Modification of Phosphatidylinositol 3-Kinase SH2 Domain Binding Properties by Abl- or Lck-mediated Tyrosine Phosphorylation at Tyr-688

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. 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 (352) 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 (487) 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. 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We the and of the to the in vitro phosphorylation with and by in the of the and phosphorylated on this was the phosphorylated We that the major site for and phosphorylation of is Tyr-688 in the C-terminal SH2 This that tyrosine phosphorylation of endogenous p85 and of the transfected p85 in or cells not Tyr-688 in the C-terminal SH2 The of Tyr-688 in the C-terminal SH2 domain to this phosphorylation may the of the SH2 a for this we that phosphorylation of the Lck SH2 domain the in the a in its binding of ligands C. Z. Jascur T. Williams S. P. Cantley L.C. Mustelin T. J. Biol. 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Cell. 1992; Full Text PDF PubMed Scopus Google Scholar), of to and the binding of upon tyrosine phosphorylation of the In the protein was also but to as as tyrosine-phosphorylated this protein to the SH2 domain, may not by phosphorylation of the C-terminal SH2 to these with the protein of not to the binding of cellular proteins to this that PI3K phosphorylated Tyr-688 in the C-terminal SH2 domain of the p85 subunit in Bcr-Abl expressing cells, by Lck in COS cells, by an protein-tyrosine in T cells, and by Abl and Lck in This phosphorylation not the activity of PI3K but was to change the binding of the SH2 are in with that of Bcr-Abl in cells tyrosine phosphorylation of PI3K in PI3K in L. P. D. Cantley L.C. Mol. Cell. Biol. 1991; PubMed Scopus Google Scholar). The of SH2 domain in p85 is by the of the two SH2 domains, in their binding to that tyrosine phosphorylation of the C-terminal SH2 domain the for some the binding of was tyrosine phosphorylation the the Tyr(P)-X-X-methionine to or or the C-terminal SH2 domain was the SH2 domain In the of ligands that to the C-terminal SH2 domain or that binding to domains We have that of the p85 proteins in T cells in the of SH2 domains in the p85 a for two SH2 domains explain why the not having the 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 (2425) Google Scholar) two Tyr(P)-X-X-leucine in in the two SH2 domains of p85 and the to physiologically relevant The binding of PI3K to and subunits T. Gilman J. Mustelin T. J. Biol. Chem. 1997; 272: 14483-14488Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 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 as to these proteins G. Vorherr T. Mustelin T. Burn P. J. Cell. Biochem. 1996; 63: 94-103Crossref PubMed Scopus (40) Google Scholar), The in the may explain why p85 binding to these receptor subunits is of stoichiometry by In the Bcr-Abl expressing cells, p85 was phosphorylated on but also with this may is that a of p85 is tyrosine and is to of the protein to the phosphorylated of p85 or to the The for SH2 domains for phosphorylation of p85 proteins in Bcr-Abl expressing cells, that are involved or in association with the protein-tyrosine for this The that the p85 SH2 domains to but upon phosphorylation of the C-terminal SH2 This explain why the of Bcr-Abl and PI3K is of We that the Lck is phosphorylated in the of its SH2 domain in activated T cells, and in cells co-transfected with or C. G. C. Williams S. D. A. G. Fischer S. Burn P. Altman A. Mustelin T. Mol. Cell. Biol. 1994; 14: PubMed Google Scholar, C. Z. Jascur T. Williams S. P. Cantley L.C. Mustelin T. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). The phosphorylation of the SH2 domain, or the of to an a strong in the of the domain for tyrosine-phosphorylated ligands C. Z. Jascur T. Williams S. P. Cantley L.C. Mustelin T. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). change was by and D.R. P. J. Biol. Chem. 1996; Full Text Full Text PDF Scopus Google Scholar) for the SH2 domain upon its phosphorylation by the growth factor In this we a to the list of SH2 domains regulated by tyrosine the C-terminal SH2 domain of PI3K p85. Tyr-688 in the region of the SH2 domain as in the Lck SH2 of the of SH2 domains proteins that but not all, tyrosine in the in the G. D. S.C. N. D. Birge R.B. D. Hanafusa H. M. M.D. L. J. Nature. 1992; PubMed Scopus Google Scholar). the SH2 domain of p85 is to that the of SH2 domains by tyrosine phosphorylation is a general for the of in their transient in We are to C. Cantley for and