The c-Src Tyrosine Kinase Regulates Signaling of the Human DF3/MUC1 Carcinoma-associated Antigen with GSK3β and β-Catenin

Abstract

The DF3/MUC1 mucin-like glycoprotein is aberrantly overexpressed in most human carcinomas. The cytoplasmic domain of MUC1 interacts with glycogen synthase kinase 3β (GSK3β) and thereby decreases binding of MUC1 and β-catenin. The present studies demonstrate that MUC1 associates with the c-Src tyrosine kinase. c-Src phosphorylates the MUC1 cytoplasmic domain at a YEKV motif located between sites involved in interactions with GSK3β and β-catenin. The results demonstrate that the c-Src SH2 domain binds directly to pYEKV and inhibits the interaction between MUC1 and GSK3β. Moreover and in contrast to GSK3β, in vitro andin vivo studies demonstrate that c-Src-mediated phosphorylation of MUC1 increases binding of MUC1 and β-catenin. The findings support a novel role for c-Src in regulating interactions of MUC1 with GSK3β and β-catenin. The DF3/MUC1 mucin-like glycoprotein is aberrantly overexpressed in most human carcinomas. The cytoplasmic domain of MUC1 interacts with glycogen synthase kinase 3β (GSK3β) and thereby decreases binding of MUC1 and β-catenin. The present studies demonstrate that MUC1 associates with the c-Src tyrosine kinase. c-Src phosphorylates the MUC1 cytoplasmic domain at a YEKV motif located between sites involved in interactions with GSK3β and β-catenin. The results demonstrate that the c-Src SH2 domain binds directly to pYEKV and inhibits the interaction between MUC1 and GSK3β. Moreover and in contrast to GSK3β, in vitro andin vivo studies demonstrate that c-Src-mediated phosphorylation of MUC1 increases binding of MUC1 and β-catenin. The findings support a novel role for c-Src in regulating interactions of MUC1 with GSK3β and β-catenin. adenomatous polyposis coli glycogen synthase kinase 3β cytoplasmic domain glutathione S-transferase polyacrylamide gel electrophoresis monoclonal antibody epidermal growth factor receptor platelet-derived growth factor receptor phosphotyrosine β-catenin, a component of the adherens junctions of mammalian epithelial cells, binds directly to the cytoplasmic domain of the transmembrane E-cadherin protein that functions in Ca2+-dependent epithelial cell-cell interactions (1Takeichi M. Annu. Rev. Biochem. 1990; 59: 237-252Crossref PubMed Scopus (1113) Google Scholar). In turn, α-catenin binds to β-catenin and thereby links the complex to the actin cytoskeleton (2Jou T. Stewart D. Stappert J. Nelson W. Marrs J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5067-5071Crossref PubMed Scopus (305) Google Scholar). 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Cell. 1996; 86: 391-399Abstract Full Text Full Text PDF PubMed Scopus (1625) Google Scholar, 15Brunner E. Peter O. Schweizer L. Basler K. Nature. 1997; 385: 829-833Crossref PubMed Scopus (447) Google Scholar). The finding that β-catenin and GSK3β interact with the cytoplasmic domain of the DF3/MUC1 mucin-like glycoprotein has supported the involvement of an additional pathway in β-catenin signaling (16Yamamoto M. Bharti A. Li Y. Kufe D. J. Biol. Chem. 1997; 272: 12492-12494Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 17Li Y. Bharti A. Chen D. Gong J. Kufe D. Mol. Cell. Biol. 1998; 18: 7216-7224Crossref PubMed Scopus (225) Google Scholar). MUC1 is highly overexpressed by human carcinomas (18Kufe D. Inghirami G. Abe M. Hayes D. Justi-Wheeler H. Schlom J. Hybridoma. 1984; 3: 223-232Crossref PubMed Scopus (502) Google Scholar). In addition, whereas MUC1 expression is restricted to the apical borders of normal secretory epithelial cells, MUC1 is aberrantly expressed by carcinoma cells at high levels throughout the cytoplasm and over the entire cell surface (18Kufe D. Inghirami G. Abe M. Hayes D. Justi-Wheeler H. Schlom J. Hybridoma. 1984; 3: 223-232Crossref PubMed Scopus (502) Google Scholar, 19Friedman E.L. Hayes D.F. Kufe D.W. Cancer Res. 1986; 46: 5189-5194PubMed Google Scholar, 20Perey L. Hayes D.F. Maimonis P. Abe M. O'Hara C. Kufe D.W. Cancer Res. 1992; 52: 2563-3568PubMed Google Scholar). The MUC1 protein consists of an N-terminal ectodomain with variable numbers of 20-amino acid tandem repeats that are subject to extensive O-glycosylation (21Gendler S. Taylor-Papadimitriou J. Duhig T. Rothbard J. Burchell J.A. J. Biol. Chem. 1988; 263: 12820-12823Abstract Full Text PDF PubMed Google Scholar, 22Siddiqui J. Abe M. Hayes D. Shani E. Yunis E. Kufe D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2320-2323Crossref PubMed Scopus (281) Google Scholar). The C-terminal region includes a transmembrane domain and a 72-amino acid cytoplasmic tail. MUC1 is subject to proteolytic cleavage and the large ectodomain containing the tandem repeats can remain complexed to the 25-kDa C-terminal subunit or undergo release from the cell surface (23Ligtenberg M. Buijs F. Vos H. Hilkens J. Cancer Res. 1992; 52: 223-232Google Scholar). β-catenin binds directly to MUC1 at a SAGNGGSSL motif in the cytoplasmic domain (16Yamamoto M. Bharti A. Li Y. Kufe D. J. Biol. Chem. 1997; 272: 12492-12494Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). Similar SXXXXXSSL sites in E-cadherin and APC are responsible for β-catenin interactions (4Rubinfield B. Souza B. Albert I. Muller O. Chamberlain S. Masiarz S. Munemitsu S. Polakis P. Science. 1993; 262: 1731-1734Crossref PubMed Scopus (1183) Google Scholar, 5Rubinfield B. Souza B. Albert I. Muller O. Munemitsu S. Polakis P. J. Biol. Chem. 1995; 270: 5549-5555Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 6Su L.-K. Vogelstein B. Kinzler K.W. Science. 1993; 262: 1734-1737Crossref PubMed Scopus (1119) Google Scholar). GSK3β also binds directly to MUC1 and phosphorylates serine in a DRSPY site adjacent to that for the β-catenin interaction (17Li Y. Bharti A. Chen D. Gong J. Kufe D. Mol. Cell. Biol. 1998; 18: 7216-7224Crossref PubMed Scopus (225) Google Scholar). GSK3β-mediated phosphorylation of MUC1 decreases the association of MUC1 and β-catenin (17Li Y. Bharti A. Chen D. Gong J. Kufe D. Mol. Cell. Biol. 1998; 18: 7216-7224Crossref PubMed Scopus (225) Google Scholar). The present studies demonstrate that the c-Src tyrosine kinase interacts directly with MUC1. A YEKV motif in the MUC1 cytoplasmic domain (CD) has been identified as a site for c-Src phosphorylation. The results demonstrate that c-Src regulates the interactions of MUC1 with GSK3β and β-catenin. Human ZR-75-1 breast carcinoma cells were grown in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum, 100 μg/ml streptomycin, 100 units/ml penicillin, and 2 mml-glutamine. 293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% heat-inactivated fetal bovine serum, 100 μg/ml streptomycin, and 100 units/ml penicillin. Subconfluent cells were disrupted on ice in lysis buffer (50 mm Tris-HCl, pH 7.6, 150 mmNaCl, 0.1% Nonidet P-40, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mm phenylmethylsulfonyl fluoride, and 1 mmdithiothreitol) for 30 min. Lysates were cleared by centrifugation at 14,000 × g for 20 min. Equal amounts of protein from cell lysates were incubated with normal mouse IgG, MAb DF3 (anti-MUC1) (18Kufe D. Inghirami G. Abe M. Hayes D. Justi-Wheeler H. Schlom J. Hybridoma. 1984; 3: 223-232Crossref PubMed Scopus (502) Google Scholar), anti-c-Src (Upstate Biotechnology, Lake Placid, NY), or the rabbit anti-DF3-E antibody prepared against a peptide derived from the MUC1 extracellular domain (HDVETQFNQYKTEAAS). After incubation for 2 h at 4 °C, the immune complexes were precipitated with protein G-agarose. The immunoprecipitates were washed with lysis buffer, separated by SDS-PAGE, and transferred to nitrocellulose membranes. The immunoblots were probed with 500 ng/ml anti-MUC1 or 1 μg/ml anti-c-Src. Reactivity was detected with horseradish peroxidase-conjugated second antibodies and chemiluminescence (ECL, Amersham Pharmacia Biotech). The MUC1/CD(Y46F) and MUC1(Y46F) mutants were generated using site-directed mutagenesis (QuikChange; Stratagene, La Jolla, CA) to change Tyr-46 to Phe. Kinase-inactive c-Src was similarly generated by mutation of Lys-295 to Arg (K295R) (24Kamps M.P. Sefton B.M. Mol. Cell. Biol. 1986; 6: 751-757Crossref PubMed Scopus (112) Google Scholar). Purified wild-type and mutant MUC1/CD proteins were incubated with 1.5 units of purified c-Src (Oncogene Research Products, Cambridge, MA) in 20 μl of kinase buffer (20 mm Tris-HCl, pH 7.6, 10 mmMgCl2, 5 mm dithiothreitol). The reaction was initiated by addition of 10 μCi [γ-32P]ATP. After incubation for 15 min at 30 °C, the reaction was stopped by addition of sample buffer and boiling for 5 min. Phosphorylated proteins were separated by SDS-PAGE and analyzed by autoradiography. Purified wild-type and mutant MUC1/CD proteins were incubated with 1.5 units of c-Src in the presence or absence of 200 μm ATP for 30 min at 30 °C. GST, GST-Src-SH3, GST-Src-SH3De90/92 (Ref. 25Shiue L. Zoller M. Brugge J. J. Biol. Chem. 1995; 270: 10498-10502Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, provided by Dr. J. Brugge, Harvard Medical School), GST-Src-SH2, or GST-β-catenin bound to glutathione beads was then added, and the reaction was incubated for 1 h at 4 °C. After washing, the proteins were subjected to SDS-PAGE and immunoblot analysis with the anti-MUC1/CD antibody that was generated against the cytoplasmic domain (17Li Y. Bharti A. Chen D. Gong J. Kufe D. Mol. Cell. Biol. 1998; 18: 7216-7224Crossref PubMed Scopus (225) Google Scholar). In other studies, GST-MUC1/CD bound to glutathione beads was incubated with 1.5 units of c-Src in the presence and absence of 200 μm ATP for 30 min at 30 °C before adding 0.1 mg of purified GSK3β (New England BioLabs) for an additional 1 h. Precipitated proteins were analyzed by immunoblotting with anti-GSK3β. ZR-75-1 or 293 cells were transiently transfected with pCMV, pCMV-MUC1, pCMV-c-Src (provided by Dr. R. Rickles, ARIAD Pharmaceuticals, Inc., Cambridge, MA) or pCMV-c-Src(K295R) using electroporation methods. Efficiency of transient transfections ranged from 40–50% of ZR-75-1 cells and 70–80% of 293 cells. Cell lysates were prepared at 48 h after transfection. To determine whether DF3/MUC1 forms a complex with c-Src, anti-MUC1 immunoprecipitates from lysates of human ZR-75-1 cells were analyzed by immunoblotting with anti-c-Src. The results demonstrate that c-Src coprecipitates with MUC1 (Fig.1 A, left). In the reciprocal experiment, analysis of anti-c-Src immunoprecipitates by immunoblotting with anti-MUC1 confirmed the association of MUC1 and c-Src (Fig. 1 A, right). Similar results have been obtained in human HeLa cells (data not shown). To assess whether the binding is direct, we incubated purified His-tagged MUC1 cytoplasmic domain (His-MUC1/CD) with a GST fusion protein that contains the c-Src SH3 domain. Analysis of the adsorbate to glutathione beads by immunoblotting with anti-MUC1/CD demonstrated binding of MUC1/CD to GST-Src SH3, and not GST or a GST-Src SH2 fusion protein (Fig.1 B). As an additional control, His-MUC1/CD was incubated with a GST fusion protein containing a mutated c-Src SH3 domain (GST-Src SH3De90/92) (26Weng Z. Rickles R. Feng S. Richard S. Shaw A. Schreiber S. Brugge J. Mol. Cell. Biol. 1995; 15: 5627-5634Crossref PubMed Scopus (110) Google Scholar). The finding that MUC1/CD binds to wild-type c-Src SH3 but not the mutant supported a direct interaction between MUC1 and c-Src (Fig. 1 C). To determine whether MUC1/CD is a substrate for c-Src, we incubated MUC1/CD with purified c-Src and [γ-32P]ATP. Analysis of the reaction products by SDS-PAGE and autoradiography demonstrated c-Src-mediated phosphorylation of MUC1/CD (Fig. 2 A). Previous studies have demonstrated that GSK3β phosphorylates MUC1/CD on Ser at a DRSPYEKV site (17Li Y. Bharti A. Chen D. Gong J. Kufe D. Mol. Cell. Biol. 1998; 18: 7216-7224Crossref PubMed Scopus (225) Google Scholar). As the adjacent YEKV sequence represents a consensus for c-Src phosphorylation, MUC1/CD was generated with a FEKV mutation (Fig. 2 B). Incubation of MUC1/CD(Y46F) with c-Src demonstrated a decrease in phosphorylation as compared with that found with wild-type MUC1/CD (Fig. 2 C). These findings indicate that c-Src phosphorylates MUC1/CD predominantly but not exclusively at the YEKV site. As the c-Src SH2 domain interacts with a preferred pYEEI sequence (27Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Lechleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2391) Google Scholar), c-Src-mediated phosphorylation of YEKV in MUC1/CD provides a potential site for c-Src SH2 binding. To determine whether the c-Src SH2 domain binds to phosphorylated MUC1/CD, we incubated MUC1/CD with c-Src and ATP and then assessed binding to GST-Src SH2. The results demonstrate that GST-Src SH2 associates with phosphorylated but not unphosphorylated MUC1/CD (Fig. 2 D). Moreover, compared with MUC1/CD, there was substantially less binding of GST-Src SH2 to the MUC1/CD(Y46F) mutant that had been incubated with c-Src and ATP (Fig. 2 D). These results support c-Src-mediated phosphorylation of MUC1/CD and thereby a direct interaction of phosphorylated MUC1/CD with the c-Src SH2 domain. As the c-Src phosphorylation site on MUC1/CD resides next to the binding and phosphorylation site for GSK3β (17Li Y. Bharti A. Chen D. Gong J. Kufe D. Mol. Cell. Biol. 1998; 18: 7216-7224Crossref PubMed Scopus (225) Google Scholar), we asked if the interaction of MUC1/CD with c-Src affects that with GSK3β. GST-MUC1/CD was incubated with c-Src and ATP before addition of GSK3β. Analysis of proteins precipitated with glutathione beads demonstrated that c-Src-mediated phosphorylation of MUC1/CD is associated with a decrease in binding of MUC1/CD and GSK3β (Fig.3 A). To assess the effects of c-Src on the interaction of MUC1/CD and GSK3β in vivo, ZR-75-1 cells were transfected to express the empty vector or c-Src. Anti-MUC1 immunoprecipitates were analyzed by immunoblotting with anti-GSK3β. The results demonstrate that c-Src also decreases the interaction of MUC1 and GSK3β in vivo (Fig.3 B). These findings indicate that GSK3β interacts with MUC1/CD by a c-Src-dependent mechanism. Phosphorylation of MUC1 by GSK3β decreases binding of MUC1 to β-catenin in vitro and in cells (17Li Y. Bharti A. Chen D. Gong J. Kufe D. Mol. Cell. Biol. 1998; 18: 7216-7224Crossref PubMed Scopus (225) Google Scholar). To determine whether c-Src-mediated phosphorylation of MUC1 affects the interaction of MUC1 with β-catenin, we incubated MUC1/CD with c-Src and ATP. Phosphorylated and unphosphorylated MUC1/CD were then incubated with GST or GST-β-catenin. Similar studies were performed with the MUC1/CD(Y46F) mutant. Analysis of proteins bound to glutathione beads by immunoblotting with anti-MUC1/CD demonstrated that c-Src-mediated phosphorylation of MUC1/CD increases binding of MUC1/CD to GST-β-catenin (Fig. 4 A). By contrast, there was no detectable binding of phosphorylated or unphosphorylated MUC1/CD to GST (Fig. 4 A). Studies performed with MUC1/CD(Y46F) demonstrated that c-Src-dependent phosphorylation of the YEKV site on MUC1/CD is necessary for the formation of MUC1/CD-β-catenin complexes (Fig. 4 A). To assess whether c-Src affects the interaction of MUC1 and β-cateninin vivo, MUC1-positive ZR-75-1 cells were transfected with pCMV or pCMV/c-Src. Anti-MUC1 immunoprecipitates prepared from the transfected cells were subjected to immunoblot analysis with anti-c-Src, anti-P-Tyr, and anti-β-catenin. The results demonstrate that c-Src associates with MUC1 in cells and induces tyrosine phosphorylation of MUC1 (Fig. 4 B, left panel). In addition, c-Src expression induced the interaction of MUC1 and β-catenin (Fig. 4 B, left panel). The finding that the MUC1 C-terminal subunit and not the large ectodomain is subject to tyrosine phosphorylation is consistent with an interaction between c-Src and MUC1/CD (Fig. 4 B, left panel). To confirm these findings, we performed immunoprecipitation studies with the anti-DF3-E antibody that was generated against the extracellular region of the C-terminal subunit. Immunoblot analysis of the precipitates demonstrated c-Src-mediated phosphorylation of the MUC1 C-terminal subunit and increased binding of MUC1/CD to β-catenin (Fig. 4 B, middle panel). By contrast, expression of a kinase-inactive c-Src(K295R) mutant resulted in less phosphorylation of MUC1 on tyrosine as compared with the control (Fig.4 B, middle panel). Moreover, expression of c-Src(K295R) was associated with a decreased interaction between MUC1/CD and β-catenin (Fig. 4 B, middle panel). To extend these findings, MUC1-negative 293 cells (17Li Y. Bharti A. Chen D. Gong J. Kufe D. Mol. Cell. Biol. 1998; 18: 7216-7224Crossref PubMed Scopus (225) Google Scholar) were transfected to express MUC1 or MUC1(Y46F) in which the CD YEKV site has been mutated to FEKV. There was a low but detectable level of MUC1 binding to endogenous c-Src (Fig. 4 C, left panel). Moreover, cotransfection of MUC1 and c-Src was associated with increased formation of MUC1-c-Src complexes (Fig. 4 C,middle panel). Cotransfection of MUC1 and c-Src was also associated with increased tyrosine phosphorylation of MUC1 and binding of MUC1 and c-Src (Fig. 4 C, middle panel). By contrast, cotransfection of MUC1(Y46F) and c-Src resulted in little binding of these proteins (Fig. 4 C, middle panel). Moreover, there was little if any tyrosine phosphorylation of MUC1(Y46F) (Fig. 4 C, middle panel). Importantly, cotransfection of MUC1 but not MUC1(Y46F) with c-Src induced the binding of MUC1 and β-catenin (Fig. 4 C,middle panel). These findings demonstrate that c-Src-mediated phosphorylation of the MUC1 YEKV site increases the interaction of MUC1 and β-catenin in cells. The present findings thus demonstrate that signaling of β-catenin and the MUC1 carcinoma-associated protein is regulated by the c-Src tyrosine kinase. Previous studies have shown that β-catenin interacts with the cytoplasmic domain of MUC1 and that GSK3β inhibits the formation of MUC1/CD-β-catenin complexes (17Li Y. Bharti A. Chen D. Gong J. Kufe D. Mol. Cell. Biol. 1998; 18: 7216-7224Crossref PubMed Scopus (225) Google Scholar). By contrast, the present work supports a model in which c-Src phosphorylates MUC1/CD and promotes the interaction of MUC1/CD and β-catenin. The c-Src kinase functions in signaling pathways activated by heterotrimeric G protein-coupled receptors (28Malarkey K. Belham C. Paul A. Graham A. Mclees A. Scott P. Plevin R. Biochem. J. 1995; 309: 361-375Crossref PubMed Scopus (266) Google Scholar) and neuronal ion channels (29Holmes T. Fadool D. Levitan I. J. Neurosci. 1996; 16: 581-590Crossref Google Scholar, 30Yu S. Yeh C. Sensi S. Gwag B. Canzoniero L. Farhangrazi Z. Ying H. Tian M. Dugan L. Choi D. Science. 1997; 278: 114-117Crossref PubMed Scopus (536) Google Scholar, 31van Hoek M. Allen C. Parsons S. Biochem. J. 1997; 326: 271-277Crossref PubMed Scopus (20) Google Scholar). c-Src also participates in the transduction of signals from the epidermal growth factor receptor (EGF-R), platelet-derived growth factor receptor (PDGF-R) and other receptor tyrosine kinases (32Biscardi J. Tice D. Parsons S. Adv. Cancer Res. 1999; 76: 61-119Crossref PubMed Google Scholar). The available evidence indicates that c-Src phosphorylates the EGF-R and thereby contributes to mitogenesis and transformation (33Maa M. Leu T. McCarley D. Schatzman R. Parsons S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6981-6985Crossref PubMed Scopus (290) Google Scholar). Mitogenesis induced by PDGF is also positively regulated by c-Src-mediated phosphorylation of the PDGF-R (34Hansen K. Johnell M. A. C. U. C. C. L. EMBO J. 1996; 15: PubMed Scopus Google Scholar). Other of c-Src that and have with the actin cytoskeleton (32Biscardi J. Tice D. Parsons S. Adv. Cancer Res. 1999; 76: 61-119Crossref PubMed Google Scholar). These findings have provided support for the involvement of c-Src in the of cell and The present studies extend these findings by that the MUC1 carcinoma-associated is also a substrate for c-Src and that interaction of MUC1 with GSK3β and β-catenin are regulated by c-Src-dependent Brugge for and Rickles for and Hilkens for