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• W2024995301 abstract "The focal adhesion adapter protein p130cas regulates adhesion and growth factor-related signaling, in part through Src-mediated tyrosine phosphorylation of p130cas. AND-34/BCAR3, one of three NSP family members, binds the p130cas carboxyl terminus, adjacent to a bipartite p130cas Src-binding domain (SBD) and induces anti-estrogen resistance in breast cancer cell lines as well as phosphorylation of p130cas. Only a subset of the signaling properties of BCAR3, specifically augmented motility, are dependent upon formation of the BCAR3-p130cas complex. Using GST pull-down and immunoprecipitation studies, we show that among NSP family members, only BCAR3 augments the ability of p130cas to bind the Src SH3 domain through an RPLPSPP motif in the p130cas SBD. Although our prior work identified phosphorylation of the serine within the p130cas RPLPSPP motif, mutation of this residue to alanine or glutamic acid did not alter BCAR3-induced Src SH3 domain binding to p130cas. The ability of BCAR3 to augment Src SH3 binding requires formation of a BCAR3-p130cas complex because mutations that reduce association between these two proteins block augmentation of Src SH3 domain binding. Similarly, in MCF-7 cells, BCAR3-induced tyrosine phosphorylation of the p130cas substrate domain, previously shown to be Src-dependent, was reduced by an R743A mutation that blocks BCAR3 association with p130cas. Immunofluorescence studies demonstrate that BCAR3 expression alters the intracellular location of both p130cas and Src and that all three proteins co-localize. Our work suggests that BCAR3 expression may regulate Src signaling in a BCAR3-p130cas complex-dependent fashion by altering the ability of the Src SH3 domain to bind the p130cas SBD. The focal adhesion adapter protein p130cas regulates adhesion and growth factor-related signaling, in part through Src-mediated tyrosine phosphorylation of p130cas. AND-34/BCAR3, one of three NSP family members, binds the p130cas carboxyl terminus, adjacent to a bipartite p130cas Src-binding domain (SBD) and induces anti-estrogen resistance in breast cancer cell lines as well as phosphorylation of p130cas. Only a subset of the signaling properties of BCAR3, specifically augmented motility, are dependent upon formation of the BCAR3-p130cas complex. Using GST pull-down and immunoprecipitation studies, we show that among NSP family members, only BCAR3 augments the ability of p130cas to bind the Src SH3 domain through an RPLPSPP motif in the p130cas SBD. Although our prior work identified phosphorylation of the serine within the p130cas RPLPSPP motif, mutation of this residue to alanine or glutamic acid did not alter BCAR3-induced Src SH3 domain binding to p130cas. The ability of BCAR3 to augment Src SH3 binding requires formation of a BCAR3-p130cas complex because mutations that reduce association between these two proteins block augmentation of Src SH3 domain binding. Similarly, in MCF-7 cells, BCAR3-induced tyrosine phosphorylation of the p130cas substrate domain, previously shown to be Src-dependent, was reduced by an R743A mutation that blocks BCAR3 association with p130cas. Immunofluorescence studies demonstrate that BCAR3 expression alters the intracellular location of both p130cas and Src and that all three proteins co-localize. Our work suggests that BCAR3 expression may regulate Src signaling in a BCAR3-p130cas complex-dependent fashion by altering the ability of the Src SH3 domain to bind the p130cas SBD. The focal adhesion protein p130cas (Crk-associated substrate) was first identified as a tyrosine-phosphorylated protein in p47 v-Crk and p60 v-Src transformed cell lines (1Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Mano H. Yazaki Y. Hirai H. A novel signaling molecule, p130, forms stable complexes in vivo with v-Crk and v-Src in a tyrosine phosphorylation-dependent manner.EMBO J. 1994; 13: 3748-3756Crossref PubMed Scopus (593) Google Scholar). p130cas contains an amino-terminal SH3 2The abbreviations used are: SH3Src homology 3SH2Src homology 2FAKfocal adhesion kinaseSrc CAconstitutively active Y528F SrcSBDSrc-binding domainNSPnovel SH2-containing proteinFATfocal adhesion-targetingGEFguanine nucleotide exchange factor. domain followed by a “substrate domain” with 15 YXXP motifs, which are the principal site of Src-mediated tyrosine phosphorylation. The p130cas SH3 domain binds to pp125 FAK and related adhesion focal tyrosine kinase, tyrosine kinases whose activity is regulated by integrin signaling (2Harte M.T. Hildebrand J.D. Burnham M.R. Bouton A.H. Parsons J.T. p130cas, a substrate associated with v-Src and v-Crk, localizes to focal adhesions and binds to focal adhesion kinase.J. Biol. Chem. 1996; 271: 13649-13655Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 3Polte T.R. Hanks S.K. Interaction between focal adhesion kinase and Crk-associated tyrosine kinase substrate p130cas.Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 10678-10682Crossref PubMed Scopus (387) Google Scholar). Src and other Src family tyrosine kinases are recruited to p130cas in part by Src SH2 domain binding to the autophosphorylation site of FAK, Tyr-397 (4Schaller M.D. Hildebrand J.D. Shannon J.D. Fox J.W. Vines R.R. Parsons J.T. Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2-dependent binding of pp60src.Mol. Cell Biol. 1994; 14: 1680-1688Crossref PubMed Scopus (1115) Google Scholar). p130cas substrate domain phosphorylation by Src in turn leads to recruitment of the SH2 domain-containing adapter protein CrkII. Integrin signaling also enhances CrkII association through its SH3 domain with the atypical Rac GDP exchange factor DOCK180, thus assembling a p130cas complex that serves as a molecular switch for cell migration (5Klemke R.L. Leng J. Molander R. Brooks P.C. Vuori K. Cheresh D.A. CAS/Crk coupling serves as a “molecular switch” for induction of cell migration.J. Cell Biol. 1998; 140: 961-972Crossref PubMed Scopus (590) Google Scholar, 6Kiyokawa E. Hashimoto Y. Kurata T. Sugimura H. Matsuda M. Evidence that DOCK180 up-regulates signals from the CrkII-p130cas complex.J. Biol. Chem. 1998; 273: 24479-24484Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 7Côté J.F. Vuori K. GEF what? Dock180 and related proteins help Rac to polarize cells in new ways.Trends Cell Biol. 2007; 17: 383-393Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). Src homology 3 Src homology 2 focal adhesion kinase constitutively active Y528F Src Src-binding domain novel SH2-containing protein focal adhesion-targeting guanine nucleotide exchange factor. Although p130cas substrate domain tyrosine phosphorylation is clearly of importance to regulation of cell motility, the precise mechanism by which Src is activated to carry out such phosphorylation remains controversial. “Pull-down” studies using glutathione S-transferase (GST)-Src SH2 and GST-Src SH3 domains have demonstrated that a carboxyl-terminal region of p130cas designated the “Src-binding domain” (SBD) contains binding sites for both the Src SH2 domain at Tyr-762 and for the Src SH3 domain at an adjacent RPLPSPP motif (8Nakamoto T. Sakai R. Ozawa K. Yazaki Y. Hirai H. Direct binding of C-terminal region of p130cas to SH2 and SH3 domains of Src kinase.J. Biol. Chem. 1996; 271: 8959-8965Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 9Cheadle C. Ivashchenko Y. South V. Searfoss G.H. French S. Howk R. Ricca G.A. Jaye M. Identification of a Src SH3 domain binding motif by screening a random phage display library.J. Biol. Chem. 1994; 269: 24034-24039Abstract Full Text PDF PubMed Google Scholar). Whereas in rat 3Y1 fibroblast cells Src SH2 domain association with p130cas required co-expression of v-Crk or v-Src, association of the Src SH3 domain with p130cas in these cells was constitutive. Mutation of the RPLPSPP motif to RLGSSPP resulted in a marked reduction in p130cas-associated kinase activity in v-Crk-transformed cells, suggesting that Src associated with the p130cas SBD may contribute to p130cas substrate domain phosphorylation (8Nakamoto T. Sakai R. Ozawa K. Yazaki Y. Hirai H. Direct binding of C-terminal region of p130cas to SH2 and SH3 domains of Src kinase.J. Biol. Chem. 1996; 271: 8959-8965Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). In contrast to these results, work by another group in COS-7 cells reported that mutation of the RPLPSPP motif to RAAASPP failed to alter the ability of transfected FAK and n-Src to induce p130cas tyrosine phosphorylation. In these studies, p130cas substrate domain phosphorylation was instead shown to require Src binding to phosphorylated FAK Tyr-397 (10Ruest P.J. Shin N.Y. Polte T.R. Zhang X. Hanks S.K. Mechanisms of CAS substrate domain tyrosine phosphorylation by FAK and Src.Mol. Cell Biol. 2001; 21: 7641-7652Crossref PubMed Scopus (131) Google Scholar). Of note, n-Src is a neuronal specific splice variant of Src that has a six-amino acid insertion in the SH3 domain that alters Src SH3 domain binding specificity. Three highly related novel SH2-containing protein (NSP) family members, NSP1, NSP2/AND-34/BCAR3 (BCAR3), and NSP3/SHEP1/CHAT (NSP3), which have an amino-terminal SH2 domain and a carboxyl-terminal domain with modest homology to the Cdc25 homology fold of Ras GDP exchange factors, bind constitutively to the carboxyl-terminal focal adhesion-targeting (FAT) domain of p130cas (11Lu Y. Brush J. Stewart T.A. NSP1 defines a novel family of adaptor proteins linking integrin and tyrosine kinase receptors to the c-Jun N-terminal kinase/stress-activated protein kinase signaling pathway.J. Biol. Chem. 1999; 274: 10047-10052Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 12Cai D. Clayton L.K. Smolyar A. Lerner A. AND-34, a novel p130cas-binding thymic stromal cell protein regulated by adhesion and inflammatory cytokines.J. Immunol. 1999; 163: 2104-2112PubMed Google Scholar, 13Dodelet V.C. Pazzagli C. Zisch A.H. Hauser C.A. Pasquale E.B. A novel signaling intermediate, SHEP1, directly couples Eph receptors to R-Ras and Rap1A.J. Biol. Chem. 1999; 274: 31941-31946Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 14Sakakibara A. Hattori S. Chat, a Cas/HEF1-associated adaptor protein that integrates multiple signaling pathways.J. Biol. Chem. 2000; 275: 6404-6410Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 15Gotoh T. Cai D. Tian X. Feig L.A. Lerner A. p130cas regulates the activity of AND-34, a novel Ral, Rap1, and R-Ras guanine nucleotide exchange factor.J. Biol. Chem. 2000; 275: 30118-30123Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 16Mace P.D. Wallez Y. Dobaczewska M.K. Lee J.J. Robinson H. Pasquale E.B. Riedl S.J. NSP-Cas protein structures reveal a promiscuous interaction module in cell signaling.Nat. Struct. Mol. Biol. 2011; 18: 1381-1387Crossref PubMed Scopus (23) Google Scholar). A random retroviral insertional mutagenesis study to identify genes whose altered expression induces anti-estrogen resistance in estrogen-dependent breast cancer cell lines identified both BCAR3 and p130cas as genes whose augmented expression confers tamoxifen resistance (17van Agthoven T. van Agthoven T.L. Dekker A. van der Spek P.J. Vreede L. Dorssers L.C. Identification of BCAR3 by a random search for genes involved in antiestrogen resistance of human breast cancer cells.EMBO J. 1998; 17: 2799-2808Crossref PubMed Scopus (96) Google Scholar, 18Brinkman A. van der Flier S. Kok E.M. Dorssers L.C. BCAR1, a human homologue of the adapter protein p130cas, and antiestrogen resistance in breast cancer cells.J. Natl. Cancer Inst. 2000; 92: 112-120Crossref PubMed Scopus (127) Google Scholar). Subsequent studies demonstrated that although all three NSP family members activate Rac and Cdc42 indirectly by a PI3K-dependent mechanism, only BCAR3 induced activation of cyclin D1 promoter luciferase constructs as well as resistance to the pure ER antagonist ICI 182,780 (19Cai D. Iyer A. Felekkis K.N. Near R.I. Luo Z. Chernoff J. Albanese C. Pestell R.G. Lerner A. AND-34/BCAR3, a GDP exchange factor whose overexpression confers antiestrogen resistance, activates Rac, PAK1, and the cyclin D1 promoter.Cancer Res. 2003; 63: 6802-6808PubMed Google Scholar, 20Felekkis K.N. Narsimhan R.P. Near R. Castro A.F. Zheng Y. Quilliam L.A. Lerner A. AND-34 activates phosphatidylinositol 3-kinase and induces anti-estrogen resistance in a SH2 and GDP exchange factor-like domain-dependent manner.Mol. Cancer Res. 2005; 3: 32-41PubMed Google Scholar, 21Near R.I. Zhang Y. Makkinje A. Vanden Borre P. Lerner A. AND-34/BCAR3 differs from other NSP homologs in induction of anti-estrogen resistance, cyclin D1 promoter activation, and altered breast cancer cell morphology.J. Cell Physiol. 2007; 212: 655-665Crossref PubMed Scopus (26) Google Scholar). As judged by tyrosine phosphorylation of paxillin and cortactin, Riggins et al. (22Riggins R.B. Quilliam L.A. Bouton A.H. Synergistic promotion of c-Src activation and cell migration by Cas and AND-34/BCAR3.J. Biol. Chem. 2003; 278: 28264-28273Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) reported that co-transfection with BCAR3 and p130cas enhances Src activation in COS-1 cells relative to transfection with p130cas alone. BCAR3 also regulates motility in both fibroblasts and breast cancer epithelial cells (22Riggins R.B. Quilliam L.A. Bouton A.H. Synergistic promotion of c-Src activation and cell migration by Cas and AND-34/BCAR3.J. Biol. Chem. 2003; 278: 28264-28273Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 23Schrecengost R.S. Riggins R.B. Thomas K.S. Guerrero M.S. Bouton A.H. Breast cancer antiestrogen resistance-3 expression regulates breast cancer cell migration through promotion of p130cas membrane localization and membrane ruffling.Cancer Res. 2007; 67: 6174-6182Crossref PubMed Scopus (56) Google Scholar, 24Vanden Borre P. Near R.I. Makkinje A. Mostoslavsky G. Lerner A. BCAR3/AND-34 can signal independent of complex formation with CAS family members or the presence of p130cas.Cell. Signal. 2011; 23: 1030-1040Crossref PubMed Scopus (18) Google Scholar). BCAR3 knock-out mice undergo post-natal ophthalmic lens rupture, suggesting a role for BCAR3 in maintaining the integrity of the lens capsule (25Near R.I. Smith R.S. Toselli P.A. Freddo T.F. Bloom A.B. Vanden Borre P. Seldin D.C. Lerner A. Loss of AND-34/BCAR3 expression in mice results in rupture of the adult lens.Mol. Vis. 2009; 15: 685-699PubMed Google Scholar). Because BCAR3 and p130cas both can regulate cell motility and breast cancer cell line estrogen-independent cell growth, formation of the BCAR3-p130cas complex would be expected to be required for BCAR3-mediated signaling. Surprisingly, in recent studies utilizing an R743A mutant form of BCAR3 that is unable to form a complex with p130cas, we found that many characterized downstream events associated with BCAR3 overexpression are independent of such complex formation (24Vanden Borre P. Near R.I. Makkinje A. Mostoslavsky G. Lerner A. BCAR3/AND-34 can signal independent of complex formation with CAS family members or the presence of p130cas.Cell. Signal. 2011; 23: 1030-1040Crossref PubMed Scopus (18) Google Scholar). Similarly, in the study by Riggins et al. (22Riggins R.B. Quilliam L.A. Bouton A.H. Synergistic promotion of c-Src activation and cell migration by Cas and AND-34/BCAR3.J. Biol. Chem. 2003; 278: 28264-28273Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) described above, BCAR3-induced Src activation was reported to occur independently of BCAR3 association with p130cas. In contrast, BCAR3-induced augmentation of cellular motility is reduced in fibroblasts expressing the complex formation-defective BCAR3 R743A (24Vanden Borre P. Near R.I. Makkinje A. Mostoslavsky G. Lerner A. BCAR3/AND-34 can signal independent of complex formation with CAS family members or the presence of p130cas.Cell. Signal. 2011; 23: 1030-1040Crossref PubMed Scopus (18) Google Scholar). The molecular mechanisms underlying BCAR3-p130cas complex-dependent and -independent signaling remain unresolved. In a study examining a BCAR3-induced reduction in p130cas PAGE migration, we determined that BCAR3 expression regulates late phase adhesion-dependent p130cas phosphorylation in an actin filament-dependent manner (26Makkinje A. Near R.I. Infusini G. Vanden Borre P. Bloom A. Cai D. Costello C.E. Lerner A. AND-34/BCAR3 regulates adhesion-dependent p130cas serine phosphorylation and breast cancer cell growth pattern.Cell. Signal. 2009; 21: 1423-1435Crossref PubMed Scopus (17) Google Scholar). Three sites of p130cas serine phosphorylation were identified, one of which was located within the previously characterized Src SH3 domain binding site, RPLPSPP. Given this observation, we sought to determine whether expression of BCAR3 could alter the ability of the Src SH3 domain to bind to p130cas. Here, we report that BCAR3 expression augments both Src SH3 domain binding to p130cas and tyrosine phosphorylation of the p130cas substrate domain in a BCAR3-p130cas complex-dependent manner. The following antibodies were used in this work: rabbit polyclonal anti-BCAR3 (Bethyl) and anti-Src (SRC2; Santa Cruz Biotechnology, Inc., Santa Cruz, CA); mouse monoclonal anti-hemagglutinin (HA) (Covance); anti-p130cas (BD Biosciences); and anti-phospho-Tyr-100, rabbit anti-phospho-p130cas (Tyr-165), and rabbit anti-phospho-p130cas (Tyr-410) (Cell Signaling Technology). Mammalian expression plasmids pUSE-Src and pUSE-SrcY528F (constitutively active Src) were purchased from Upstate Biotechnology. Vectors expressing wild type and mutant HA-BCAR3 and NSP proteins were described previously (19Cai D. Iyer A. Felekkis K.N. Near R.I. Luo Z. Chernoff J. Albanese C. Pestell R.G. Lerner A. AND-34/BCAR3, a GDP exchange factor whose overexpression confers antiestrogen resistance, activates Rac, PAK1, and the cyclin D1 promoter.Cancer Res. 2003; 63: 6802-6808PubMed Google Scholar, 21Near R.I. Zhang Y. Makkinje A. Vanden Borre P. Lerner A. AND-34/BCAR3 differs from other NSP homologs in induction of anti-estrogen resistance, cyclin D1 promoter activation, and altered breast cancer cell morphology.J. Cell Physiol. 2007; 212: 655-665Crossref PubMed Scopus (26) Google Scholar, 24Vanden Borre P. Near R.I. Makkinje A. Mostoslavsky G. Lerner A. BCAR3/AND-34 can signal independent of complex formation with CAS family members or the presence of p130cas.Cell. Signal. 2011; 23: 1030-1040Crossref PubMed Scopus (18) Google Scholar, 26Makkinje A. Near R.I. Infusini G. Vanden Borre P. Bloom A. Cai D. Costello C.E. Lerner A. AND-34/BCAR3 regulates adhesion-dependent p130cas serine phosphorylation and breast cancer cell growth pattern.Cell. Signal. 2009; 21: 1423-1435Crossref PubMed Scopus (17) Google Scholar). Plasmids used for expression of HA-p130cas and HA-p130cas (RLGS) proteins were as described (15Gotoh T. Cai D. Tian X. Feig L.A. Lerner A. p130cas regulates the activity of AND-34, a novel Ral, Rap1, and R-Ras guanine nucleotide exchange factor.J. Biol. Chem. 2000; 275: 30118-30123Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). The MCF-7 cell lines stably transduced with HA-NSP-1, HA-BCAR3, and HA-NSP-3 have been described previously (21Near R.I. Zhang Y. Makkinje A. Vanden Borre P. Lerner A. AND-34/BCAR3 differs from other NSP homologs in induction of anti-estrogen resistance, cyclin D1 promoter activation, and altered breast cancer cell morphology.J. Cell Physiol. 2007; 212: 655-665Crossref PubMed Scopus (26) Google Scholar). MCF-7, BCAR3/MCF-7 (II-6 cells), and COS-7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Mediatech, Inc.) supplemented with 10% heat-treated fetal calf serum (Biomeda), 2.2 mm l-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin (27Cai D. Felekkis K.N. Near R.I. O'Neill G.M. van Seventer J.M. Golemis E.A. Lerner A. The GDP exchange factor AND-34 is expressed in B cells, associates with HEF1, and activates Cdc42.J. Immunol. 2003; 170: 969-978Crossref PubMed Scopus (35) Google Scholar). Transient transfection of cultured cells were performed by mixing plasmid DNA with Lipofectamine 2000 (ratio 1:3 (μg of DNA/μl of Lipofectamine)) in quantities needed to treat duplicate 100-mm culture plates with a subconfluent growth of cells. After 18 h of growth in transfection medium, cells were washed with ice-cold phosphate-buffered saline (PBS). Cell lysates were prepared by scraping cell monolayers on duplicate plates into 750 μl of buffer A (20 mm Hepes, pH 7.4, 10% glycerol, 1% Triton X-100, 150 mm NaCl, 50 mm β-glycerophosphate, 2 mm EDTA, 2 mm EGTA, 1 mm sodium orthovanadate, 1 mm benzamidine, 1 mm DTT, and 1 mm PMSF) on ice. Cell lysates were clarified of Triton X-100-insoluble debris by centrifuging samples at 20,800 × g for 30 min at 5 °C. Protein concentrations of the clarified cell lysates were measured using Bradford reagent (Bio-Rad). For immunoprecipitation, pull-down assays, and expression analysis, lysates were normalized to approximately equal concentration of protein. For immunoprecipitation, 500 μg of cellular protein was mixed end-over-end with 10–20 μl of protein G-Sepharose (GE Healthcare) and designated antibodies for 3 h at 5 °C. Sepharose beads were collected by centrifuging samples at 1000 × g and discarding the supernatants. Sample pellets were washed three times with ice-cold buffer A (see above), suspended in 2× SDS-PAGE sample buffer, and boiled for 5 min. Heat-denatured protein samples were separated using SDS-PAGE, Western transfer, and immunoblotting methods described previously (26Makkinje A. Near R.I. Infusini G. Vanden Borre P. Bloom A. Cai D. Costello C.E. Lerner A. AND-34/BCAR3 regulates adhesion-dependent p130cas serine phosphorylation and breast cancer cell growth pattern.Cell. Signal. 2009; 21: 1423-1435Crossref PubMed Scopus (17) Google Scholar). pGEX plasmids encoding the SH2 and SH3 domains of Src were gifts from Dr. Todd Holmes (University of California, Irvine). A pGEX plasmid encoding the SH3 domain of p130cas was from Dr. Kathrin Kirsch (Boston University School of Medicine). The SH3 domain of mouse p85α was amplified from expression vector pRSV-Myc-p85α, a gift from Dr. Zhijun Luo (Boston University School of Medicine), using Pfu DNA polymerase (Stratagene), forward primer (5′-AGTGCAGAGGGCTACCAG-3′) and reverse primer (5′-TGAAATTCTTTTCCTTCCAATG-3′). p85α SH3 forward and reverse primers also had BamHI and NotI restriction enzyme sequences at their 5′ termini, respectively. The PCR product was digested with BamHI-NotI (New England Biolabs) and ligated into BamHI-NotI-digested pET-6P-1 (Invitrogen). Plasmids encoding GST and GST-tagged recombinant SH3 and SH2 domain proteins were used to transform Escherichia coli strain DH5α (Invitrogen) as recommended by the manufacturer. Transformed bacteria were cultured in LB containing 100 μg/ml ampicillin to midphase of linear growth, upon which time isopropyl-1-thiol-β-galactopyranoside was added to the culture to a concentration of 0.5 mm to induce recombinant protein expression. Isopropyl-1-thiol-β-galactopyranoside-treated bacterial cultures were grown at room temperature overnight. Bacteria were harvested by centrifugation, and bacterial pellets were lysed using a lytic, non-mechanical method. Bacterial pellets were suspended in lysis buffer B (25 mm Tris-HCl buffer, pH 7.6, containing 100 mm NaCl, 1% Triton X-100, 0.1 mm EDTA, and 1 mm DTT). Egg white lysozyme (1 mg/ml; Sigma-Aldrich) was added, and the mixtures were incubated on ice for 45 min. MgCl2 (10 mm) and DNase I (25 μg/ml) were added and incubated on ice for 20 min. The cytoplasmic fraction of bacterial lysates were prepared by centrifugation at 100,000 × g and mixed with glutathione-Sepharose 4B (GE Healthcare) resin for 3 h. After mixing, Sepharose resins were washed sequentially with buffer C (50 mm Tris-HCl, pH 7.4, 100 mm NaCl, 1% Triton X-100, 10% glycerol, 1 mm EDTA, 1 mm EGTA, 1 mm DTT, 1 mm PMSF, and 1 mm benzamidine), high salt buffer D (100 mm Tris-HCl, pH 7.6, 1 m NaCl, 1% Triton X-100, 10% glycerol, 1 mm EDTA, 1 mm EGTA, 1 mm DTT, 1 mm PMSF, and 1 mm benzamidine), and buffer E (20 mm Hepes, 100 mm NaCl, 0.1% Triton X-100, 1 mm EDTA, 1 mm EGTA, 1 mm DTT, 1 mm benzamidine, and 1 mm PMSF). Proteins bound to washed Sepharose resins were eluted with 0.2 m reduced glutathione (Sigma) in buffer E. Eluants were transferred to dialysis tubing (Spectra/Por®; molecular mass cut-off 12–14 kDa), dialyzed overnight against two changes of buffer C containing 50% glycerol, and kept at −20 °C for long term storage. Protein concentrations of the dialysates were determined using Bio-Rad protein assay reagent. Purity of the protein in the dialysates was analyzed by SDS-PAGE and assessed to be >97% by Coomassie Blue staining. Protein lysates prepared from non-transfected, transiently transfected, or transduced cells were normalized to equal concentrations of 1 mg of protein/ml in lysis buffer. GST pull-down samples were assembled by suspending 10–20 μl of GSH-Sepharose beads with 1 ml of protein lysate (1 mg) to which 15 μg of designated GST-tagged protein was added. Samples were tumbled end-over-end for 2 h at 5 °C. Proteins complexed to GSH-Sepharose beads were recovered by centrifuging samples at 1000 × g for 5 min at 5 °C. Supernatants of the samples were discarded, and Sepharose pellets were washed three times in lysis buffer and three times in lysis buffer containing 250 mm NaCl and suspended in 2× SDS-PAGE sample buffer. Samples were heat-denatured at 100 °C for 5 min and applied to wells of SDS-polyacrylamide gels prepared with 8 and 12% discontinuous resolving acrylamide gels, ⅔ and ⅓ length, respectively. After electrophoretic separation, proteins in the 8% acrylamide resolving gels were Western transferred onto PVDF membranes for immunoblot analysis as described above, and proteins retained in the 12% acrylamide resolving gels were stained with Coomassie Blue G-250 (Sigma-Aldrich). The insert from the plasmid pUSE-Src wild type and Y528F was subcloned by PCR and inserted into the NotI-BamHI sites of lentiviral vector pHAGE2-FullEF1a-ZsGreen-IRES-dTomatoW (23Schrecengost R.S. Riggins R.B. Thomas K.S. Guerrero M.S. Bouton A.H. Breast cancer antiestrogen resistance-3 expression regulates breast cancer cell migration through promotion of p130cas membrane localization and membrane ruffling.Cancer Res. 2007; 67: 6174-6182Crossref PubMed Scopus (56) Google Scholar). This construct plus packaging plasmids were transfected into HEK-293T cells in 10-cm plates at 50–70% confluence using Fugene 6 (Roche Applied Science). Medium containing packaged phage particles was harvested, and phage particles were concentrated by centrifugation (SW28 Beckman rotor 1.5 h at 16,500 rpm). For lentiviral transduction, 50 μl of the concentrated lentiviral supernatants was added with Polybrene (final concentration 8 μg/ml) to MCF-7 and BCAR3/MCF-7 cells cultured in 6-well plates, followed by centrifugation of the plates at 800 × g for 45 min. The transduced cells were trypsinized and resuspended in DMEM with 3% FCS and sorted for dTomato in a MoFlo apparatus (Beckman Coulter). Src overexpression was confirmed by Western analysis. For immunofluorescence studies, cell lines were cultured on fibronectin-coated coverslips (BD Biosciences) as described previously and fixed using 3.7% paraformaldehyde at 37 °C (24Vanden Borre P. Near R.I. Makkinje A. Mostoslavsky G. Lerner A. BCAR3/AND-34 can signal independent of complex formation with CAS family members or the presence of p130cas.Cell. Signal. 2011; 23: 1030-1040Crossref PubMed Scopus (18) Google Scholar). Following incubation with primary antibodies, including anti-c-Src polyclonal antibodies, anti-p130cas mouse monoclonal antibody, and anti-HA mouse monoclonal antibody, cells were probed with Alexa Fluor 350 goat anti-rabbit and Alexa Fluor 488 goat anti-mouse secondary antibodies (Invitrogen). All antibodies were diluted 1:100 in TBS with 2% BSA. After incubation with the antibodies, cells were rinsed several times in TBS prior to being mounted on slides with either Prolong Gold anti-fade reagent with DAPI or Slow Fade Gold without DAPI (Invitrogen) and allowed to cure overnight. Imaging was performed on either a Nikon TE2000-E microscope or a Zeiss LSM 710 Live Duo scan confocal microscope. Images were captured using NIS Elements (Nikon) software and processed using ImageJ (National Institutes of Health) and Photoshop (Adobe). To examine the effect of BCAR3 expression on Src domain interaction with p130cas, purified GST, GST-Src SH2 domain, or GST-Src SH3 domain were incubated with lysates from MCF-7 cells or a stable HA-BCAR3 transfectant of MCF-7, BCAR3/MCF-7 cells. Only GST-Src SH3 associated with endogenous p130cas, and this occurred only in the presence of HA-BCAR3 expression in BCAR3/MCF-7 cells (Fig. 1A). As expected, given that BCAR3 induces phosphorylation of p130cas that results in reduced mobility in low bisacrylamide gels, the predominant form of endogenous p130cas brought down by GST-Src SH3 domain in BCAR3/MCF-7 cells migrated more slowly than the endogenous predominant form of p130cas present in MCF7 cells (Fig. 1A) (26Makkinje A. Near R.I. Infusini G. Vanden Borre P. Bloom A. Cai D. Costello C.E. Lerner A. AND-34/BCAR3 regulates adhesion-dependent p130cas serine phosphorylation and breast cancer cell growth pattern.Cell. Signal. 2009; 21: 1423-1435Crossref PubMed Scopus (17) Google Scholar). To confirm BCAR3-induced association of the Src SH3 domain with p130cas using overexpressed epitope-tagged p130cas, a GST pull-down assay was performed on MCF-7 or BCAR3/MCF-7 cells transiently transfected with HA-tagged p130cas. GST-Src SH3 robustly associated with HA-tagged p130cas only in lysates from BCAR3/MCF-7 ce" @default.
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