Roles of Gab1 and SHP2 in Paxillin Tyrosine Dephosphorylation and Src Activation in Response to Epidermal Growth Factor

• DOI: 10.1074/jbc.m312575200
• Publication Date: 2004-02-20T22:08:01Z
• Authors: Yuan Ren; Songshu Meng; Lin Mei; Z. Joe Zhao; Richard Jove; Jie Wu
• Keywords: Dephosphorylation; Paxillin

Epidermal growth factor (EGF) induces paxillin tyrosine dephosphorylation and Src activation, but the signaling pathways that mediate these responses were largely undefined. We found Gab1, a docking protein for SHP2 protein-tyrosine phosphatase in EGF-stimulated cells, was associated with paxillin. dephosphorylated caused dissociation of Csk, negative regulator Src, from had no effect on paxillin-Src association. A lower level Tyr-530 phosphorylation detected paxillin-associated cells. Expression an binding defective mutant Gab1 (Gab1FF) or catalytically inactive (SHP2DN) prevented activation induced by EGF. Importantly, Gab1FF blocked paxillin-SHP2 complex formation, dephosphorylation, Erk cell migration Inhibition kinase activity abrogated migration. Together, results reveal recruits to dephosphorylate paxillin, leading Csk is effector involved 1The abbreviations used are: EGF, epidermal factor; PTPase, phosphatase; Dox, doxycycline; Tyr(P), phosphotyrosine; C-terminal kinase; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; SH2, Src-homology-2; Erk, extracellular signal-regulated GST, glutathione S-transferase; Mek, mitogen-activated kinase/extracellular kinase. receptor are known have functional cross-talk (1Kassenbrock C.K. Hunter S. Garl P. Johnson G.L. Anderson S.M. J. Biol. Chem. 2002; 277: 24967-24975Abstract Full Text PDF PubMed Scopus (98) Google Scholar, 2Belsches-Jablonski A.P. Biscardi J.S. Peavy D.R. Tice D.A. Romney Parsons S.J. Oncogene. 2001; 20: 1465-1475Crossref (162) 3Olayioye M.A. Badache A. Daly J.M. Hynes N.E. Exp. Cell Res. 267: 81-87Crossref (47) 4Olayioye Beuvink I. Horsch K. N. 1999; 274: 17209-17218Abstract (312) 5Broome T. 1996; 271: 16798-16806Abstract (117) 6Erpel Alonso G. 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Munoz-Antonia Mei 275: 13842-13848Abstract (118) 35Yart Laffargue Mayeux Chretien Peres Tonks Payrastre Chap Raynal 276: 8856-8864Abstract (126) pleckstrin-homology domain-containing becomes tyrosine-phosphorylated factors cytokines (36Gu Neel 122-130Abstract (322) Gab1-SHP2 association necessary (37Cunnick Doupnik 24380-24387Abstract (134) 38Cai Khavari P.A. 159: 103-112Crossref (75) we recently fusion (28Cunnick provide evidence here recruiting Furthermore, our show Primary Antibodies—Polyclonal antibodies SHP2, total Erk1/2, FLAG tag Santa Cruz Biotechnology. Mouse monoclonal (RC20H) BD Biosciences. Polyclonal either Upstate biotechnology (rabbit) Biotechnology (goat). mouse antibody (clone 327) Oncogene Sciences. against phosphorylated Signaling. active Erk1/2 Promega. DNA Constructs—A single-vector doxycycline (Dox)-inducible system, pSTAR (39Zeng Q. Tan Y.H. Hong Anal. 259: 187-194Crossref (22) 40Dorsey Cunnick Mane Blood. 99: 1388-1397Crossref (55) Scholar), provided Dr. Wanjin Hong. (Gab1FF), Tyr-627 Tyr-659 Phe, Human cDNA (41Ahmad Banville Fisher E.H. Shen S-H. 90: 2197-2201Crossref (196) 42Zhao Wright J.H. Diltz C.D. Krebs E.G. 270: 11765-11769Abstract (82) Scholar) subcloned into pcDNA3.1-FLAG vector (34Cunnick SHP2DN double mutations site Cys residue (Cys-459) Ser general acid Asp (Asp-425) Ala made sequential PCR using primers confer mutations. verified analysis. Coding sequences FLAG-tagged SHP2DN, respectively, generate pSTAR-Gab1FF pSTAR-SHP2DN. pGEX-GST-Gab1CT cloning coding (amino acids 592-694) BamHI EcoRI pGEX2T. Culture Establishment Dox-inducible Lines—MDA-MB-468 breast A431 epidermoid obtained American Type Collection maintained medium (DMEM) containing 10% serum (FCS) 37 °C/5% CO2. To establish lines MDA-MB-468 transfected pSTAR-Gab1FF, pSTAR-SHP2DN, linearized PvuI digestion. Transfected cultured tetracycline free 10%FCS (Clontech) presence 0.4 mg/ml G418. G418-resistant colonies empty pooled (MpSTAR) control. Individual colonies/lines pSTAR-Gab1FF- pSTAR-SHP2DN-transfected (24-36 lines/each) screened immunoblot analysis lysates. Immunoprecipitation Immunoblot Analysis—Near-confluent serum-starved 0.1% BSA 18 h then stimulated mock-treated indicated figure legends. Cells lysed Buffer (50 mm Tris-HCl, pH 7.5, 150 NaCl, EDTA, EGTA, 25 NaF, 5 sodium pyrophosphate, Na3VO4, 2 μg/ml aprotinin, leupeptin, 100 phenylmethylsulfonyl fluoride, dithiothreitol, 20 mmp-nitrophenyl phosphate, 1% Triton X-100). For experiments involving analysis, 0.5 μm inhibitor, PD180970 (43Dorsey Lanehart Huang Kraker Bhalla Leukemia (Baltimore). 1589-1595Crossref (41) included lysis buffer. Precleared supernatants incubated specific Immune complexes collected G-agarose A-agarose. Immunoprecipitated separated 8% SDS-polyacrylamine gels transferred nitrocellulose membranes. performed essentially described previously (44Cunnick Standley Turkson Fry D.W. 273: 14468-14475Abstract (144) Tyrosine Dephosphorylation Assay—A GST (45Mei Doherty Huganir R.L. 269: 12254-12262Abstract expressed Escherichia coli BL21 affinity-purified glutathione-agarose. measured hydrolysis p-nitrophenyl phosphate 80-μl reaction mixtures 50 Hepes, 7.0, 10 various amounts 30 °C min. stopped 80 μl m NaOH. absorbance 405 nm (A405) determined 96-well plates. One unit defined amount produces A405 this assay. assay, immunoprecipitated confluent h. immunoprecipitates different SHP2-PTPase dithiothreitol volume After reaction, washed ml 3 times resolved SDS-polyacrylamide gel electrophoresis. examined immunoblotting anti-phosphotyrosine (RC20H). Kinase Assay—Cells B (25 5% glycerol, deoxycholate, SDS, X-100, Na3VO4). lysate anti-Src 327. immune assay 15 min 30-μl mixture 7.4, MgCl2, μg GST-Gab1CT, [γ-32P]ATP (5,000 cpm/pmol). terminated 4× SDS-gel loading buffer heat denaturation. resolving gel, processed autoradiography. decay radioactivity, presented immunoblotting. Migration Assay—Transwell (46Krueger Keshamouni V.G. Atanaskova Reddy 4209-4218Crossref (146) 47Ren Anticancer 3231-3236PubMed Transwell culture insert polycarbonate coated overnight rat tail type I collagen (10 μg/ml) 4 air-dried. without Dox 48 detached plates trypsin digestion, number determined. FCS (0.6 ml) placed chamber 24-well (5 × 104) 0.2 upper triplicate ng/ml absence PP1 μm), (0.5 U0126 μm). incubation °C, CO2 8 h, member surface mechanically removed cotton swab. migrated side fixed stained HEMA3 reagents (Fisher) enumerated under microscope three randomly chosen 1.2 0.8-mm fields. Stimulates Paxillin—To verify others after stimulation. antibody. Fig. shows high levels detectable within 2.5 maximal (90%) occurred stimulation (Fig. 1A). lesser (40%) more transient, consistent, decrease treatment 1B). Control immunoblots (Fig B, bottom panels) equal present samples. determine change affects paxillin-Csk association, analyzed co-immunoprecipitation before treatment. As 1C, decreased 1C). confirm question about transmitted Binds Paxillin, Is Recruited Stimulation—During study Gab1-interacting novel Gab1-binding (data not shown). illustrated 2A (also 4C), Gab1-paxillin resting further increased immunoprecipitates.Fig. 4Gab1-SHP2 SHP2-paxillin formation dephosphorylation.A, pSTAR-Gab1FF. Gab1FF. Among 24 analyzed, lines, MG12 MG24, showed pSTAR-transfected (a pool colonies, MpSTAR), MG12, MG24 (+) (-) (2 days, treated ng/ml, min). lysates (IB) (pY), indicated. α-, anti-. C, anti-FLAG immunoprecipitate Immunoprecipitates (IP) (top panel), (middle (bottom panel). arrowhead middle panel indicates position corresponding where would if membrane.View Large Image Figure ViewerDownload Hi-res image Download (PPT) minimal residual markedly 2A). correlates recruitment 2B). raise recruit Dephosphorylates Vitro—To evaluate be tested catalytic (SHP2-PTPase). 3A GST-SHP2 resulted concentration-dependent manner. control apparent vitro 3A, inset). result demonstrates PTPase. next loss Src. incubation, remove dissociated complex. c-Src, (control). 3B, suggesting dependent phosphorylation. contrast, did affect independent Thus, lead separation kinase, Necessary Recruitment Response EGF—Data do prove mediated Gab1. twenty-four derived pSTAR-Gab1FF-transfected (MG12 MG24) 4A). expression. vector-transfected MpSTAR) serum-starved, mock-treated,