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

• DOI: 10.1074/jbc.273.7.3994
• Publication Date: 2002-07-26T14:47:21Z
• Authors: Maria von Willebrand; Scott Williams; Manju Saxena; Jennifer Gilman; Pankaj Tailor; Thomas Jascur; Gustavo P. Amaran...; Douglas R. Green; Tomas Mustelin
• Keywords: 0301 basic medicine; Fusion Proteins, bcr-abl; Transfection; Gene Expression Regulation, Neoplastic; src Homology Domains; Phosphatidylinositol 3-Kinases; 03 medical and health sciences; Lymphocyte Specific Protein Tyrosine Kinase p56(lck); Tumor Cells, Cultured; Humans; Tyrosine; Phosphorylation; Phosphotyrosine

In cells expressing the oncogenic Bcr-Abl tyrosine kinase, regulatory p85 subunit of phosphatidylinositol 3-kinase is phosphorylated on residues. We report that this phosphorylation event readily catalyzed by Abl and Lck protein-tyrosine kinases in vitro, or a catalytically activated Lck-Y505F co-transfected COS cells, endogenous transfected Jurkat T upon triggering their cell antigen receptor. Using these systems, we have mapped major site to Tyr-688 C-terminal SH2 domain p85. Tyrosine vitro vivo was not associated with detectable change enzymatic activity heterodimer, but correlated strong reduction binding some, all, phosphoproteins domains This provides an additional candidate list regulated may explain why association some cellular ligands transient lower stoichiometry than anticipated. Phosphatidylinositol 3-kinases (PI3Ks) 1The abbreviations used are: PI3K, 3-kinase; HA, hemagglutinin; Tyr(P), phosphotyrosine; SH2, Src homology 2 region; SH3, 3 mAb, monoclonal antibody; TPCK,l-1-tosylamido-2-phenylethyl chloromethyl ketone; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis. are family enzymes involved multiplicity functions, including proliferation 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) 3Kaplan D.R. Whitman M. Schaffhausen B. Pallas D.C. White Roberts T.M. Cell. 1987; 50: 1021-1029Abstract Full Text PDF (487) Scholar), lymphocyte activation (4Gold M.R. Chan V.W.-F. Turck C. DeFranco A.L. J. Immunol. 1992; 148: 2012-2022PubMed 5Jascur T. Gilman Mustelin Biol. Chem. 1997; 272: 14483-14488Abstract (55) 6von Willebrand Jascur Bonnefoy-Bérard N. Yano H. Altman A. Matsuda Y. Eur. 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Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa 72: 767-778Abstract (2425) or, cases, Tyr(P)-X-X-leucine (20Exley Varticovski Peter Sancho Terhorst 1994; 15140-15146Abstract 21Zenner Vorherr Burn 63: 94-103Crossref (40) physiologically relevant include tyrosine-phosphorylated CD28 (22Pages Ragueneau Rottapel Truneh Nunes Imbert Olive Nature. 369: 327-329Crossref (351) subunits receptor 23Carrera A.C. Rodriguez-Borlado Martinez-Alonso Merida 19435-19440Abstract CD5 (24Dennehy K.M. Broszeit Garnett Durrheim Spruyt L.L. Beyers 27: 679-686Crossref (42) CD7 (25Lee D.M. Patel D.D. Pendergast A.M. Haynes B.F. Int. 8: 1195-1203Crossref (24) c-Cbl proto-oncogene product (26Meisner Conway B.R. Hartley Czech M.P. 15: 3571-3578Crossref (213) addition causing subcellular relocation cause allosteric p110 subunit, which bound between (15Hu 27Dhand Hara Bax Yonezawa Kasuga 511-521Crossref (300) 28Holt K.H. Olson Moye-Rowley W.S. Pessin 42-49Crossref 29Klippel Hu Q. 5560-5566Crossref (89) Several modes regulation been demonstrated, it likely they act concert regulate production 3-phosphorylated inositol phospholipids response variety stimuli. interacts GTP-bound Ras adjacent p85-binding NH2terminus (30Rodriguez-Viciana Warne P.H. Downward 370: 527-532Crossref (1749) 31Rodriguez-Viciana 2442-2451Crossref (504) Active enhances intact data indicate acts downstream (32Hu Klippel Muslin A.J. Fantl W.J. 100-102Crossref (520) isoforms shown undergo both serine threonine (33Reif 10780-10788Abstract 34Dhand Roche N.F. Truong Vicendo 522-533Crossref (429) has occur many different such platelet-derived insulin (35Hayashi Nishioka Kamohara Kanai Ishii Fukui Shibasaki Takenawa Kido Katsunuma Ebina 7107-7117Abstract B ligation interleukin-2 (36Karnitz L.M. Sutor S.L. Abraham R.T. Exp. Med. 179: 1799-1808Crossref (74) transformed fusion kinase (37Amarante-Mendes G.P. W.K. Green Cell Death Differ. 4: 541-555Crossref (23) 38Gotoh Miyazawa Ohyashiki Toyama Leukemia. 115-120PubMed 39Skorski Kanakaraj Nieborowska-Skorska Ratajczak M.Z. Wen S.-C. Zon Gewirtz Perussia Calabretta Blood. 86: 726-736Crossref 40Varticovski Daley G.Q. Jackson Baltimore 11: 1107-1113Crossref (192) sites tyrosines 368, 508, 607 insulin-stimulated physiological function remained unknown. seems be required PI3K. Instead, reported correlate dissociation from (41Zhang W. Johnson J.D. Rutter Proc. Natl. Acad. Sci. U. 90: 11317-11321Crossref (16) studied hematopoietic occurs at least domain. does detectably affect per se, causes properties modify cells. Antibodies against anti-Tyr(P) mAb 4G10 were Upstate Biotechnology Inc. (Lake Placid, NY) anti-HA epitope 12CA5 Boehringer Mannheim (Indianapolis, IN). hybridoma produces OKT3 anti-CD3ε American Type Collection. TPCK-treated trypsin Worthington Biochemicals (NJ). Recombinant (Abl-K) New England Biolabs (Beverly, MA) purified expression plasmids before (42Tailor Couture 5371-5376Abstract (96) 43Williams Deckert 245: 84-90Crossref (45) recently described cloning HA-tagged constructs (5Jascur N-terminal hemagglutinin (HA) tag, contain following amino acids bovine p85α: N-SH2 (329–439), C-SH2 (563–724), NC (329–439 + 563–724), NiC (329–724), p85ΔiSH2 (1–439 wild-type (1–724). GST containing same fragments ligated into glutathioneS-transferase (GST) vector pGEX-4T-2 (Pharmacia, Sweden) standard procedures. Expression induced using glutathione-Sepharose beads (Pharmacia). leukemia simian virus 40 large antigen-expressing variant, J-TAg (kind gift Dr. Karin), kept logarithmic RPMI 1640 supplemented 10% heat-inactivated fetal calf serum, l-glutamine, antibiotics. HL-60/Bcr-Abl obtained retroviral transfection HL-60 promyelocytic pSRαMSVp185bcr-abl tkneo elsewhere COS-1 maintained Dulbecco's modified Eagle's medium serum. 15 × 106 HL-60, total 10–20 μg electroporation 960 microfarads 240 V. Typically, construct. Empty added control samples make constant amount each sample. Cells harvested days after electroporation. lipofection 10 grown 48 h prior experiments 44Couture Baier 91: 5301-5305Crossref (118) 45Couture Oetken Telford Marie-Cardine Baier-Bitterlich 5249-5258Crossref 46Couture Russo F.O. 271: 24294-24299Abstract (36) 47Couture Songyang Tailor 24880-24884Abstract (72) These procedures 42Tailor All steps carried out 0–4 °C. lysed 20 mm Tris/HCl, pH 7.5, 150 mmNaCl, 5 EDTA 1% Nonidet P-40, 1 Na3V04, μg/ml aprotinin leupeptin, phenylmethylsulfonyl fluoride, 100 soybean inhibitor clarified centrifugation 13,000 g min. lysates preabsorbed agarose-conjugated goat anti-rabbit IgG G-Sepharose. then incubated antibody 2–4 h, followed Immune complexes washed three times lysis buffer, once buffer 0.5 m NaCl, again either suspended SDS sample further assays. Proteins separated SDS-PAGE transferred onto nitrocellulose filters. antisera 1:500–1:2000 dilution blots developed enhanced chemiluminescence technique (ECL kit, Amersham) according manufacturer's instructions. dissolved (10 mmHEPES 0.1% Triton X-100, MgCl2, dithiothreitol, 0.1 mmNa3VO4). After mmcold ATP μm μCi [γ-32P]ATP μl units Abl-K, reaction 30 min °C overnight room temperature (for higher phosphorylation). stopped adding cold buffer. latter case, mixture extensively precleared lysates. done (48Tailor von 237: 736-742Crossref (20) prepared above, cleared centrifugation, preadsorbed beads. removal beads, ice subsequently five eluted resolved SDS-PAGE, analyzed immunoblotting above. assay lipid immunoprecipitated 49von 24: 234-238Crossref (41) contained EGTA, (stock solution mg/ml water), [γ-32P]ATP. Reaction products ascending chromatography Silica thin layer plates CHCl3, CH3OH, 25% NH4OH/H2O (90:90:9:19) autoradiography. GST-p85-NiC presence gels, filter, exposed film, correct band excised. filter piece blocked digested detail Luo co-workers (50Luo Hurley T.R. Sefton B.M. Oncogene. 1990; 5: 921-923PubMed resulting phosphopeptides electrophoresis cellulose 1.9 27 n-butanol/pyridine/acetic acid/water (75:50:15:60), film 35 h. induces fibroblasts (51Clark S.S. McLaughlin Timmonis Ben-Neriah Dow Rovera S.D.W. 1988; 239: 775-777Crossref (214) 52Daley Van Etten 247: 824-830Crossref (1938) 53Elefanty A.G. Hariharan I.K. Cory 9: 1069-1078Crossref (284) 54Shtivelman Lifshitz Gale R.P. Roe B.A. 1985; 315: 550-551Crossref (1310) confers resistance apoptosis (55McGahon Bissonnette Schmitt Cotter T.G. 83: 1179-1187Crossref elevated amounts At fraction included among substrates phosphorylation. When parental antibodies immunoblotted mAbs, no could detected (Fig. 1, lane 1), even if pretreated pervanadate increase Tyr(P) content (lanes 3). contrast, when anti-p85 immunoprecipitates stably (HL-60/Bcr-Abl cells) parallel, sharp, very prominent, 85 kDa (in several phosphoproteins) seen 4). Reprobing revealed all equal p85, co-migrated precisely Tyr(P)-containing 4. result independent experiments. To investigate whether low level any effect first resting immune complex recombinant half assayed analysis immunoblotting. Abl-K found strongly phosphorylate 2, left), had (Fig.2, right, lanes 2). An blot confirmed A comparison versusTyr(P) Figs. similar experiments, demonstrated considerably vivo. Thus, more unlikely study activity, activity. 5-fold However, accompanied present center, panel, Anti-p85 types 6), immunoblot completely unphosphorylated while co-immunoprecipitated heavily 6). Despite difference, cases. there marginal Bcr-Abl-expressing probably due blot. conclude closely regardless status. Apparently, directly measurable degree. identify part molecule transiently set truncated constructs, 3), tyrosine, lacking inter-SH2 (p85ΔiSH2) without inter-SH2-region (NiC NC, respectively). individual (N-SH2 C-SH2) did long exposures. expressed comparable levels judged tag 3, panel). Since together protein, main requirement correlates co-immunoprecipitation single domains. proper interaction proteins, perhaps itself, important efficient previously system potential role Scholar,44Couture full-length versions (56Amrein K.E. 85: 4247-4251Crossref (189) 57Marth C.S. Ziegler S.F. Tinker Overell R.W. Krebs E.G. Perlmutter R.M. 540-550Crossref P