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• W2094366341 abstract "The adapter molecule p130Cas (Cas) plays a role in cellular processes such as proliferation, survival, cell adhesion, and migration. The ability of Cas to promote migration has been shown to be dependent upon its carboxyl terminus, which contains a bipartite binding site for the protein tyrosine kinase c-Src (Src). The association between Src and Cas enhances Src kinase activity, and like Cas, Src plays an important role in cell proliferation and migration. In this study, we show that Src and Cas function cooperatively to promote cell migration in a manner that depends upon kinase-active Src. Another carboxyl-terminal binding partner of Cas, AND-34/BCAR3 (AND-34), functions synergistically with Cas to enhance Src activation and cell migration. The carboxyl-terminal guanine nucleotide exchange factor domain of AND-34, as well as the activity of its putative target Rap1, contribute to these events. A mechanism through which AND-34 may regulate Cas-dependent cell migration is suggested by the finding that Cas becomes redistributed from focal adhesions to lamellipodia located at the leading edge of AND-34 overexpressing cells. These data thus provide insight into how Cas and AND-34 may function together to stimulate Src signaling pathways and promote cell migration. The adapter molecule p130Cas (Cas) plays a role in cellular processes such as proliferation, survival, cell adhesion, and migration. The ability of Cas to promote migration has been shown to be dependent upon its carboxyl terminus, which contains a bipartite binding site for the protein tyrosine kinase c-Src (Src). The association between Src and Cas enhances Src kinase activity, and like Cas, Src plays an important role in cell proliferation and migration. In this study, we show that Src and Cas function cooperatively to promote cell migration in a manner that depends upon kinase-active Src. Another carboxyl-terminal binding partner of Cas, AND-34/BCAR3 (AND-34), functions synergistically with Cas to enhance Src activation and cell migration. The carboxyl-terminal guanine nucleotide exchange factor domain of AND-34, as well as the activity of its putative target Rap1, contribute to these events. A mechanism through which AND-34 may regulate Cas-dependent cell migration is suggested by the finding that Cas becomes redistributed from focal adhesions to lamellipodia located at the leading edge of AND-34 overexpressing cells. These data thus provide insight into how Cas and AND-34 may function together to stimulate Src signaling pathways and promote cell migration. p130Cas (Cas) has been implicated in a wide range of cellular processes, including proliferation, survival, cell adhesion and migration, oncogenic transformation, and potentially metastasis (for review see Refs. 1O'Neill G.M. Fashena S.J. Golemis E.A. Trends Cell Biol. 2000; 10: 111-119Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar and 2Bouton A.H. Riggins R.B. Bruce-Staskal P.J. Oncogene. 2001; 20: 6448-6458Crossref PubMed Scopus (167) Google Scholar). Cas was initially identified as a phosphotyrosine (pTyr) 1The abbreviations used are: pTyr, phosphotyrosine; ECM, extracellular matrix; FN, fibronectin; PTK, protein tyrosine kinase; SH, Src homology; NSP, novel SH2-containing protein; GEF, guanine nucleotide exchange factor; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; mAb, monoclonal antibody; TR, Texas Red; mCT, minimal carboxyl terminus; YFP, yellow fluorescent protein; WT, wild-type; KD, kinase-dead; dHLH, divergent helix-loop-helix; IP, immunoprecipitation; IB, immunoblotting.1The abbreviations used are: pTyr, phosphotyrosine; ECM, extracellular matrix; FN, fibronectin; PTK, protein tyrosine kinase; SH, Src homology; NSP, novel SH2-containing protein; GEF, guanine nucleotide exchange factor; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; mAb, monoclonal antibody; TR, Texas Red; mCT, minimal carboxyl terminus; YFP, yellow fluorescent protein; WT, wild-type; KD, kinase-dead; dHLH, divergent helix-loop-helix; IP, immunoprecipitation; IB, immunoblotting.-containing protein in cells transformed by the v-src and v-crk oncogenes (3Reynolds A.B. Kanner S.B. Wang H.C. Parsons J.T. Mol. Cell. Biol. 1989; 9: 3951-3958Crossref PubMed Scopus (135) Google Scholar, 4Matsuda M. Mayer B.J. Fukui Y. Hanafusa H. Science. 1990; 248: 1537-1539Crossref PubMed Scopus (285) Google Scholar). It was subsequently shown to be comprised of multiple protein-protein interaction modules, consistent with its predicted role as an adapter molecule (5Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Mano H. Yazaki Y. Hirai H. EMBO J. 1994; 13: 3748-3756Crossref PubMed Scopus (593) Google Scholar). Cas is a component of focal adhesions, which are molecular complexes that form at sites of cell attachment to the extracellular matrix (ECM). The presence of Cas in focal adhesions is consistent with a role for Cas in regulating the actin cytoskeleton and cell migration (6Polte T.R. Hanks S.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10678-10682Crossref PubMed Scopus (387) Google Scholar, 7Petch L.A. Bockholt S.M. Bouton A. Parsons J.T. Burridge K. J. Cell Sci. 1995; 108: 1371-1379Crossref PubMed Google Scholar, 8Harte M.T. Hildebrand J.D. Burnham M.R. Bouton A.H. Parsons J.T. J. Biol. Chem. 1996; 271: 13649-13655Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 9Harte M.T. Macklem M. Weidow C.L. Parsons J.T. Bouton A.H. Biochim. Biophys. Acta. 2000; 1499: 34-48Crossref PubMed Scopus (25) Google Scholar). One of the major initiating events for cell migration is engagement of integrin receptors by ECM ligands. In this way, integrins directly link ECM components to the actin cytoskeleton (for review see Ref. 10Miranti C.K. Brugge J.S. Nat. Cell Biol. 2002; 4: 83-90Crossref PubMed Scopus (691) Google Scholar). Integrins also recruit cellular proteins, including kinases and adapter molecules such as Cas, to newly formed focal complexes and mature focal adhesions (for review see Ref. 11Critchley D.R. Curr. Opin. Cell Biol. 2000; 12: 133-139Crossref PubMed Scopus (494) Google Scholar). Ultimately, many of the signaling molecules that participate in cell adhesion and migration impact the Rho family of small GTPases, which modify the actin cytoskeleton by regulating actin polymerization and the molecular composition of adhesion sites (12Ridley A.J. J. Cell Sci. 2001; 114: 2713-2722Crossref PubMed Google Scholar). Elucidating the functions of focal adhesion proteins that participate in the regulation of these small GTPases is critical for understanding mechanisms that govern cell motility. Several pieces of evidence suggest that Cas plays a critical role in cell migration. Consistent with its presence in focal adhesions, Cas becomes tyrosine phosphorylated in response to integrin ligation by numerous ECM components, including fibronectin (FN), collagen, laminin, and vitronectin (8Harte M.T. Hildebrand J.D. Burnham M.R. Bouton A.H. Parsons J.T. J. Biol. Chem. 1996; 271: 13649-13655Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 13Nojima Y. Morino N. Mimura T. Hamasaki K. Furuya H. Sakai R. Sato T. Tachibana K. Morimoto C. Yazaki Y. et al.J. Biol. Chem. 1995; 270: 15398-15402Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 14Vuori K. Ruoslahti E. J. Biol. Chem. 1995; 270: 22259-22262Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 15Polte T.R. Hanks S.K. J. Biol. Chem. 1997; 272: 5501-5509Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). In addition, overexpression of Cas has been shown to enhance migration of FG pancreatic carcinoma cells and COS cells (16Klemke R.L. Leng J. Molander R. Brooks P.C. Vuori K. Cheresh D.A. J. Cell Biol. 1998; 140: 961-972Crossref PubMed Scopus (590) Google Scholar). Conversely, Cas–/– mouse embryo fibroblasts do not migrate as efficiently as their wild-type counterparts unless they are engineered to express ectopic Cas (17Honda H. Nakamoto T. Sakai R. Hirai H. Biochem. Biophys. Res. Commun. 1999; 262: 25-30Crossref PubMed Scopus (102) Google Scholar, 18Cho S.Y. Klemke R.L. J. Cell Biol. 2000; 149: 223-236Crossref PubMed Scopus (240) Google Scholar). Recent studies (19Huang J. Hamasaki H. Nakamoto T. Honda H. Hirai H. Saito M. Takato T. Sakai R. J. Biol. Chem. 2002; 277: 27265-27272Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) have shown that the carboxyl terminus of Cas is required to fully rescue this defect in cell migration. The Cas carboxyl terminus contains a bipartite binding site for the nonreceptor protein tyrosine kinase (PTK) c-Src (Src) (20Nakamoto T. Sakai R. Ozawa K. Yazaki Y. Hirai H. J. Biol. Chem. 1996; 271: 8959-8965Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). This PTK is not only a mediator of cell proliferation and survival (for review see Refs. 21Bjorge J.D. Jakymiw A. Fujita D.J. Oncogene. 2000; 19: 5620-5635Crossref PubMed Scopus (338) Google Scholar and 22Frame M.C. Biochim. Biophys. Acta. 2002; 1602: 114-130PubMed Google Scholar), but it also plays an essential role in cell migration. Fibroblasts derived from mice lacking three Src family members (Src, Yes, and Fyn) are deficient in migration, and this defect can be rescued by re-expression of Src alone (23Klinghoffer R.A. Sachsenmaier C. Cooper J.A. Soriano P. EMBO J. 1999; 18: 2459-2471Crossref PubMed Scopus (646) Google Scholar). In addition, the kinase activity of Src has been shown to be essential for integrin-mediated cell adhesion, spreading, and migration (24Cary L.A. Klinghoffer R.A. Sachsenmaier C. Cooper J.A. Mol. Cell. Biol. 2002; 22: 2427-2440Crossref PubMed Scopus (129) Google Scholar, 25Li L. Okura M. Imamoto A. Mol. Cell. Biol. 2002; 22: 1203-1217Crossref PubMed Scopus (78) Google Scholar). Evidence suggests that Src family kinases mediate integrin-dependent Cas phosphorylation (23Klinghoffer R.A. Sachsenmaier C. Cooper J.A. Soriano P. EMBO J. 1999; 18: 2459-2471Crossref PubMed Scopus (646) Google Scholar, 26Hamasaki K. Mimura T. Morino N. Furuya H. Nakamoto T. Aizawa S.I. Morimoto C. Yazaki Y. Hirai H. Nojima Y. Biochem. Biophys. Res. Commun. 1996; 222: 338-343Crossref PubMed Scopus (116) Google Scholar, 27Vuori K. Hirai H. Aizawa S. Ruoslahti E. Mol. Cell. Biol. 1996; 16: 2606-2613Crossref PubMed Google Scholar, 28Schlaepfer D.D. Broome M.A. Hunter T. Mol. Cell. Biol. 1997; 17: 1702-1713Crossref PubMed Scopus (399) Google Scholar) and that Src is the primary PTK responsible for phosphorylation of Cas (29Ruest P.J. Shin N.Y. Polte T.R. Zhang X. Hanks S.K. Mol. Cell. Biol. 2001; 21: 7641-7652Crossref PubMed Scopus (131) Google Scholar). Interestingly, Cas is not only a substrate of Src, but also a potent enhancer of Src kinase activity (30Burnham M.R. Bruce-Staskal P.J. Harte M.T. Weidow C.L. Ma A. Weed S.A. Bouton A.H. Mol. Cell. Biol. 2000; 20: 5865-5878Crossref PubMed Scopus (109) Google Scholar, 31Xing L. Ge C. Zeltser R. Maskevitch G. Mayer B.J. Alexandropoulos K. Mol. Cell. Biol. 2000; 20: 7363-7377Crossref PubMed Scopus (46) Google Scholar). Src is typically found in a closed or kinase-inactive conformation, held in place by repressive intramolecular interactions involving its Src homology-2 (SH2) and -3 (SH3) domains (for review see Ref. 32Hubbard S.R. Nat. Struct. Biol. 1999; 6: 711-714Crossref PubMed Scopus (50) Google Scholar). Because Cas contains motifs that interact with the SH2 and SH3 domains, it can prevent the adoption of this autoinhibitory conformation by directly binding to Src. We and others (30Burnham M.R. Bruce-Staskal P.J. Harte M.T. Weidow C.L. Ma A. Weed S.A. Bouton A.H. Mol. Cell. Biol. 2000; 20: 5865-5878Crossref PubMed Scopus (109) Google Scholar, 31Xing L. Ge C. Zeltser R. Maskevitch G. Mayer B.J. Alexandropoulos K. Mol. Cell. Biol. 2000; 20: 7363-7377Crossref PubMed Scopus (46) Google Scholar, 33Pellicena P. Miller W.T. J. Biol. Chem. 2001; 276: 28190-28196Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) have shown that the formation of Src/Cas complexes leads to a significant increase in PTK activity, as well as serum- and anchorage-independent growth. Interactions between Src and Cas may therefore initiate signaling cascades that lead to a number of cellular processes, including cell migration. In addition to Src, members of a second group of proteins called NSPs (novel SH2-containing proteins) have also been shown to bind to the carboxyl terminus of Cas (34Lu Y.M. Brush J. Stewart T.A. J. Biol. Chem. 1999; 274: 10047-10052Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). These proteins contain an amino-terminal SH2 domain and a carboxyl-terminal domain with sequence homology to the Cdc25 family of guanine nucleotide exchange factors (GEFs) for Ras family GTPases. The association between Cas and one of these proteins, Chat (Nsp3), has been shown recently (35Sakakibara A. Ohba Y. Kurokawa K. Matsuda M. Hattori S. J. Cell Sci. 2002; 115: 4915-4924Crossref PubMed Scopus (41) Google Scholar) to be involved in the regulation of cell adhesion. A second family member, AND-34/BCAR3 (Nsp2), hereafter referred to as AND-34, also binds to the carboxyl terminus of Cas (36Cai D.P. Clayton L.K. Smolyar A. Lerner A. J. Immunol. 1999; 163: 2104-2112PubMed Google Scholar, 37Gotoh T. Cai D. Tian X. Feig L.A. Lerner A. J. Biol. Chem. 2000; 275: 30118-30123Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Murine AND-34 has been shown to exhibit GEF activity toward the small GTPases RalA, Rap1, and R-Ras (37Gotoh T. Cai D. Tian X. Feig L.A. Lerner A. J. Biol. Chem. 2000; 275: 30118-30123Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Because all three of these GTPases have been reported to function in various aspects of cell adhesion, migration, and/or proliferation (for review see Refs. 38Bos J.L. EMBO J. 1998; 17: 6776-6782Crossref PubMed Scopus (287) Google Scholar, 39Bos J.L. de Rooij J. Reedquist K.A. Nat. Rev. Mol. Cell. Biol. 2001; 2: 369-377Crossref PubMed Scopus (512) Google Scholar, 40Reuther G.W. Der C.J. Curr. Opin. Cell Biol. 2000; 12: 157-165Crossref PubMed Scopus (346) Google Scholar), it is interesting to speculate that AND-34 may contribute to some of the functions attributed to the carboxyl terminus of Cas- and Src/Cas-dependent signaling pathways. In this study, we focused on determining whether Cas and AND-34 function cooperatively to regulate cell signaling pathways that lead to Src activation and cell migration. The use of strategies involving protein overexpression is supported for these studies by the fact that both Src and Cas are highly expressed in a number of naturally occurring cellular contexts, including human breast tumors (41Biscardi J.S. Belsches A.P. Parsons S.J. Mol. Carcinog. 1998; 21: 261-272Crossref PubMed Scopus (139) Google Scholar, 42Biscardi J.S. Tice D.A. Parsons S.J. Adv. Canc. Res. 1999; 76: 61-119Crossref PubMed Google Scholar, 43van der Flier S. Brinkman A. Look M.P. Kok E.M. Meijer-van Gelder M.E. Klijn J.G. Dorssers L.C. Foekens J.A. J. Natl. Cancer Inst. 2000; 92: 120-127Crossref PubMed Scopus (106) Google Scholar). Moreover, the human homologues of both Cas (BCAR1) and AND-34 (BCAR3) were identified in a screen for genes that promoted resistance to the antiestrogen tamoxifen when overexpressed in breast cancer cells (43van der Flier S. Brinkman A. Look M.P. Kok E.M. Meijer-van Gelder M.E. Klijn J.G. Dorssers L.C. Foekens J.A. J. Natl. Cancer Inst. 2000; 92: 120-127Crossref PubMed Scopus (106) Google Scholar, 44van Agthoven T. van Agthoven T.L. Dekker A. van der Spek P.J. Vreede L. Dorssers L.C. EMBO J. 1998; 17: 2799-2808Crossref PubMed Scopus (96) Google Scholar). We found that Cas promotes haptotactic and chemotactic migration in a manner that depends upon kinase-active Src and that AND-34 functions synergistically with Cas to enhance both cell migration and Src activation. The carboxyl-terminal GEF domain of AND-34 was found to contribute to these activities, as was one of the reported targets of this GEF activity, Rap1. Immunolocalization studies suggest that AND-34 may participate in these processes by relocalizing Cas to lamellipodia located at the leading edge of migrating cells. Together, these data indicate that Cas and AND-34 can function together to stimulate Src signaling pathways and promote cell migration. Cells and Antibodies—COS-1 and REF-52 cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 1× penicillin/streptomycin. C3H10T½-5H (c-Src overexpressors) and C3H10T½-KD cells were a generous gift from S. J. Parsons (University of Virginia) (45Luttrell D.K. Luttrell L.M. Parsons S.J. Mol. Cell. Biol. 1988; 8: 497-501Crossref PubMed Scopus (128) Google Scholar, 46Wilson L.K. Parsons S.J. Oncogene. 1990; 5: 1471-1480PubMed Google Scholar). These cell lines were maintained in DMEM supplemented with 10% FCS, 1× penicillin/streptomycin, and 800 μg/ml geneticin (G418; Invitrogen). Cas B polyclonal antisera has been described previously (47Bouton A.H. Burnham M.R. Hybridoma. 1997; 16: 403-411Crossref PubMed Scopus (27) Google Scholar). Myc monoclonal antibody (mAb) 9E10 was generated by the Lymphocyte Culture Center at the University of Virginia or purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rap1 polyclonal antibody was also obtained from Santa Cruz Biotechnology, Inc. RalA mAb was purchased from Transduction Laboratories (San Diego, CA). pTyr mAb 4G10 was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Phospho-specific antibodies for cortactin (pY421) were obtained from BioSource International (Camarillo, CA), and cortactin mAb 4F11 was generously provided by J. T. Parsons (University of Virginia) (48Kanner S.B. Reynolds A.B. Vines R.R. Parsons J.T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3328-3332Crossref PubMed Scopus (397) Google Scholar). FLAG M2 affinity resin and FLAG M5 mAb were purchased from Sigma. Src mAb 2–17 was purchased from Quality Biotech Inc. (Camden, NJ). Protein A-Sepharose CL-4B and horseradish peroxidase-conjugated anti-mouse and anti-rabbit immunoglobulins were obtained from Amersham Biosciences. Rabbit anti-mouse immunoglobulin, fluorescein isothiocyanate-conjugated goat anti-mouse and anti-rabbit, and Texas Red (TR)-conjugated goat anti-mouse were purchased from Jackson ImmunoResearch (West Grove, PA). TR-phalloidin was obtained from Molecular Probes (Eugene, OR). Plasmid Construction and Mutagenesis—pRK5 constructs encoding the genes for Myc-tagged full-length Cas, deletion of the minimal carboxyl terminus (ΔmCT; deletion of amino acids 693–874), and the minimal carboxyl terminus (mCT; amino acids 693–874) have been described previously (9Harte M.T. Macklem M. Weidow C.L. Parsons J.T. Bouton A.H. Biochim. Biophys. Acta. 2000; 1499: 34-48Crossref PubMed Scopus (25) Google Scholar, 49Burnham M.R. Harte M.T. Bouton A.H. Mol. Carcinog. 1999; 26: 20-31Crossref PubMed Scopus (20) Google Scholar). Single point mutations substituting a proline residue for leucine 791 (L791P) in full-length Cas and mCT, as well as mutation of leucine 762 (L762P) in mCT, were generated using QuikChange site-directed mutagenesis (Stratagene). The mCT double mutant (L762/791P) was generated by subjecting mCT-L791P to a second round of mutagenesis. All mutations were confirmed by automated DNA sequence analysis. Yellow fluorescent protein (YFP)-tagged Cas was constructed by inserting the complete Cas cDNA as a BamHI/XbaI fragment into pLP-EYFP-C1 using the Creator™ cloning and expression system (Clontech). pCDNA-c-Src was generously supplied by S. J. Parsons (University of Virginia) (50Tice D.A. Biscardi J.S. Nickles A.L. Parsons S.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1415-1420Crossref PubMed Scopus (397) Google Scholar), and pCDNA3FLAG2AB-paxillin has been described previously (30Burnham M.R. Bruce-Staskal P.J. Harte M.T. Weidow C.L. Ma A. Weed S.A. Bouton A.H. Mol. Cell. Biol. 2000; 20: 5865-5878Crossref PubMed Scopus (109) Google Scholar). FLAG-tagged AND-34 was constructed in pcDNA3 by replacing the polylinker of pcDNA3 with that of pFLAG-CMV2 (pFLAG3). The expressed sequence tag AI115884 was found to contain the entire coding sequence of the mouse BCAR3 homologue, AND-34. The first 24 amino acids of AND-34 were deleted to allow use of an NcoI site to blunt into the newly created pFLAG3 vector, resulting in pFLAG3-AND-34. AND-34 deleted for the GEF-containing carboxyl terminus (ΔGEF) was created from wild-type cDNA by synthesizing a PCR product using a 5′ primer that contained a unique EcoRI site just upstream of codon 25 and a 3′ primer containing a unique XbaI site just downstream of codon 593. The PCR product was digested with EcoRI and XbaI and ligated in-frame into pCDNA3FLAG2AB in place of the analogous restriction fragment. A constitutively active RalA construct (pCAG-Ral72L) was generously provided by L. Feig (Tufts University) (51Urano T. Emkey R. Feig L.A. EMBO J. 1996; 15: 810-816Crossref PubMed Scopus (300) Google Scholar), and constitutively active R-Ras (pCGN-R-Ras87L) was a gift from A. Cox (University of North Carolina) (52Huff S.Y. Quilliam L.A. Cox A.D. Der C.J. Oncogene. 1997; 14: 133-143Crossref PubMed Scopus (43) Google Scholar). Dominant-negative and constitutively active Rap1B constructs (pCMV-Rap1N17 and pCMV-Rap1V12) were kindly provided by P. Stork (Oregon Health Sciences Center) (53Vossler M.R. Yao H. York R.D. Pan M.G. Rim C.S. Stork P.J. Cell. 1997; 89: 73-82Abstract Full Text Full Text PDF PubMed Scopus (944) Google Scholar). Transfection and Protein Expression—Transient transfections in COS-1 and REF-52 cells were performed according to manufacturer specifications using Superfect™ purchased from Qiagen, and C3H10T½ cell lines were transfected using LipofectAMINE PLUS (Invitrogen). In those cases where cells were co-transfected with two or more constructs, immunofluorescence was performed to confirm that >90% of transfected cells expressed both proteins. Cells were lysed 24 h post-transfection in modified radioimmune precipitation assay buffer (150 mm NaCl, 50 mm Tris, pH 7.5, 1% Igepal CA-630, 0.5% deoxycholate) supplemented with protease and phosphatase inhibitors (100 μm leupeptin, 1 mm phenylmethylsulfonyl fluoride, 0.15 unit/ml aprotinin, 1 mm sodium orthovanadate) as described previously (54Burnham M.R. Harte M.T. Richardson A. Parsons J.T. Bouton A.H. Oncogene. 1996; 12: 2467-2472PubMed Google Scholar). Protein concentrations were determined using the BCA assay from Pierce. Immunoprecipitation and Immunoblotting—For Myc immunoprecipitates, cell lysate was incubated with mAb 9E10 overnight at 4 °C with rotation, and the immune complexes were recovered by a 1-h incubation with protein A-Sepharose beads that had been preincubated with rabbit anti-mouse immunoglobulin. For FLAG immunoprecipitates, cell lysates were incubated with M2 resin (25 μl/mg protein) for 1 h at 4 °C with rotation. Complexes were washed twice in modified radioimmune precipitation assay buffer and twice in Tris-buffered saline, resuspended in 2× Laemmli sample buffer, and boiled for 5 min. Proteins were resolved by SDS-PAGE, transferred to nitrocellulose, and incubated with antibodies as indicated. Proteins were detected by horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin followed by enhanced chemiluminescence (PerkinElmer Life Sciences). Cell Migration—C3H10T½ cell lines were transfected with the indicated constructs and cultured overnight in low-serum migration medium (DMEM containing 0.05% FCS). The following day, cells were trypsinized, counted, and a total of 105 cells in 500 μl of migration medium were loaded into the top of a modified Boyden chamber (24-well, 8.0-μm BioCoat insert; BD Biosciences). For migration toward FN (Sigma), the underside of the insert membrane was preincubated with FN overnight at 4 °C (20 μg/ml in phosphate-buffered saline), and the bottom chamber was filled with migration medium. For migration toward serum, uncoated inserts were placed in wells filled with DMEM containing 10% FCS. Cells were allowed to migrate for2hat37 °C, and the nonmigratory cells were then removed from the top of the membrane with cotton swabs. The underside of the membrane was fixed in 3% paraformaldehyde for 20 min at room temperature and mounted on glass slides using VectaShield (Vector Laboratories Inc., Burlingame, CA). Migrated cells were counted using a Nikon fluorescence microscope, and the ratio of fluorescent cells to total cells (visualized by phase microscopy) was used to determine percent migration. This was divided by the percent transfection for each condition, resulting in the migratory index (55Slack J.K. Catling A.D. Eblen S.T. Weber M.J. Parsons J.T. J. Biol. Chem. 1999; 274: 27177-27184Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). The log values for each group of data were analyzed by single factor analysis of variance. When significant differences were found between groups at the 5% level, they were compared using the Student's t test assuming unequal variance. The data are shown as the mean relative migratory index (normalized to vector-transfected cells) ± S.D. Immunofluorescence—For immunolocalization of AND-34 wild-type and mutant constructs, REF-52 cells were transfected as described above. The following day, cells were replated on FN-coated coverslips (20 μg/ml) for 4–5 h. The cells were then fixed in 3% paraformaldehyde for 20 min at room temperature and permeabilized using 0.4% Triton X-100 in phosphate-buffered saline for 5 min at room temperature. Cells were incubated with FLAG M5 mAb in 2% bovine serum albumin/phosphate-buffered saline for 1 h, followed by fluorescein isothiocyanate-conjugated goat anti-mouse and TR-conjugated phalloidin for 1 h. For Cas/AND-34 colocalization, transfected REF-52 cells were plated on FN-coated coverslips for 4–5 h and then fixed, permeabilized, incubated with FLAG M5 and Cas B polyclonal antisera, and subsequently incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit and TR-conjugated goat anti-mouse antibodies. Cells were visualized through a Nikon fluorescence microscope and photographed using a cooled CCD camera controlled by Inovision Isee software. Cell Migration Is Induced by Overexpression of Src and Cas—We have shown previously (30Burnham M.R. Bruce-Staskal P.J. Harte M.T. Weidow C.L. Ma A. Weed S.A. Bouton A.H. Mol. Cell. Biol. 2000; 20: 5865-5878Crossref PubMed Scopus (109) Google Scholar) that Src and Cas form stable molecular complexes under conditions in which they are both overexpressed. Using a cell system derived from C3H10T½-5H murine fibroblasts, which stably overexpress wild-type (WT) Src at levels 16-fold greater than endogenous Src (45Luttrell D.K. Luttrell L.M. Parsons S.J. Mol. Cell. Biol. 1988; 8: 497-501Crossref PubMed Scopus (128) Google Scholar), we demonstrated that one functional consequence of these interactions is serum- and anchorage-independent proliferation (30Burnham M.R. Bruce-Staskal P.J. Harte M.T. Weidow C.L. Ma A. Weed S.A. Bouton A.H. Mol. Cell. Biol. 2000; 20: 5865-5878Crossref PubMed Scopus (109) Google Scholar). Because both Src and Cas have been shown to also play a role in cell adhesion and migration, we used the C3H10T½-5H model to determine whether these molecules functioned cooperatively in these processes. Src-overexpressing C3H10T½-5H cells were transiently transfected with plasmids encoding YFP or YFP-Cas and analyzed 24 h later for their ability to migrate toward FN or serum (Fig. 1, WT). The relative migratory index represents the percent of migrated fluorescent cells corrected for transfection efficiency and normalized to vector-transfected cells. Cas was found to promote a significant 2-fold increase in migration toward FN and serum in the presence of overexpressed WT Src. To determine whether this augmentation of cell migration by Cas was dependent on the kinase activity of overexpressed Src, a similar experiment was performed using C3H10T½-KD fibroblasts stably overexpressing kinase-dead (KD) Src (46Wilson L.K. Parsons S.J. Oncogene. 1990; 5: 1471-1480PubMed Google Scholar). Cas was unable to promote migration toward either FN or serum in cells expressing KD Src. This deficiency did not appear to be because of an inability of Cas to associate with KD Src, because KD Src-Cas complexes were readily detectable in these cell lysates (data not shown). Thus, it appears from these data that Cas-dependent cell migration requires Src kinase activity. AND-34 Synergizes with Cas to Promote Src Activation and Cell Migration—Coincident with their effect on cell migration, interactions between Src and Cas result in the activation of Src, leading to tyrosine phosphorylation of several Src substrates and the promotion of adhesion- and serum-independent growth (30Burnham M.R. Bruce-Staskal P.J. Harte M.T. Weidow C.L. Ma A. Weed S.A. Bouton A.H. Mol. Cell. Biol. 2000; 20: 5865-5878Crossref PubMed Scopus (109) Google Scholar). A recently identified Cas binding partner, AND-34, also demonstrates pro-proliferative effects when overexpressed in breast cancer cell lines grown in the presence of the antiestrogen tamoxifen (44van Agthoven T. van Agthoven T.L. Dekker A. van der Spek P.J. Vreede L. Dorssers L.C. EMBO J. 1998; 17: 2799-2808Crossref PubMed Scopus (96) Google Scholar). To determine whether AND-34 participates in Cas-mediated Src activation and substrate phosphorylation, COS-1 cells were transiently co-transfected with plasmids encoding WT Src, Myc-tagged Cas, and/or FLAG-tagged AND-34. PTK activity was determined 24 h post-transfection by measuring the level of pTyr on the Src substrate paxillin, which was expressed as a FLAG-tagged construct, together with the other proteins. Consistent w" @default.
• W2094366341 created "2016-06-24" @default.
• W2094366341 creator A5032949456 @default.
• W2094366341 creator A5052385827 @default.
• W2094366341 creator A5054473487 @default.
• W2094366341 date "2003-07-01" @default.
• W2094366341 modified "2023-09-29" @default.
• W2094366341 title "Synergistic Promotion of c-Src Activation and Cell Migration by Cas and AND-34/BCAR3" @default.
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• W2094366341 doi "https://doi.org/10.1074/jbc.m303535200" @default.
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