera A.C. Rodriguez-Borlado L. Martinez-Alonso C. Merida I. J. Biol. Chem. 1994; 269: 19435-19440Abstract Full Text PDF PubMed Google Scholar), CD5 (24Dennehy K.M. Broszeit R. Garnett D. Durrheim G.A. Spruyt L.L. Beyers A.D. Eur. J. Immunol. 1997; 27: 679-686Crossref PubMed Scopus (42) Google Scholar), CD7 (25Lee D.M. Patel D.D. Pendergast A.M. Haynes B.F. Int. Immunol. 1996; 8: 1195-1203Crossref PubMed Scopus (23) Google Scholar), and the c-Cbl proto-oncogene product (26Meisner H. Conway B.R. Hartley D. Czech M.P. Mol. Cell. Biol. 1995; 15: 3571-3578Crossref PubMed Scopus (212) Google Scholar). In addition to causing a subcellular relocation of PI3K, these SH2 ligands cause an allosteric activation of the catalytic p110 subunit, which is bound to the region between the two SH2 domains of p85 (15Hu P. Mondino A. Skolnik E.Y. Schlessinger J. Mol. Cell. Biol. 1993; 13: 7677-7688Crossref PubMed Scopus (234) Google Scholar, 27Dhand R. Hara K. Hiles I. Bax B. Gout I. Panayotou G. Fry M.J. Yonezawa K. Kasuga M. Waterfield M.D. EMBO J. 1994; 13: 511-521Crossref PubMed Scopus (295) Google Scholar, 28Holt K.H. Olson A.L. Moye-Rowley W.S. Pessin J.E. Mol. Cell. Biol. 1994; 14: 42-49Crossref PubMed Google Scholar, 29Klippel A. Escobedo J.A. Hu Q. Williams L.T. Mol. Cell. Biol. 1993; 13: 5560-5566Crossref PubMed Scopus (87) Google Scholar).Several additional modes of PI3K regulation have been demonstrated, and it is likely that they act in concert to regulate the production of 3-phosphorylated inositol phospholipids in response to a variety of stimuli. The catalytic p110 interacts with activated GTP-bound Ras proteins through a region adjacent to its p85-binding NH2terminus (30Rodriguez-Viciana 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 (1716) Google Scholar, 31Rodriguez-Viciana P. Warne P.H. Vanhaesebroeck B. Waterfield M.D. Downward J. EMBO J. 1996; 15: 2442-2451Crossref PubMed Scopus (497) Google Scholar). Active Ras enhances PI3K activity in intact cells (30Rodriguez-Viciana 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 (1716) Google Scholar, 31Rodriguez-Viciana P. Warne P.H. Vanhaesebroeck B. Waterfield M.D. Downward J. EMBO J. 1996; 15: 2442-2451Crossref PubMed Scopus (497) Google Scholar), but some data indicate that Ras also acts downstream of PI3K (32Hu Q. Klippel A. Muslin A.J. Fantl W.J. Williams L.T. Science. 1995; 268: 100-102Crossref PubMed Scopus (516) Google Scholar). In T cells, the two p85 isoforms have been shown to undergo phosphorylation on both serine and threonine (33Reif K. Gout I. Waterfield M.D. Cantrell D.A. J. Biol. Chem. 1993; 268: 10780-10788Abstract Full Text PDF PubMed Google Scholar, 34Dhand R. Hiles I. Panayotou G. Roche S. Fry M.J. Gout I. Totty N.F. Truong O. Vicendo P. Yonezawa K. Kasuga M. Courtneidge S.A. Waterfield M.D. EMBO J. 1994; 13: 522-533Crossref PubMed Scopus (414) Google Scholar). Tyrosine phosphorylation of the p85 subunit has been shown to occur in many different systems, such as in response to platelet-derived growth factor (3Kaplan D.R. Whitman M. Schaffhausen B. Pallas D.C. White M. Cantley L.C. Roberts T.M. Cell. 1987; 50: 1021-1029Abstract Full Text PDF PubMed Scopus (407) Google Scholar), insulin (35Hayashi H. Nishioka Y. Kamohara S. Kanai F. Ishii K. Fukui Y. Shibasaki F. Takenawa T. Kido H. Katsunuma N. Ebina Y. J. Biol. Chem. 1993; 268: 7107-7117Abstract Full Text PDF PubMed Google Scholar), B cell antigen receptor ligation (4Gold M.R. Chan V.W.-F. Turck C. DeFranco A.L. J. Immunol. 1992; 148: 2012-2022PubMed Google Scholar), interleukin-2 (36Karnitz L.M. Sutor S.L. Abraham R.T. J. Exp. Med. 1994; 179: 1799-1808Crossref PubMed Scopus (72) Google Scholar), and in cells transformed by the Bcr-Abl fusion protein-tyrosine kinase (37Amarante-Mendes G.P. Jascur T. Nishioka W.K. Mustelin T. Green D.R. Cell Death Differ. 1997; 4: 541-555Crossref Scopus (23) Google Scholar, 38Gotoh A. Miyazawa K. Ohyashiki K. Toyama K. Leukemia. 1994; 8: 115-120PubMed Google Scholar, 39Skorski T. Kanakaraj P. Nieborowska-Skorska M. Ratajczak M.Z. Wen S.-C. Zon G. Gewirtz A.M. Perussia B. Calabretta B. Blood. 1995; 86: 726-736Crossref PubMed Google Scholar, 40Varticovski L. Daley G.Q. Jackson P. Baltimore D. Cantley L.C. Mol. Cell. Biol. 1991; 11: 1107-1113Crossref PubMed Scopus (180) Google Scholar). The sites of phosphorylation in p85 have been mapped to tyrosines 368, 508, and 607 in insulin-stimulated cells (35Hayashi H. Nishioka Y. Kamohara S. Kanai F. Ishii K. Fukui Y. Shibasaki F. Takenawa T. Kido H. Katsunuma N. Ebina Y. J. Biol. Chem. 1993; 268: 7107-7117Abstract Full Text PDF PubMed Google Scholar), but the physiological function of this phosphorylation has remained unknown. Tyrosine phosphorylation of p85 seems not to be required for the enzymatic activity of PI3K. Instead, tyrosine phosphorylation of p85 has been reported to correlate with the dissociation of PI3K from the activated insulin receptor kinase (41Zhang W. Johnson J.D. Rutter W.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11317-11321Crossref PubMed Scopus (15) Google Scholar).We have studied the tyrosine phosphorylation of p85 in hematopoietic cells, and report that phosphorylation occurs at least at Tyr-688 in the C-terminal SH2 domain. This event does not detectably affect the catalytic activity of PI3K per se, but causes a change in the binding properties of the SH2 domain. This change is likely to modify the function of PI3K in intact cells.DISCUSSIONTaken together, our findings indicate that PI3K can be phosphorylated at Tyr-688 in the C-terminal SH2 domain of the p85 subunit in Bcr-Abl expressing HL-60 cells, by active Lck in COS cells, by an unidentified receptor-activated protein-tyrosine kinase in T cells, and by both Abl and Lck in vitro. This phosphorylation does not measurably affect the lipid kinase activity of PI3K (at least at physiological stoichiometry), but was found to change the ligand binding properties of the SH2 domain(s). These results are in agreement with previous observations showing that expression of Bcr-Abl in NIH 3T3 cells induces tyrosine phosphorylation of PI3K without any significant increase in PI3K products in vivo(40Varticovski L. Daley G.Q. Jackson P. Baltimore D. Cantley L.C. Mol. Cell. Biol. 1991; 11: 1107-1113Crossref PubMed Scopus (180) Google Scholar).The analysis of SH2 domain function in p85 is complicated by the tandem arrangement of the two SH2 domains, resulting in their cooperative binding to many ligands. Our results indicate that tyrosine phosphorylation of the C-terminal SH2 domain reduced the affinity for some ligands, while the binding of others was unchanged. Two alternative explanations could be envisualized: either tyrosine phosphorylation changed the ligand selection from the classical Tyr(P)-X-X-methionine to something more or less different, or only the C-terminal SH2 domain was inhibited, while the N-terminal SH2 domain remained unchanged. In the latter case, only of those ligands that bind exclusively to the C-terminal SH2 domain or that require simultaneous binding to both domains will bind less strongly. We have previously observed that expression of the HA-tagged p85 proteins in T cells resulted in the co-immunoprecipitation of phospho-TCRζ only when both SH2 domains were present in the p85 protein. Such a requirement for two SH2 domains would explain why the TCRζ binds despite not having the optimal Tyr(P)-X-X-methionine (19Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Leichleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2373) Google Scholar) motif. Apparently, two Tyr(P)-X-X-leucine motifs in tandem in TCRζ can bind the two SH2 domains of p85 simultaneously and thereby increase the affinity to physiologically relevant levels. The binding of PI3K to TCRζ and CD3 subunits (5Jascur T. Gilman J. Mustelin T. J. Biol. Chem. 1997; 272: 14483-14488Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 20Exley M. Varticovski L. Peter M. Sancho J. Terhorst C. J. Biol. Chem. 1994; 269: 15140-15146Abstract Full Text PDF PubMed Google Scholar, 23Carrera A.C. Rodriguez-Borlado L. Martinez-Alonso C. Merida I. J. Biol. Chem. 1994; 269: 19435-19440Abstract Full Text PDF PubMed Google Scholar), as well as to isolated phosphopeptides derived from these proteins (21Zenner G. Vorherr T. Mustelin T. Burn P. J. Cell. Biochem. 1996; 63: 94-103Crossref PubMed Scopus (38) Google Scholar), has been reported. The findings reported in the present paper may explain why p85 binding to these receptor subunits is of low stoichiometry when assessed by co-immunoprecipitation.In the Bcr-Abl expressing cells, p85 was phosphorylated on tyrosine, but also co-immunoprecipitated with several Tyr(P)-containing proteins. While this may seem conflicting, it is clear that only a fraction of p85 is tyrosine phosphorylated, and it is impossible to judge if any of the co-immunoprecipitating protein bound to the phosphorylated minority of p85 molecules or (more likely) to the unphosphorylated majority. The requirement for both SH2 domains for efficient phosphorylation of p85 proteins in Bcr-Abl expressing cells, suggests that both are involved directly or indirectly in association with the protein-tyrosine kinase responsible for this phosphorylation. The simplest model predicts that the p85 SH2 domains bind directly to Bcr-Abl, but dissociate from it upon phosphorylation of the C-terminal SH2 domain. This would explain why the co-immunoprecipitation of Bcr-Abl and PI3K is of very low stoichiometry.We recently reported that the Lck kinase is phosphorylated at Tyr-192 in the EF loop of its SH2 domain in activated T cells, and in COS-1 cells co-transfected with either Syk or Zap (45Couture C. Baier G. Oetken C. Williams S. Telford D. Marie-Cardine A. Baier-Bitterlich G. Fischer S. Burn P. Altman A. Mustelin T. Mol. Cell. Biol. 1994; 14: 5249-5258Crossref PubMed Google Scholar, 47Couture C. Songyang Z. Jascur T. Williams S. Tailor P. Cantley L.C. Mustelin T. J. Biol. Chem. 1996; 271: 24880-24884Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The phosphorylation of the SH2 domain, or the mutation of Tyr-192 to an acidic residue, caused a strong decline in the affinity of the domain for tyrosine-phosphorylated ligands (47Couture C. Songyang Z. Jascur T. Williams S. Tailor P. Cantley L.C. Mustelin T. J. Biol. Chem. 1996; 271: 24880-24884Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). A similar change was reported by Stover and co-workers (65Stover D.R. Furet P. Lydon N.B. J. Biol. Chem. 1996; 21: 12481-12487Abstract Full Text Full Text PDF Scopus (78) Google Scholar) for the c-Src SH2 domain upon its phosphorylation at Tyr-213 by the platelet-derived growth factor kinase. In this paper, we add a third example to the list of SH2 domains regulated by tyrosine phosphorylation, namely the C-terminal SH2 domain of PI3K p85. Interestingly, Tyr-688 resides in the same region of the SH2 domain as Tyr-192 in the Lck SH2 domain. Comparison of the amino acid sequences of SH2 domains from different proteins shows that many, but not all, contain tyrosine residues in the corresponding location in the EF loop (66Waksman G. Kominos D. Robertson S.C. Pant N. Baltimore D. Birge R.B. Cowburn D. Hanafusa H. Mayer B.J. Overduin M. Resh M.D. Rios C.B. Silverman L. Kuriyan J. Nature. 1992; 358: 646-653Crossref PubMed Scopus (573) Google Scholar). Notably, the N-terminal SH2 domain of p85 does not. Thus, it is tempting to speculate that the regulation of SH2 domains by tyrosine phosphorylation is a more general mechanism for the termination of SH2-ligand interactions, perhaps in part explaining their transient nature in intact cells. Phosphatidylinositol 3-kinases (PI3Ks) 1The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; HA, hemagglutinin; Tyr(P), phosphotyrosine; SH2, Src homology 2 region; SH3, Src homology 3 region; mAb, monoclonal antibody; TPCK,l-1-tosylamido-2-phenylethyl chloromethyl ketone; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis.1The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; HA, hemagglutinin; Tyr(P), phosphotyrosine; SH2, Src homology 2 region; SH3, Src homology 3 region; mAb, monoclonal antibody; TPCK,l-1-tosylamido-2-phenylethyl chloromethyl ketone; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis. are a family of enzymes involved in a multiplicity of cellular functions, including cell proliferation and transformation (1Auger K.R. Cantley L.C. Cancer Cells. 1991; 3: 263-275PubMed Google Scholar, 2Coughlin S.R. Escobedo J.A. Williams L.T. Science. 1989; 243: 1191-1194Crossref PubMed Scopus (287) Google Scholar, 3Kaplan D.R. Whitman M. Schaffhausen B. Pallas D.C. White M. Cantley L.C. Roberts T.M. Cell. 1987; 50: 1021-1029Abstract Full Text PDF PubMed Scopus (407) Google Scholar), lymphocyte activation (4Gold M.R. Chan V.W.-F. Turck C. DeFranco A.L. J. Immunol. 1992; 148: 2012-2022PubMed Google Scholar, 5Jascur T. Gilman J. Mustelin T. J. Biol. Chem. 1997; 272: 14483-14488Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 6von Willebrand M. Jascur T. Bonnefoy-Bérard N. Yano H. Altman A. Matsuda Y. Mustelin T. Eur. J. Biochem. 1996; 235: 828-835Crossref PubMed Scopus (64) Google Scholar, 7Ward S.G. Ley S.C. MacPhee C. Cantrell D.A. Eur. J. Immunol. 1992; 22: 45-49Crossref PubMed Scopus (96) Google Scholar), G protein signaling (8Stoyanov B. Volinia S. Hanck T. Rubio I. Loubtchenkov M. Malek D. Stoyanova S. Vanhaesebroeck B. Dhand R. Nürnberg B. Gierschik P. Seedorf K. Hsuan J.J. Waterfield M.D. Wetzker R. Science. 1995; 269: 690-693Crossref PubMed Scopus (638) Google Scholar), DNA repair (9Savitsky K. Bar-Shira A. Gilad S. Rotman G. Ziv Y. Vanagaite L. Tagle D.A. Smith S. Uziel T. Sfez S. Ashkenazi M. Pecker I. Frydman M. Harnik R. Patanjali S.R. Simmons A. Clines G.A. Sartiel A. Gatti R.A. Chessa L. Sanal O. Lavin M.F. Jaspers N.G.J. Taylor A.M.R. Arlett C.F. Miki T. Weissman S.M. Lovett M. Collins F.S. Shiloh Y. Science. 1995; 268: 1749-1753Crossref PubMed Scopus (2355) Google Scholar), intracellular vesicle trafficking (10Schu P.V. Takegawa K. Fry M.J. Stack J.H. Waterfield M.D. Emr S.D. Science. 1993; 260: 88-91Crossref PubMed Scopus (802) Google Scholar, 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 (306) Google Scholar), and inhibition of programmed cell death (12Minshall C. Arkins S. Freund G.G. Kelley K.W. J. Immunol. 1996; 156: 939-947PubMed Google Scholar, 13Yao R. Cooper G.M. Science. 1995; 267: 2003-2006Crossref PubMed Scopus (1288) Google Scholar). The currently best characterized type of PI3K is the heterodimeric enzymes that consist of a 110-kDa catalytic subunit (p110α or p110β; Refs. 14Hiles I. Otsu M. Volinia S. Fry M.J. Gout I. Dhand R. Panayotou G. Ruiz-Larrea F. Thompson A. Totty N. Hsuan J. Courtneidge S.A. Parker P.J. Waterfield M.D. Cell. 1992; 70: 419-425Abstract Full Text PDF PubMed Scopus (539) Google Scholar and 15Hu P. Mondino A. Skolnik E.Y. Schlessinger J. Mol. Cell. Biol. 1993; 13: 7677-7688Crossref PubMed Scopus (234) Google Scholar) and a 85-kDa regulatory subunit (p85α or p85β; Refs. 16Escobedo J.A. Navankasattusas S. Kavanaugh W.M. Milfay D. Fried V.A. Williams L.T. Cell. 1991; 65: 75-82Abstract Full Text PDF PubMed Scopus (373) Google Scholar, 17Otsu M. Hiles I. Gout I. Fry M.J. Ruiz-Larrea F. Panayotou G. Thompson A. Dhand R. Hsuan J. Totty N. Smith A.D. Morgan S.J. Courtneidge S.A. Parker P.J. Waterfield M.D. Cell. 1991; 65: 91-97Abstract Full Text PDF PubMed Scopus (539) Google Scholar, 18Skolnik E.Y. Margolis B. Mohammadi M. Lowenstein E. Fischer R. Drepps A. Ullrich A. Schlessinger J. Cell. 1991; 65: 83-90Abstract Full Text PDF PubMed Scopus (436) Google Scholar), and that are utilized for signaling by activated growth factor, cytokine, and antigen receptors. In these heterodimeric PI3Ks, the p85 subunit also functions as an adaptor protein that mediates protein-protein interactions through its two Src homology 2 (SH2) domains, one SH3 domain, two proline-rich sequences, and a region with similarity to the breakpoint cluster region gene (16Escobedo J.A. Navankasattusas S. Kavanaugh W.M. Milfay D. Fried V.A. Williams L.T. Cell. 1991; 65: 75-82Abstract Full Text PDF PubMed Scopus (373) Google Scholar, 17Otsu M. Hiles I. Gout I. Fry M.J. Ruiz-Larrea F. Panayotou G. Thompson A. Dhand R. Hsuan J. Totty N. Smith A.D. Morgan S.J. Courtneidge S.A. Parker P.J. Waterfield M.D. Cell. 1991; 65: 91-97Abstract Full Text PDF PubMed Scopus (539) Google Scholar, 18Skolnik E.Y. Margolis B. Mohammadi M. Lowenstein E. Fischer R. Drepps A. Ullrich A. Schlessinger J. Cell. 1991; 65: 83-90Abstract Full Text PDF PubMed Scopus (436) Google Scholar). The two SH2 domains of p85 are involved in recruitment of PI3K to activated growth factor receptors (3Kaplan D.R. Whitman M. Schaffhausen B. Pallas D.C. White M. Cantley L.C. Roberts T.M. Cell. 1987; 50: 1021-1029Abstract Full Text PDF PubMed Scopus (407) Google Scholar, 16Escobedo J.A. Navankasattusas S. Kavanaugh W.M. Milfay D. Fried V.A. Williams L.T. Cell. 1991; 65: 75-82Abstract Full Text PDF PubMed Scopus (373) Google Scholar) or other proteins having the general motif phosphotyrosine Tyr(P)-X-X-methionine (19Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Leichleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2373) Google Scholar), or, in some cases, Tyr(P)-X-X-leucine (20Exley M. Varticovski L. Peter M. Sancho J. Terhorst C. J. Biol. Chem. 1994; 269: 15140-15146Abstract Full Text PDF PubMed Google Scholar, 21Zenner G. Vorherr T. Mustelin T. Burn P. J. Cell. Biochem. 1996; 63: 94-103Crossref PubMed Scopus (38) Google Scholar). In T cells, the physiologically relevant ligands for p85 include tyrosine-phosphorylated CD28 (22Pages F. Ragueneau M. Rottapel R. Truneh A. Nunes J. Imbert J. Olive D. Nature. 1994; 369: 327-329Crossref PubMed Scopus (345) Google Scholar), subunits of the T cell antigen receptor (20Exley M. Varticovski L. Peter M. Sancho J. Terhorst C. J. Biol. Chem. 1994; 269: 15140-15146Abstract Full Text PDF PubMed Google Scholar, 21Zenner G. Vorherr T. Mustelin T. Burn P. J. Cell. Biochem. 1996; 63: 94-103Crossref PubMed Scopus (38) Google Scholar, 23Carrera A.C. Rodriguez-Borlado L. Martinez-Alonso C. Merida I. J. Biol. Chem. 1994; 269: 19435-19440Abstract Full Text PDF PubMed Google Scholar), CD5 (24Dennehy K.M. Broszeit R. Garnett D. Durrheim G.A. Spruyt L.L. Beyers A.D. Eur. J. Immunol. 1997; 27: 679-686Crossref PubMed Scopus (42) Google Scholar), CD7 (25Lee D.M. Patel D.D. Pendergast A.M. Haynes B.F. Int. Immunol. 1996; 8: 1195-1203Crossref PubMed Scopus (23) Google Scholar), and the c-Cbl proto-oncogene product (26Meisner H. Conway B.R. Hartley D. Czech M.P. Mol. Cell. Biol. 1995; 15: 3571-3578Crossref PubMed Scopus (212) Google Scholar). In addition to causing a subcellular relocation of PI3K, these SH2 ligands cause an allosteric activation of the catalytic p110 subunit, which is bound to the region between the two SH2 domains of p85 (15Hu P. Mondino A. Skolnik E.Y. Schlessinger J. Mol. Cell. Biol. 1993; 13: 7677-7688Crossref PubMed Scopus (234) Google Scholar, 27Dhand R. Hara K. Hiles I. Bax B. Gout I. Panayotou G. Fry M.J. Yonezawa K. Kasuga M. Waterfield M.D. EMBO J. 1994; 13: 511-521Crossref PubMed Scopus (295) Google Scholar, 28Holt K.H. Olson A.L. Moye-Rowley W.S. Pessin J.E. Mol. Cell. Biol. 1994; 14: 42-49Crossref PubMed Google Scholar, 29Klippel A. Escobedo J.A. Hu Q. Williams L.T. Mol. Cell. Biol. 1993; 13: 5560-5566Crossref PubMed Scopus (87) Google Scholar). Several additional modes of PI3K regulation have been demonstrated, and it is likely that they act in concert to regulate the production of 3-phosphorylated inositol phospholipids in response to a variety of stimuli. The catalytic p110 interacts with activated GTP-bound Ras proteins through a region adjacent to its p85-binding NH2terminus (30Rodriguez-Viciana 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 (1716) Google Scholar, 31Rodriguez-Viciana P. Warne P.H. Vanhaesebroeck B. Waterfield M.D. Downward J. EMBO J. 1996; 15: 2442-2451Crossref PubMed Scopus (497) Google Scholar). Active Ras enhances PI3K activity in intact cells (30Rodriguez-Viciana 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 (1716) Google Scholar, 31Rodriguez-Viciana P. Warne P.H. Vanhaesebroeck B. Waterfield M.D. Downward J. EMBO J. 1996; 15: 2442-2451Crossref PubMed Scopus (497) Google Scholar), but some data indicate that Ras also acts downstream of PI3K (32Hu Q. Klippel A. Muslin A.J. Fantl W.J. Williams L.T. Science. 1995; 268: 100-102Crossref PubMed Scopus (516) Google Scholar). In T cells, the two p85 isoforms have been shown to undergo phosphorylation on both serine and threonine (33Reif K. Gout I. Waterfield M.D. Cantrell D.A. J. Biol. Chem. 1993; 268: 10780-10788Abstract Full Text PDF PubMed Google Scholar, 34Dhand R. Hiles I. Panayotou G. Roche S. Fry M.J. Gout I. Totty N.F. Truong O. Vicendo P. Yonezawa K. Kasuga M. Courtneidge S.A. Waterfield M.D. EMBO J. 1994; 13: 522-533Crossref PubMed Scopus (414) Google Scholar). Tyrosine phosphorylation of the p85 subunit has been shown to occur in many different systems, such as in response to platelet-derived growth factor (3Kaplan D.R. Whitman M. Schaffhausen B. Pallas D.C. White M. Cantley L.C. Roberts T.M. Cell. 1987; 50: 1021-1029Abstract Full Text PDF PubMed Scopus (407) Google Scholar), insulin (35Hayashi H. Nishioka Y. Kamohara S. Kanai F. Ishii K. Fukui Y. Shibasaki F. Takenawa T. Kido H. Katsunuma N. Ebina Y. J. Biol. Chem. 1993; 268: 7107-7117Abstract Full Text PDF PubMed Google Scholar), B cell antigen receptor ligation (4Gold M.R. Chan V.W.-F. Turck C. DeFranco A.L. J. Immunol. 1992; 148: 2012-2022PubMed Google Scholar), interleukin-2 (36Karnitz L.M. Sutor S.L. Abraham R.T. J. Exp. Med. 1994; 179: 1799-1808Crossref PubMed Scopus (72) Google Scholar), and in cells transformed by the Bcr-Abl fusion protein-tyrosine kinase (37Amarante-Mendes G.P. Jascur T. Nishioka W.K. Mustelin T. Green D.R. Cell Death Differ. 1997; 4: 541-555Crossref Scopus (23) Google Scholar, 38Gotoh A. Miyazawa K. Ohyashiki K. Toyama K. Leukemia. 1994; 8: 115-120PubMed Google Scholar, 39Skorski T. Kanakaraj P. Nieborowska-Skorska M. Ratajczak M.Z. Wen S.-C. Zon G. Gewirtz A.M. Perussia B. Calabretta B. Blood. 1995; 86: 726-736Crossref PubMed Google Scholar, 40Varticovski L. Daley G.Q. Jackson P. Baltimore D. Cantley L.C. Mol. Cell. Biol. 1991; 11: 1107-1113Crossref PubMed Scopus (180) Google Scholar). The sites of phosphorylation in p85 have been mapped to tyrosines 368, 508, and 607 in insulin-stimulated cells (35Hayashi H. Nishioka Y. Kamohara S. Kanai F. Ishii K. Fukui Y. Shibasaki F. Takenawa T. Kido H. Katsunuma N. Ebina Y. J. Biol. Chem. 1993; 268: 7107-7117Abstract Full Text PDF PubMed Google Scholar), but the physiological function of this phosphorylation has remained unknown. Tyrosine phosphorylation of p85 seems not to be required for the enzymatic activity of PI3K. Instead, tyrosine phosphorylation of p85 has been reported to correlate with the dissociation of PI3K from the activated insulin receptor kinase (41Zhang W. Johnson J.D. Rutter W.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11317-11321Crossref PubMed Scopus (15) Google Scholar). We have studied the tyrosine phosphorylation of p85 in hematopoietic cells, and report that phosphorylation occurs at least at Tyr-688 in the C-terminal SH2 domain. This event does not detectably affect the catalytic activity of PI3K per se, but causes a change in the binding properties of the SH2 domain. This change is likely to modify the function of PI3K in intact cells. DISCUSSIONTaken together, our findings indicate that PI3K can be phosphorylated at Tyr-688 in the C-terminal SH2 domain of the p85 subunit in Bcr-Abl expressing HL-60 cells, by active Lck in COS cells, by an unidentified receptor-activated protein-tyrosine kinase in T cells, and by both Abl and Lck in vitro. This phosphorylation does not measurably affect the lipid kinase activity of PI3K (at least at physiological stoichiometry), but was found to change the ligand binding properties of the SH2 domain(s). These results are in agreement with previous observations showing that expression of Bcr-Abl in NIH 3T3 cells induces tyrosine phosphorylation of PI3K without any significant increase in PI3K products in vivo(40Varticovski L. Daley G.Q. Jackson P. Baltimore D. Cantley L.C. Mol. Cell. Biol. 1991; 11: 1107-1113Crossref PubMed Scopus (180) Google Scholar).The analysis of SH2 domain function in p85 is complicated by the tandem arrangement of the two SH2 domains, resulting in their cooperative binding to many ligands. Our results indicate that tyrosine phosphorylation of the C-terminal SH2 domain reduced the affinity for some ligands, while the binding of others was unchanged. Two alternative explanations could be envisualized: either tyrosine phosphorylation changed the ligand selection from the classical Tyr(P)-X-X-methionine to something more or less different, or only the C-terminal SH2 domain was inhibited, while the N-terminal SH2 domain remained unchanged. In the latter case, only of those ligands that bind exclusively to the C-terminal SH2 domain or that require simultaneous binding to both domains will bind less strongly. We have previously observed that expression of the HA-tagged p85 proteins in T cells resulted in the co-immunoprecipitation of phospho-TCRζ only when both SH2 domains were present in the p85 protein. Such a requirement for two SH2 domains would explain why the TCRζ binds despite not having the optimal Tyr(P)-X-X-methionine (19Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Leichleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2373) Google Scholar) motif. Apparently, two Tyr(P)-X-X-leucine motifs in tandem in TCRζ can bind the two SH2 domains of p85 simultaneously and thereby increase the affinity to physiologically relevant levels. The binding of PI3K to TCRζ and CD3 subunits (5Jascur T. Gilman J. Mustelin T. J. Biol. Chem. 1997; 272: 14483-14488Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 20Exley M. Varticovski L. Peter M. Sancho J. Terhorst C. J. Biol. Chem. 1994; 269: 15140-15146Abstract Full Text PDF PubMed Google Scholar, 23Carrera A.C. Rodriguez-Borlado L. Martinez-Alonso C. Merida I. J. Biol. Chem. 1994; 269: 19435-19440Abstract Full Text PDF PubMed Google Scholar), as well as to isolated phosphopeptides derived from these proteins (21Zenner G. Vorherr T. Mustelin T. Burn P. J. Cell. Biochem. 1996; 63: 94-103Crossref PubMed Scopus (38) Google Scholar), has been reported. The findings reported in the present paper may explain why p85 binding to these receptor subunits is of low stoichiometry when assessed by co-immunoprecipitation.In the Bcr-Abl expressing cells, p85 was phosphorylated on tyrosine, but also co-immunoprecipitated with several Tyr(P)-containing proteins. While this may seem conflicting, it is clear that only a fraction of p85 is tyrosine phosphorylated, and it is impossible to judge if any of the co-immunoprecipitating protein bound to the phosphorylated minority of p85 molecules or (more likely) to the unphosphorylated majority. The requirement for both SH2 domains for efficient phosphorylation of p85 proteins in Bcr-Abl expressing cells, suggests that both are involved directly or indirectly in association with the protein-tyrosine kinase responsible for this phosphorylation. The simplest model predicts that the p85 SH2 domains bind directly to Bcr-Abl, but dissociate from it upon phosphorylation of the C-terminal SH2 domain. This would explain why the co-immunoprecipitation of Bcr-Abl and PI3K is of very low stoichiometry.We recently reported that the Lck kinase is phosphorylated at Tyr-192 in the EF loop of its SH2 domain in activated T cells, and in COS-1 cells co-transfected with either Syk or Zap (45Couture C. Baier G. Oetken C. Williams S. Telford D. Marie-Cardine A. Baier-Bitterlich G. Fischer S. Burn P. Altman A. Mustelin T. Mol. Cell. Biol. 1994; 14: 5249-5258Crossref PubMed Google Scholar, 47Couture C. Songyang Z. Jascur T. Williams S. Tailor P. Cantley L.C. Mustelin T. J. Biol. Chem. 1996; 271: 24880-24884Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The phosphorylation of the SH2 domain, or the mutation of Tyr-192 to an acidic residue, caused a strong decline in the affinity of the domain for tyrosine-phosphorylated ligands (47Couture C. Songyang Z. Jascur T. Williams S. Tailor P. Cantley L.C. Mustelin T. J. Biol. Chem. 1996; 271: 24880-24884Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). A similar change was reported by Stover and co-workers (65Stover D.R. Furet P. Lydon N.B. J. Biol. Chem. 1996; 21: 12481-12487Abstract Full Text Full Text PDF Scopus (78) Google Scholar) for the c-Src SH2 domain upon its phosphorylation at Tyr-213 by the platelet-derived growth factor kinase. In this paper, we add a third example to the list of SH2 domains regulated by tyrosine phosphorylation, namely the C-terminal SH2 domain of PI3K p85. Interestingly, Tyr-688 resides in the same region of the SH2 domain as Tyr-192 in the Lck SH2 domain. Comparison of the amino acid sequences of SH2 domains from different proteins shows that many, but not all, contain tyrosine residues in the corresponding location in the EF loop (66Waksman G. Kominos D. Robertson S.C. Pant N. Baltimore D. Birge R.B. Cowburn D. Hanafusa H. Mayer B.J. Overduin M. Resh M.D. Rios C.B. Silverman L. Kuriyan J. Nature. 1992; 358: 646-653Crossref PubMed Scopus (573) Google Scholar). Notably, the N-terminal SH2 domain of p85 does not. Thus, it is tempting to speculate that the regulation of SH2 domains by tyrosine phosphorylation is a more general mechanism for the termination of SH2-ligand interactions, perhaps in part explaining their transient nature in intact cells. Taken together, our findings indicate that PI3K can be phosphorylated at Tyr-688 in the C-terminal SH2 domain of the p85 subunit in Bcr-Abl expressing HL-60 cells, by active Lck in COS cells, by an unidentified receptor-activated protein-tyrosine kinase in T cells, and by both Abl and Lck in vitro. This phosphorylation does not measurably affect the lipid kinase activity of PI3K (at least at physiological stoichiometry), but was found to change the ligand binding properties of the SH2 domain(s). These results are in agreement with previous observations showing that expression of Bcr-Abl in NIH 3T3 cells induces tyrosine phosphorylation of PI3K without any significant increase in PI3K products in vivo(40Varticovski L. Daley G.Q. Jackson P. Baltimore D. Cantley L.C. Mol. Cell. Biol. 1991; 11: 1107-1113Crossref PubMed Scopus (180) Google Scholar). The analysis of SH2 domain function in p85 is complicated by the tandem arrangement of the two SH2 domains, resulting in their cooperative binding to many ligands. Our results indicate that tyrosine phosphorylation of the C-terminal SH2 domain reduced the affinity for some ligands, while the binding of others was unchanged. Two alternative explanations could be envisualized: either tyrosine phosphorylation changed the ligand selection from the classical Tyr(P)-X-X-methionine to something more or less different, or only the C-terminal SH2 domain was inhibited, while the N-terminal SH2 domain remained unchanged. In the latter case, only of those ligands that bind exclusively to the C-terminal SH2 domain or that require simultaneous binding to both domains will bind less strongly. We have previously observed that expression of the HA-tagged p85 proteins in T cells resulted in the co-immunoprecipitation of phospho-TCRζ only when both SH2 domains were present in the p85 protein. Such a requirement for two SH2 domains would explain why the TCRζ binds despite not having the optimal Tyr(P)-X-X-methionine (19Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Leichleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2373) Google Scholar) motif. Apparently, two Tyr(P)-X-X-leucine motifs in tandem in TCRζ can bind the two SH2 domains of p85 simultaneously and thereby increase the affinity to physiologically relevant levels. The binding of PI3K to TCRζ and CD3 subunits (5Jascur T. Gilman J. Mustelin T. J. Biol. Chem. 1997; 272: 14483-14488Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 20Exley M. Varticovski L. Peter M. Sancho J. Terhorst C. J. Biol. Chem. 1994; 269: 15140-15146Abstract Full Text PDF PubMed Google Scholar, 23Carrera A.C. Rodriguez-Borlado L. Martinez-Alonso C. Merida I. J. Biol. Chem. 1994; 269: 19435-19440Abstract Full Text PDF PubMed Google Scholar), as well as to isolated phosphopeptides derived from these proteins (21Zenner G. Vorherr T. Mustelin T. Burn P. J. Cell. Biochem. 1996; 63: 94-103Crossref PubMed Scopus (38) Google Scholar), has been reported. The findings reported in the present paper may explain why p85 binding to these receptor subunits is of low stoichiometry when assessed by co-immunoprecipitation. In the Bcr-Abl expressing cells, p85 was phosphorylated on tyrosine, but also co-immunoprecipitated with several Tyr(P)-containing proteins. While this may seem conflicting, it is clear that only a fraction of p85 is tyrosine phosphorylated, and it is impossible to judge if any of the co-immunoprecipitating protein bound to the phosphorylated minority of p85 molecules or (more likely) to the unphosphorylated majority. The requirement for both SH2 domains for efficient phosphorylation of p85 proteins in Bcr-Abl expressing cells, suggests that both are involved directly or indirectly in association with the protein-tyrosine kinase responsible for this phosphorylation. The simplest model predicts that the p85 SH2 domains bind directly to Bcr-Abl, but dissociate from it upon phosphorylation of the C-terminal SH2 domain. This would explain why the co-immunoprecipitation of Bcr-Abl and PI3K is of very low stoichiometry. We recently reported that the Lck kinase is phosphorylated at Tyr-192 in the EF loop of its SH2 domain in activated T cells, and in COS-1 cells co-transfected with either Syk or Zap (45Couture C. Baier G. Oetken C. Williams S. Telford D. Marie-Cardine A. Baier-Bitterlich G. Fischer S. Burn P. Altman A. Mustelin T. Mol. Cell. Biol. 1994; 14: 5249-5258Crossref PubMed Google Scholar, 47Couture C. Songyang Z. Jascur T. Williams S. Tailor P. Cantley L.C. Mustelin T. J. Biol. Chem. 1996; 271: 24880-24884Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The phosphorylation of the SH2 domain, or the mutation of Tyr-192 to an acidic residue, caused a strong decline in the affinity of the domain for tyrosine-phosphorylated ligands (47Couture C. Songyang Z. Jascur T. Williams S. Tailor P. Cantley L.C. Mustelin T. J. Biol. Chem. 1996; 271: 24880-24884Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). A similar change was reported by Stover and co-workers (65Stover D.R. Furet P. Lydon N.B. J. Biol. Chem. 1996; 21: 12481-12487Abstract Full Text Full Text PDF Scopus (78) Google Scholar) for the c-Src SH2 domain upon its phosphorylation at Tyr-213 by the platelet-derived growth factor kinase. In this paper, we add a third example to the list of SH2 domains regulated by tyrosine phosphorylation, namely the C-terminal SH2 domain of PI3K p85. Interestingly, Tyr-688 resides in the same region of the SH2 domain as Tyr-192 in the Lck SH2 domain. Comparison of the amino acid sequences of SH2 domains from different proteins shows that many, but not all, contain tyrosine residues in the corresponding location in the EF loop (66Waksman G. Kominos D. Robertson S.C. Pant N. Baltimore D. Birge R.B. Cowburn D. Hanafusa H. Mayer B.J. Overduin M. Resh M.D. Rios C.B. Silverman L. Kuriyan J. Nature. 1992; 358: 646-653Crossref PubMed Scopus (573) Google Scholar). Notably, the N-terminal SH2 domain of p85 does not. Thus, it is tempting to speculate that the regulation of SH2 domains by tyrosine phosphorylation is a more general mechanism for the termination of SH2-ligand interactions, perhaps in part explaining their transient nature in intact cells. We are grateful to Dr. Lewis C. Cantley for valuable discussions and advice." @default.
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W2124186695 title "Modification of Phosphatidylinositol 3-Kinase SH2 Domain Binding Properties by Abl- or Lck-mediated Tyrosine Phosphorylation at Tyr-688" @default.
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