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

1998; Elsevier BV; Volume: 273; Issue: 7 Linguagem: Inglês

10.1074/jbc.273.7.3994

ISSN

1083-351X

Autores

Maria von Willebrand, Scott Williams, Manju Saxena, Jennifer Gilman, Pankaj Tailor, Thomas Jascur, Gustavo P. Amarante‐Mendes, Douglas R. Green, Tomas Mustelin,

Tópico(s)

Mast cells and histamine

Resumo

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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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 (434) 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 (609) 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 (496) 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 (487) 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 (434) 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 (2425) 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 (40) 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 (351) 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 (40) 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 (24) 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 (213) 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 (239) 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 (300) 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 (89) 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 (1749) Google Scholar, 31Rodriguez-Viciana P. Warne P.H. Vanhaesebroeck B. Waterfield M.D. Downward J. EMBO J. 1996; 15: 2442-2451Crossref PubMed Scopus (504) 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 (1749) Google Scholar, 31Rodriguez-Viciana P. Warne P.H. Vanhaesebroeck B. Waterfield M.D. Downward J. EMBO J. 1996; 15: 2442-2451Crossref PubMed Scopus (504) 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 (520) 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 (429) 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 (487) 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 (74) 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 (192) 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 (16) 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. Antibodies against p85 of PI3K and the anti-Tyr(P) mAb 4G10 were from Upstate Biotechnology Inc. (Lake Placid, NY) and the anti-HA epitope mAb 12CA5 was from Boehringer Mannheim (Indianapolis, IN). The hybridoma that produces the OKT3 anti-CD3ε mAb was from American Type Cell Collection. Phosphatidylinositol was from Upstate Biotechnology Inc. and TPCK-treated trypsin was from Worthington Biochemicals (NJ). Recombinant Abl protein-tyrosine kinase domain (Abl-K) was from New England Biolabs (Beverly, MA) and purified Lck from Upstate Biotechnology Inc. The protein-tyrosine kinase expression plasmids have been used before (42Tailor P. Gilman J. Williams S. Couture C. Mustelin T. J. Biol. Chem. 1997; 272: 5371-5376Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 43Williams S. Couture C. Gilman J. Jascur T. Deckert M. Altman A. Mustelin T. Eur. J. Biochem. 1997; 245: 84-90Crossref PubMed Scopus (45) Google Scholar), and we recently described the cloning of the HA-tagged p85 constructs (5Jascur T. Gilman J. Mustelin T. J. Biol. Chem. 1997; 272: 14483-14488Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). In addition to an N-terminal hemagglutinin (HA) tag, the constructs contain the following amino acids of bovine p85α: N-SH2 (329–439), C-SH2 (563–724), NC (329–439 + 563–724), NiC (329–724), p85ΔiSH2 (1–439 + 563–724), and wild-type p85 (1–724). GST fusion proteins containing the same fragments of p85 were ligated into the glutathioneS-transferase (GST) fusion vector pGEX-4T-2 (Pharmacia, Sweden) by standard procedures. Expression was induced and the fusion proteins were purified using glutathione-Sepharose beads (Pharmacia). Jurkat T leukemia cells, and their simian leukemia virus 40 large T antigen-expressing variant, J-TAg (kind gift from Dr. M. Karin), were kept at logarithmic growth in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, l-glutamine, and antibiotics. HL-60/Bcr-Abl cells were obtained by retroviral transfection of HL-60 promyelocytic leukemia cells with pSRαMSVp185bcr-abl tkneo as described elsewhere (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). COS-1 cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum. 15 × 106 J-TAg cells, HL-60, or HL-60/Bcr-Abl were transfected with a total of 10–20 μg of DNA by electroporation at 960 microfarads and 240 V. Typically, cells were transfected with 15 μg of HA-tagged p85 construct. Empty vector was added to control samples to make a constant amount of DNA in each sample. Cells were harvested two days after electroporation. COS-1 cells were transfected by lipofection with 10 μg of DNA and grown for 48 h prior to the experiments as described (42Tailor P. Gilman J. Williams S. Couture C. Mustelin T. J. Biol. Chem. 1997; 272: 5371-5376Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 44Couture C. Baier G. Altman A. Mustelin T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5301-5305Crossref PubMed Scopus (118) Google Scholar, 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, 46Couture C. Deckert M. Williams S. Russo F.O. Altman A. Mustelin T. J. Biol. Chem. 1996; 271: 24294-24299Abstract Full Text Full Text PDF PubMed Scopus (36) 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). These procedures were as reported before (5Jascur T. Gilman J. Mustelin T. J. Biol. Chem. 1997; 272: 14483-14488Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 42Tailor P. Gilman J. Williams S. Couture C. Mustelin T. J. Biol. Chem. 1997; 272: 5371-5376Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 46Couture C. Deckert M. Williams S. Russo F.O. Altman A. Mustelin T. J. Biol. Chem. 1996; 271: 24294-24299Abstract Full Text Full Text PDF PubMed Scopus (36) 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). All steps were carried out at 0–4 °C. Cells were lysed in 20 mm Tris/HCl, pH 7.5, 150 mmNaCl, 5 mm EDTA containing 1% Nonidet P-40, 1 mm Na3V04, 10 μg/ml aprotinin and leupeptin, 1 mm phenylmethylsulfonyl fluoride, and 100 μg/ml soybean trypsin inhibitor and clarified by centrifugation at 13,000 × g for 10 min. The clarified lysates were preabsorbed on agarose-conjugated goat anti-rabbit IgG or protein G-Sepharose. The lysates were then incubated with antibody for 2–4 h, followed by agarose-conjugated goat anti-rabbit IgG or protein G-Sepharose. Immune complexes were washed three times in lysis buffer, once in lysis buffer with 0.5 m NaCl, again in lysis buffer, and either suspended in SDS sample buffer or washed further for kinase assays. Proteins were separated by SDS-PAGE and transferred onto nitrocellulose filters. The antisera were used at 1:500–1:2000 dilution and the blots developed by the enhanced chemiluminescence technique (ECL kit, Amersham) according to the manufacturer's instructions. The GST fusion proteins were dissolved in kinase buffer (10 mmHEPES pH 7.5, 0.1% Triton X-100, 20 mm MgCl2, 1 mm dithiothreitol, and 0.1 mmNa3VO4). After addition of 2 mmcold ATP or 1 μm ATP and 10 μCi of [γ-32P]ATP and 1 μl of purified Lck or 100 units of Abl-K, the reaction was carried out for 30 min at 30 °C or overnight at room temperature (for higher stoichiometry of phosphorylation). The reaction was stopped by adding SDS sample buffer or by adding cold lysis buffer. In the latter case, the mixture was incubated with glutathione-Sepharose beads for 1 h, and the beads washed extensively before they were added to precleared cellular lysates. These experiments were done as before (48Tailor P. Jascur T. Williams S. von Willebrand M. Couture C. Mustelin T. Eur. J. Biochem. 1996; 237: 736-742Crossref PubMed Scopus (20) Google Scholar). Cell lysates were prepared as above, cleared by centrifugation, and preadsorbed to glutathione-Sepharose beads. After removal of the beads, the lysates were incubated on ice with 5 μg of GST fusion protein and glutathione-Sepharose beads for 2 h, which were subsequently washed five times with lysis buffer. The bound proteins were eluted in SDS sample buffer, resolved by SDS-PAGE, and analyzed by immunoblotting as above. The assay for lipid kinase activity of immunoprecipitated PI3K was as before (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, 49von Willebrand M. Baier G. Couture C. Burn P. Mustelin T. Eur. J. Immunol. 1994; 24: 234-238Crossref PubMed Scopus (41) Google Scholar). The assay mixture contained 20 mm Tris/HCl, pH 7.5, 100 mm NaCl, 0.5 mm EGTA, 10 μg of phosphatidylinositol (stock solution at 20 mg/ml in water), 20 mm MgCl2, and 10 μCi of [γ-32P]ATP. Reaction products were analyzed by ascending chromatography on Silica gel thin layer plates in CHCl3, CH3OH, 25% NH4OH/H2O (90:90:9:19) followed by autoradiography. GST-p85-NiC protein phosphorylated by Lck in the presence of [γ-32P]ATP was resolved on 10% SDS gels, transferred onto a nitrocellulose filter, exposed to film, and the correct band excised. The filter piece was blocked and digested with TPCK-treated trypsin as described in detail by Luo and co-workers (50Luo K. Hurley T.R. Sefton B.M. Oncogene. 1990; 5: 921-923PubMed Google Scholar). The resulting phosphopeptides were separated by electrophoresis on cellulose thin layer plates at pH 1.9 for 27 min followed by ascending chromatography in n-butanol/pyridine/acetic acid/water (75:50:15:60), and exposed to film for 35 h. Bcr-Abl induces transformation of fibroblasts and hematopoietic cells (51Clark S.S. McLaughlin J. Timmonis M. Pendergast A.M. Ben-Neriah Y. Dow L. Rovera G. Smith S.D.W. Science. 1988; 239: 775-777Crossref PubMed Scopus (214) Google Scholar, 52Daley G.Q. Van Etten R.A. Baltimore D. Science. 1990; 247: 824-830Crossref PubMed Scopus (1938) Google Scholar, 53Elefanty A.G. Hariharan I.K. Cory S. EMBO J. 1990; 9: 1069-1078Crossref PubMed Scopus (284) Google Scholar, 54Shtivelman E. Lifshitz B. Gale R.P. Roe B.A. Nature. 1985; 315: 550-551Crossref PubMed Scopus (1310) Google Scholar) and confers resistance to apoptosis (55McGahon A. Bissonnette R. Schmitt M. Cotter K.M. Green D.R. Cotter T.G. Blood. 1994; 83: 1179-1187Crossref PubMed Google Scholar). Cells expressing the Bcr-Abl tyrosine kinase contain elevated amounts of proteins phosphorylated on tyrosine residues. At least in some Bcr-Abl expressing cells, such as HL-60 cells transfected with Bcr-Abl (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), a fraction of the p85 subunit is included among these substrates for enhanced tyrosine phosphorylation. When PI3K was immunoprecipitated from parental HL-60 cells with antibodies against the p85 subunit and immunoblotted with anti-Tyr(P) mAbs, no phosphorylation of p85 could be detected (Fig. 1, lane 1), even if the cells were pretreated with 100 μm pervanadate to increase intracellular Tyr(P) content (lanes 2 and 3). In contrast, when anti-p85 immunoprecipitates prepared from HL-60 cells stably transfected with Bcr-Abl (HL-60/Bcr-Abl cells) were analyzed in parallel, a sharp, but not very prominent, band at 85 kDa (in addition to several other phosphoproteins) was seen (Fig. 1, lane 4). Reprobing of the same filter with anti-p85 revealed that all immunoprecipitates contained equal amounts of p85, which co-migrated precisely with the Tyr(P)-containing 85-kDa band in lane 4. This result was obtained in several independent experiments. To investigate whether this low level tyrosine phosphorylation of p85 in HL-60/Bcr-Abl cells has any effect on the catalytic activity of PI3K, we first immunoprecipitated PI3K from resting Jurkat T cells, and phosphorylated the immune complex in vitro with recombinant Abl-K, after which half of the sample was assayed for PI3K activity and the other half separated by SDS-PAGE for analysis by immunoblotting. Abl-K was found to strongly phosphorylate p85 (Fig. 2, left), but this phosphorylation had no effect on the catalytic activity of PI3K (Fig.2, right, lanes 1 and 2). An anti-p85 blot of the same filter confirmed that the samples contained the same amount of PI3K. A comparison of the amount of p85 protein versusTyr(P) content between Figs. 1 and 2 and several similar experiments, demonstrated that the stoichiometry of tyrosine phosphorylation was considerably higher in vitro than in vivo. Thus, the lower level of tyrosine phosphorylation of p85 in intact cells is even more unlikely to affect the catalytic activity of PI3K. To further study the effect of in vivo phosphorylation on PI3K activity, we assayed anti-Tyr(P) and anti-p85 immunoprecipitates from wild-type HL-60 and HL-60/Bcr-Abl cells for PI3K activity. The anti-Tyr(P) immunoprecipitates contained 5-fold higher PI3K activity from HL-60/Bcr-Abl cells than from parental HL-60 cells (Fig. 2, right, lanes 3 and 4). However, this increase was accompanied by a similar increase in the amount of p85 protein present in these immunoprecipitates (Fig. 2, center, lower panel, lanes 3 and 4). Anti-p85 immunoprecipitates from both cell types contained equal amounts of p85 protein (Fig. 2, center, lower panel, lanes 5 and 6), but an anti-Tyr(P) immunoblot of the same filter revealed that p85 from wild-type HL-60 cells was completely unphosphorylated while p85 and several co-immunoprecipitated proteins were heavily tyrosine phosphorylated from Bcr-Abl expressing cells (Fig. 2, center, lanes 5 and 6). Despite this difference, the PI3K activity in these immunoprecipitates was similar in both cases. In some experiments, there was a marginal increase in PI3K activity in anti-p85 immunoprecipitates from Bcr-Abl-expressing cells, probably due to the binding of phosphoproteins to the SH2 domains of p85, as seen in the anti-Tyr(P) blot. We conclude from these experiments that PI3K activity correlated closely with the amount of p85 protein regardless of its phosphorylation status. Apparently, tyrosine phosphorylation of p85 is not required for enzymatic activity and at the stoichiometry that it occurs in intact cells it does not directly affect the enzymatic activity of PI3K to any measurable degree. To identify in which part of the molecule p85 is tyrosine phosphorylated in HL-60/Bcr-Abl cells, we transiently transfected these cells with a set of HA-tagged truncated p85 constructs, which were subsequently immunoprecipitated and analyzed by anti-Tyr(P) immunoblotting. In these experiments (Fig. 3), wild-type p85 was phosphorylated on tyrosine, as was p85 lacking the inter-SH2 domain (p85ΔiSH2) and the two constructs containing both SH2 domains with or without the inter-SH2-region (NiC and NC, respectively). In contrast, the individual SH2 domain proteins (N-SH2 and C-SH2) did not contain detectable Tyr(P) even on very long exposures. All constructs were expressed at comparable levels as judged by anti-HA tag immunoblotting (Fig. 3, lower panel). Since the N-SH2 and C-SH2 proteins together contain the same amino acids as the NC protein, we conclude that the main phosphorylation occurs in one or both of these two domains, but that both are required for phosphorylation in intact HL-60/Bcr-Abl cells. This requirement correlates with the co-immunoprecipitation of several cellular phosphoproteins with all constructs having both SH2 domains (Fig. 3), but not the single SH2 domains. Thus, proper interaction of p85 with other proteins, perhaps including Bcr-Abl itself, is important for efficient phosphorylation. We have previously used a transient COS-1 cell transfection system to study potential interactions between protein-tyrosine kinases and the role of phosphorylation sites (42Tailor P. 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