Differential Signaling after β1 Integrin Ligation Is Mediated Through Binding of CRKL to p120 and p110

• DOI: 10.1074/jbc.272.22.14320
• Publication Date: 2002-07-26T14:57:46Z
• Authors: Martin Sattler; Ravi Salgia; Gautam Shrikhande; Shalini Verma; Naoki Uemura; Susan F. Law; Erica A. Golemis; James D. Griffin
• Keywords: Adapter molecule crk

CRKL is an SH2-SH3-SH3 adapter protein that a major substrate of the BCR/ABL oncogene. The function in normal cells unknown. In transformed by we have previously shown associated with two focal adhesion proteins, tensin and paxillin, suggesting could be involved integrin signaling. hematopoietic cell lines, MO7e H9, found rapidly associates tyrosine-phosphorylated proteins after cross-linking β1 integrins fibronectin or anti-β1 monoclonal antibodies. CRKL-binding megakaryocytic was identified as p120CBL, cellular homolog v-Cbl oncoprotein. However, lymphoid H9 line, p110HEF1. both cases, this binding mediated SH2 domain. Interestingly, although express p120CBLand p110HEF1, induces tyrosine phosphorylation p120CBL (but not p110HEF1) p110HEF1 p120CBL) cells. types, constitutively complexed to C3G, SOS, c-ABL through its SH3 domains, stoichiometry these complexes does change upon ligation. Thus, different types SH3-associated may form multimeric depending on whether shift association from contribute functional outcomes "outside-in" signaling Integrins play role movement apoptosis also act costimulatory molecules. receptors are α/β heterodimeric transmembrane mediate cell-cell cell-extracellular matrix interactions. Activation leads formation adhesions where cytoplasmic domains connected actin-containing cytoskeleton components, thereby providing link between extracellular environment intracellular elements. Tyrosine early event receptor stimulation believed initiate series events involving protein-protein interactions leading changes viability, proliferation, other functions various (1Schwartz M.A. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell Dev. Biol. 1995; 11: 549-599Crossref PubMed Scopus (1475) Google Scholar, 2Clark E.A. Brugge J.S. Science. 268: 233-239Crossref (2824) Scholar). One kinase localized activated ligation has been p125FAK (3Lipfert L. Haimovich B. Cobb B.S. Parsons J.T. J. 1992; 119: 905-912Crossref (633) This negative regulatory (4Ilic D. Furuta Y. Kanazawa S. Takeda N. Sobue K. Nakatsuji Nomura Fujimoto Okada M. Yamamoto T. Aizawa Nature. 377: 539-544Crossref (1602) Also, another non-receptor (related kinase) partially actin (5Avraham London R. Fu Ota Hiregowdara Li Jiang Pasztor L.M. White R.A. Groopman J.E. Avraham H. Chem. 270: 27742-27751Abstract Full Text PDF (326) 6Li J.Z. Rogers Raja Blood. 1996; 88: 417-428Crossref Recently, investigators begun identify ligands. For example, human B line Nalm-6 β2 megakaryoblastic (7Blake T.J. Shapiro Morse H.D. Langdon W.Y. Oncogene. 1991; 6: 653-657PubMed 8Manié S.N. Sattler Astier A. Phifer Canty Morimoto C. Druker B.J. Salgia Griffin J.D. Freedman A.S. Exp. Hematol. 1997; 25: 45-50PubMed many signal transduction pathways kinases, molecules key mediating transient We paxillin oncogenic (9Salgia Uemura Okuda J.-L. Pisick E. de Jong Heisterkamp Chen L.B. Groffen 29145-29150Abstract (129) 39-kDa one (10ten Hoeve Morris 1993; 8: 2469-2474PubMed high homology c-CRK-II belongs CRK family which includes v-CRK, c-CRK-II, c-CRK-I (11Mayer Hamaguchi Hanafusa 1988; 332: 272-275Crossref (581) 12Reichman C.T. Mayer Keshav Growth & Differ. 3: 451-460PubMed 13Matsuda Tanaka Nagata Kojima Kurata Shibuya Mol. Cell. 12: 3482-3489Crossref (250) specifically bind c-ABL, C3G (14Feller S.M. Knudsen EMBO 1994; 13: 2341-2351Crossref (334) 15Matsuda Hashimoto Muroya Hasegawa Nakamura Hattori 14: 5495-5500Crossref (184) 16Knudsen Feller 269: 32781-32787Abstract 17Tanaka Morishita Matuoka Takenawa Nagashima Matsuda Proc. Natl. Acad. Sci. U. 91: 3443-3447Crossref (367) 18Feller 10: 1465-1473PubMed 19Sattler Durstin Xu G. J.L. Prasad K.V. 839-846PubMed domain kinases (19Sattler 20de ten 21468-21471Abstract (128) Scholar), binds inducibly epidermal growth factor (21Fukazawa Miyake Band V. 271: 14554-14559Abstract (145) Scholar) T (22Reedquist K.A. Fukazawa Panchamoorthy Shoelson S.E. 8435-8442Abstract (166) study, examined involvement H9. resulted rapid single protein. Surprisingly, however, apparent molecular mass lines. cells, cases domain, while including did appear affected stimulation. These results indicate can interact more than pathway (obtained Dr. Steve Clark, Genetics Institute, Cambridge, MA) maintained Dulbecco's modified Eagle's medium (Mediatech, Washington, C.), 10 ng/ml granulocyte-macrophage colony-stimulating (Genetics Institute), 20% (v/v) fetal calf serum (PAA Laboratories Inc., Newport Beach, CA) at 37 °C 10% CO2. BCR/ABL-expressing MO7/p210 generated transfection pGD vector containing sequence for p210BCR/ABL cDNA George Daley, MIT, MA). studies, were washed phosphate-buffered saline (DPBS) 1The abbreviations used are: DPBSDulbecco's salineGSTglutathioneS-transferase deprived factors 20 h serum-free 1% (w/v) bovine albumin (Sigma). Jerome Ritz, Dana-Farber Cancer Institute) RPMI 1640 (Mediatech) Inc.) 5% Starved prepared washing DPBS 2 medium. glutathioneS-transferase first incubated 15 min ice antibodies against CD29/β1 (4B4, obtained Morimoto, CD3 (OKT3, Coulter Corp., Miami, FL), irrelevant antibody (3C11C8, anti-interferon-γ murine antibody) then stimulated using affinity-purified rabbit anti-mouse Ig (Dako Carpinteria, min. α subunit cross-linking, starved 30 α4 (8F2 B5G10 Hemler, α5 (2H6 A5-PUJ2 Hemler) described above. Either (washed three times starvation resuspended medium) (Life Technologies, same fashion. lysates (23Sattler Frank D.A. Kaushansky 23: 1040-1048PubMed Western blotting chemiluminescence technique performed Immunochemical detection blots utilized 4G10 (kindly provided Druker, Oregon Health Science University, Portland, OR). Polyclonal antisera (Santa Cruz Biotechnology, Santa Cruz, CA), Biotechnology), p110HEF1(HEF1 SB) (24Law S.F. Estojak Wang B.L. Mysliwiec Kruh Golemis 16: 3327-3337Crossref (221) mouse (AB-3 Oncogene Science, Manhasset, NY) (the elsewhere (25Uemura J-L. Leukemia. 376-385Crossref (36) only blotting) study. pGEX SH3-SH3 Groffen, Children's Hospital, UCLA, Los Angeles, CA. GST-fusion expressed Escherichia coli (DH-5α) isopropyl-1-thio-β-d-galactopyranoside induction isolated sonicated bacterial glutathione-Sepharose beads (Pharmacia Biotech according manufacturer's directions. (0.5 × 106 cells/sample) α1 (TS2/7), α2 (A2–2E10), α3 (A3–2F5), (B5G10), (A5-PUJ2), α6 (A6-ELE) (all anti-α (4B4), (3C11C8) once DPBS. Cells fluorescein isothiocyanate-conjugated goat (Southern Biotechnology Assoc., Birmingham, AL) additional subsequently twice before analysis Epics XL flow cytometer (Coulter Corp.) analysis. Using established techniques (26ten Kaartinen Fioretos Haataja Voncken J.W. Res. 54: 2563-2567PubMed far-Western brief, immunoprecipitated transferred SDS-PAGE Immobilon-P (polyvinylidene difluoride) membrane (Millipore) blocked nonfat dry milk 0.1% Tween saline, pH 7.4. specific direct vitro evaluated probing visualized combination anti-GST Biotechnology) horseradish peroxidase-coupled IgG chemiluminescence. To investigate potential looked coprecipitate CRKL, since following determine if investigated lines known differences initial experiments fibronectin, natural ligand some VLA-4 (α4β1) VLA-5 (α5β1), well induced prominent 120-kDa phosphoprotein (Fig. 1 A, left panel). therefore asked coprecipitating p120CBL. blot stripped, immunoblotting upper right when immunoprecipitation reversed (data shown). lower panel Fig.1 A demonstrates equal amounts loaded each lane. associate proteins. 110-kDa coprecipitated B, react Based presence multiple motifs (Tyr-X-X-Pro), possible coprecipitation CRKL. comparable loaded. demonstrate activation induce CRKL-p120CBL complex CRKL-p110HEF1 detect significant any time points tested 0 60 Since observed differential respectively, differentially Stimulation cross-link compared unstimulated A,left Mock include masses 145, 120, 95, 70, 40 kDa, whereas 110 95 kDa tyrosine-phosphorylated. treated pattern virtually identical integrin-induced pattern. phosphotyrosine immunoprecipitations but (Fig.2 middle contrast, recently cloned p130CAS-related addition, again selectively) respectively increased likely interaction 1). attributed expression B). addition antiserum recognized 95-kDa identity time. our preliminary data suggest it domain-containing p95EFS/SIN(27Ishino Ohba Sasaki 2331-2338PubMed 28Alexandropoulos Baltimore Genes 1341-1355Crossref failure tyrosine-phosphorylate due defects address issue, examined. immunoprecipitates expressing untransfected demonstrating response C). D). stimuli cross-linking. Tyrosine-phosphorylated selectively E). Cross-linking produced similar results; very small further expression. had α4, α5, integrins. Expression α1, α2, negligible F). Overall activate distinct above appears require adjacent sought mechanism segments precipitated unstimulated) (Fig.3 A). stripped reprobed GST-CRKL-SH3 GST-CRKL-SH2 3 constitutive SOS ABL-SH3 stimulation, GST-CRKL precipitations do indirect. examine Cellular β integrin-stimulated anti-p120CBL anti-CRKL antibody. Fig. C shows GST alone immunoprecipitations. Direct band probe blot. set 140 160 kDa; changed phosphotyrosine, no induced. linked SH3-binding 4demonstrates alter SOS. observe detectable biological effects vary widely cell, ranging proliferation apoptosis. When cross-linked such initiated. cytoskeleton: cytoskeletal actin, vinculin, talin, p125FAK, tensin, phosphorylation, p21RAS pathway. Overall, outside-in important part signals sent microenvironment influence behavior mechanisms understood. complexity studying system related family) heterogeneously coupled fact share ligand. interest how augment type. It would anticipated potentially Despite prediction, there few examples even fewer integrin-activated directly event. study (an domains) larger effort understand During studies MO7e, noted antibody, most prominently tyrosine-phosphophorylated (8Manié despite abundant (29Bazzoni Carlesso Hemler M.E. Clin. Invest. 98: 521-528Crossref (111) 30Nojima Tachibana Sato Schlossman Immunol. 161: 8-13Crossref (49) unexpected result made interesting finding p130CAS. Again, Like copies (phospho Tyr-X-X-Pro) 24Law combined observations integrin-mediated signaling, participate upstream molecule (p120CBL phosphorylated (probably phospho Tyr-X-X-Pro represent sites domains). provides possibility "switch" couple effects. contrast molecules, bound CRKII domain; others they guanine-exchange activity p21RAS; guanine exchange p21RAP1. specificity (31Gotoh Kitayama Noda Takai Kaibuchi Matsui Hatase O. Takahashi 15: 6746-6753Crossref (338) p21RAP1 regulate, least part, RAF kinase. similarity CDC25 (17Tanaka preferentially N-terminal (16Knudsen exact unknown, transcriptional (32Welch P.J. J.Y. 75: 779-790Abstract (371) possibly certain DNA damage (33Yuan Z.M. Huang Y.Y. Whang Sawyers Weichselbaum Kharbanda Kufe 382: 272-274Crossref (212) site terminus (34Van Etten Jackson P.K. Sanders M.C. Matsudaira P.T. Janmey P.A. 124: 325-340Crossref (245) either evidence demonstrated protooncoprotein (for Casitas B-lineage lymphoma) v-Cbl, oncoprotein CAS NS-1 retrovirus (35Langdon Hyland C.D. Grumont R.J. Virol. 1989; 63: 5420-5424Crossref 36Langdon Hartley Klinken S.P. Ruscetti S.K. 86: 1168-1172Crossref (290) pre-B lymphomas myelogenous leukemias mice (37Fredrickson T.N. Hoffman P.M. Inst. 1984; 72: 447-454PubMed Sli-1 Caenorhabditis elegans regulator Let-23 (38Yoon C.H. Lee J.H. Jongeward G.D. Sternberg P.W. 1102-1105Crossref (282) (39Donovan J.A. Wange R.L. Samelson L.E. 22921-22924Abstract (40Cory Lovering R.C. Hinshelwood Maccarthymorrogh Levinsky Kinnon Med. 182: 611-615Crossref (126) activation, FC-γ (41Marcilla Rivero-Lezcano O.M. Agarwal Robbins K.C. 9115-9120Abstract (125) 42Tanaka Neff Baron Levy J.B. 14347-14351Abstract (165) 43Galisteo M.L. Dikic I. Batzer A.G. Schlessinger 20242-20245Abstract (174) 44Meisner Conway B.R. Czech M.P. 3571-3578Crossref (213) 45Odai Iwamatsu Hanazono Mitani Yazaki Hirai 10800-10805Abstract mammalian known, several reported. active phosphatidylinositol 3-kinase antigen receptor-stimulated interacts GRB2, NCK, SRC LYN FYN 46Rivero-Lezcano Sameshima Marcilla 17363-17366Abstract derived HuT 78 expresses full-size c-CBL C-terminal truncation (47Blake 7: 757-762PubMed full-length p120CBLafter indicating intact. enhancer filamentation 1) tissue-specific during cloning genes morphological Saccharomyces cerevisiae. pseudohyphae shares 64% p130CAS similarly v-ABL docking structurally p130CAS, nucleus periphery organization cytoskeleton. 48Andoniou C.E. Thien 1981-1989PubMed consistent findings Our activating pathways. probably α4β1 (VLA4) α5β1 (VLA5)) Further, chains selective P110HEF1, respectively. consists four isoforms (A, C, D) differ their domains. isoform β1A ubiquitously substituted muscle β1D thus (49Belkin A.M. Zhidkova N.I. Balzac F. Altruda Tomatis Maier Tarone Koteliansky V.E. Burridge 132: 211-226Crossref (192) β1B minor coexpressed tissues (50Balzac Belkin Balabanov Y.V. Silengo 121: 171-178Crossref (85) might negatively regulate mobility (51Balzac Retta Albini Melchiorri Geuna 127: 557-565Crossref (66) β1C colocalize cause arrest inhibit synthesis transfected fibroblasts (52Meredith Jr., Takada Fornaro Languino L.R. Schwartz 1570-1572Crossref (117) CRKL-related Nalm-6, 2M. Sattler, unpublished data. suggests mutually exclusive. process currently investigating possibility.