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The c-Src Tyrosine Kinase Regulates Signaling of the Human DF3/MUC1 Carcinoma-associated Antigen with GSK3beta and beta -Catenin*

Yongqing Li, Hiroaki Kuwahara, Jian Ren, Gengyun Wen, and Donald KufeDagger

From the Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115

Received for publication, October 24, 2000, and in revised form, January 9, 2001



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

The DF3/MUC1 mucin-like glycoprotein is aberrantly overexpressed in most human carcinomas. The cytoplasmic domain of MUC1 interacts with glycogen synthase kinase 3beta (GSK3beta ) and thereby decreases binding of MUC1 and beta -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 GSK3beta and beta -catenin. The results demonstrate that the c-Src SH2 domain binds directly to pYEKV and inhibits the interaction between MUC1 and GSK3beta . Moreover and in contrast to GSK3beta , in vitro and in vivo studies demonstrate that c-Src-mediated phosphorylation of MUC1 increases binding of MUC1 and beta -catenin. The findings support a novel role for c-Src in regulating interactions of MUC1 with GSK3beta and beta -catenin.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

beta -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 (1). In turn, alpha -catenin binds to beta -catenin and thereby links the complex to the actin cytoskeleton (2). Formation of the cadherin-catenin complex is essential for adherens junction function (3). In the cytosol, beta -catenin binds directly to the adenomatous polyposis coli (APC)1 tumor suppressor (4-6). Phosphorylation of APC and beta -catenin by GSK3beta increases the formation of APC-beta -catenin complexes (7) and targets beta -catenin for ubiquitination and degradation by the 26 S proteosome (8-10). Cells that express certain APC mutants or are APC deficient thus exhibit increased levels of cytosolic beta -catenin (11). Other studies have shown that beta -catenin forms complexes with members of the T-cell factor/leukocyte-enhancing factor (Tcf/LEF-1) family of transcription factors (12-14) and functions in the activation of gene expression (13-15).

The finding that beta -catenin and GSK3beta interact with the cytoplasmic domain of the DF3/MUC1 mucin-like glycoprotein has supported the involvement of an additional pathway in beta -catenin signaling (16, 17). MUC1 is highly overexpressed by human carcinomas (18). 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 (18-20). 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 (21, 22). 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 (23). beta -catenin binds directly to MUC1 at a SAGNGGSSL motif in the cytoplasmic domain (16). Similar SXXXXXSSL sites in E-cadherin and APC are responsible for beta -catenin interactions (4-6). GSK3beta also binds directly to MUC1 and phosphorylates serine in a DRSPY site adjacent to that for the beta -catenin interaction (17). GSK3beta -mediated phosphorylation of MUC1 decreases the association of MUC1 and beta -catenin (17).

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 GSK3beta and beta -catenin.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

Cell Culture-- 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 mM L-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.

Lysate Preparation-- Subconfluent cells were disrupted on ice in lysis buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.1% Nonidet P-40, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol) for 30 min. Lysates were cleared by centrifugation at 14,000 × g for 20 min.

Immunoprecipitation and Immunoblotting-- Equal amounts of protein from cell lysates were incubated with normal mouse IgG, MAb DF3 (anti-MUC1) (18), 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).

Preparation of MUC1 and c-Src Mutants-- 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) (24).

In Vitro Phosphorylation-- 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 mM MgCl2, 5 mM dithiothreitol). The reaction was initiated by addition of 10 µCi [gamma -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.

Binding Studies-- 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. 25, provided by Dr. J. Brugge, Harvard Medical School), GST-Src-SH2, or GST-beta -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 (17). 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 GSK3beta (New England BioLabs) for an additional 1 h. Precipitated proteins were analyzed by immunoblotting with anti-GSK3beta .

Transient Transfection Studies-- 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.


    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

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. 1A, 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. 1A, 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. 1B). 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) (26). 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. 1C).



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Fig. 1.   Interaction of MUC1 with c-Src. A, lysates from ZR-75-1 cells were subjected to immunoprecipitation with anti-MUC1 (MAb DF3; left panel) or anti-c-Src (right panel). Mouse IgG was used as a control. The immunoprecipitates and lysates not subjected to immunoprecipitation were analyzed by immunoblotting with anti-c-Src (left panel) and anti-MUC1 (right panel). B, purified MUC1/CD was incubated with GST, GST-Src-SH2, or GST-Src-SH3 for 1 h at 4 °C. Proteins precipitated with glutathione-Sepharose 4B beads were subjected to SDS-PAGE and immunoblot analysis with anti-MUC1/CD. C, purified MUC1/CD was incubated with GST, GST-Src-SH3, or GST-Src-SH3De90/92 (deletion of amino acids 90/92). Adsorbates to glutathione beads were subjected to immunoblot analysis with anti-MUC1/CD (left panel). The gel was stained with Coomassie Blue to assess loading of the wild-type and mutant SH3 domains (right panel).

To determine whether MUC1/CD is a substrate for c-Src, we incubated MUC1/CD with purified c-Src and [gamma -32P]ATP. Analysis of the reaction products by SDS-PAGE and autoradiography demonstrated c-Src-mediated phosphorylation of MUC1/CD (Fig. 2A). Previous studies have demonstrated that GSK3beta phosphorylates MUC1/CD on Ser at a DRSPYEKV site (17). As the adjacent YEKV sequence represents a consensus for c-Src phosphorylation, MUC1/CD was generated with a FEKV mutation (Fig. 2B). Incubation of MUC1/CD(Y46F) with c-Src demonstrated a decrease in phosphorylation as compared with that found with wild-type MUC1/CD (Fig. 2C). 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 (27), 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. 2D). 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. 2D). 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.



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Fig. 2.   Phosphorylation of MUC1 by c-Src in vitro. A, GST or GST-MUC1/CD were incubated with c-Src and [gamma -32P]ATP. The reaction products were analyzed by SDS-PAGE and autoradiography. B, schematic representation of wild-type and mutant forms of MUC1/CD. TR, tandem repeat; TM, transmembrane; CD, cytoplasmic domain. Numbers (1-72) reflect amino acids in the CD. Underlined codons and amino acids are those that differ from the wild type. C, purified MUC1/CD and the MUC1/CD(Y46F) mutant were incubated with purified c-Src and [gamma -32P]ATP. As a control, MUC1/CD was incubated with only [gamma -32P]ATP. The reaction products were analyzed by SDS-PAGE and autoradiography (upper panel). Equal loading of the MUC1/CD proteins was assessed by Coomassie Blue staining (lower panel). D, purified MUC1/CD or MUC1/CD(Y46F) was incubated with c-Src in the presence or absence of ATP for 30 min at 30 °C. GST-Src-SH2 was added, and the reaction was incubated for 1 h at 4 °C. Proteins precipitated with glutathione beads were separated by SDS-PAGE and subjected to immunoblot analysis with anti-MUC1/CD (upper panel) and anti-P-Tyr (lower panel).

As the c-Src phosphorylation site on MUC1/CD resides next to the binding and phosphorylation site for GSK3beta (17), we asked if the interaction of MUC1/CD with c-Src affects that with GSK3beta . GST-MUC1/CD was incubated with c-Src and ATP before addition of GSK3beta . 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 GSK3beta (Fig. 3A). To assess the effects of c-Src on the interaction of MUC1/CD and GSK3beta 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-GSK3beta . The results demonstrate that c-Src also decreases the interaction of MUC1 and GSK3beta in vivo (Fig. 3B). These findings indicate that GSK3beta interacts with MUC1/CD by a c-Src-dependent mechanism.



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Fig. 3.   Effect of c-Src-mediated phosphorylation of MUC1/CD on the interaction of MUC1 and GSK3beta . A, GST-MUC1/CD bound to glutathione beads was incubated with c-Src in the presence or absence of ATP for 1 h at 30 °C before adding GSK3beta for an additional 1 h. Proteins precipitated with the beads were separated by SDS-PAGE and subjected to immunoblot analysis with anti-GSK3beta . B, ZR-75-1 cells were transiently transfected with pCMV or pCMV/c-Src by electroporation. After 48 h, the cells were harvested, and lysates were subjected to immunoprecipitation (IP) with anti-MUC1. The immunoprecipitates were analyzed by immunoblotting with anti-c-Src and anti-GSK3beta .

Phosphorylation of MUC1 by GSK3beta decreases binding of MUC1 to beta -catenin in vitro and in cells (17). To determine whether c-Src-mediated phosphorylation of MUC1 affects the interaction of MUC1 with beta -catenin, we incubated MUC1/CD with c-Src and ATP. Phosphorylated and unphosphorylated MUC1/CD were then incubated with GST or GST-beta -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-beta -catenin (Fig. 4A). By contrast, there was no detectable binding of phosphorylated or unphosphorylated MUC1/CD to GST (Fig. 4A). 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-beta -catenin complexes (Fig. 4A). To assess whether c-Src affects the interaction of MUC1 and beta -catenin in 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-beta -catenin. The results demonstrate that c-Src associates with MUC1 in cells and induces tyrosine phosphorylation of MUC1 (Fig. 4B, left panel). In addition, c-Src expression induced the interaction of MUC1 and beta -catenin (Fig. 4B, 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. 4B, 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 beta -catenin (Fig. 4B, 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. 4B, middle panel). Moreover, expression of c-Src(K295R) was associated with a decreased interaction between MUC1/CD and beta -catenin (Fig. 4B, middle panel). To extend these findings, MUC1-negative 293 cells (17) 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. 4C, left panel). Moreover, cotransfection of MUC1 and c-Src was associated with increased formation of MUC1-c-Src complexes (Fig. 4C, 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. 4C, middle panel). By contrast, cotransfection of MUC1(Y46F) and c-Src resulted in little binding of these proteins (Fig. 4C, middle panel). Moreover, there was little if any tyrosine phosphorylation of MUC1(Y46F) (Fig. 4C, middle panel). Importantly, cotransfection of MUC1 but not MUC1(Y46F) with c-Src induced the binding of MUC1 and beta -catenin (Fig. 4C, middle panel). These findings demonstrate that c-Src-mediated phosphorylation of the MUC1 YEKV site increases the interaction of MUC1 and beta -catenin in cells.



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Fig. 4.   c-Src-mediated phosphorylation of the MUC1 YEKV site increases binding of MUC1 to beta -catenin. A, purified MUC1/CD and MUC1/CD(Y46F) were incubated with (+) or without (-) purified c-Src and ATP for 30 min at 30 °C. The MUC1/CD and MUC1/CD(Y46F) proteins were then incubated with GST or GST-beta -catenin for 1 h at 4 °C. Proteins precipitated with glutathione beads were subjected to immunoblot analysis with anti-MUC1/CD (upper panel) and anti-beta -catenin (lower panel). B, ZR-75-1 cells were transiently transfected with pCMV, pCMV-c-Src or pCMV-c-Src(K295R). After 48 h, the cells were harvested, and lysates were subjected to immunoprecipitation (IP) with anti-MUC1 (left) or anti-DF3-E (middle). The immunoprecipitates were analyzed by immunoblotting (IB) with anti-c-Src (upper panels), anti-P-Tyr (middle panels), and anti-beta -catenin (lower panels). Densitometric scanning of the anti-P-Tyr signals has demonstrated a 3.1 ± 1.5-fold (mean ± S.E. of three separate experiments) increase of c-Src-mediated phosphorylation of MUC1 as compared with that of control. Lysates not subjected to immunoprecipitation were also assayed by immunoblotting with anti-c-Src (right). C, 293 cells were transiently transfected with MUC1, MUC1+c-Src, or MUC1(Y46F)+c-Src. After 48 h, the cells were harvested, and lysates were subjected to immunoprecipitation with IgG or anti-MUC1. Left, the immunoprecipitates were analyzed by immunoblotting with anti-c-Src (upper panel), and lysates not subjected to immunoprecipitation were analyzed with anti-MUC1 (lower panel). Middle, the immunoprecipitates were analyzed by immunoblotting with anti-c-Src (upper panel), anti-P-Tyr (middle panel), and anti-beta -catenin (lower panel). Right, lysates not subjected to immunoprecipitation were assayed by immunoblotting with anti-MUC1 or anti-c-Src (upper and lower panels).

The present findings thus demonstrate that signaling of beta -catenin and the MUC1 carcinoma-associated protein is regulated by the c-Src tyrosine kinase. Previous studies have shown that beta -catenin interacts with the cytoplasmic domain of MUC1 and that GSK3beta inhibits the formation of MUC1/CD-beta -catenin complexes (17). By contrast, the present work supports a model in which c-Src phosphorylates MUC1/CD and promotes the interaction of MUC1/CD and beta -catenin. The c-Src kinase functions in signaling pathways activated by heterotrimeric G protein-coupled receptors (28) and neuronal ion channels (29-31). 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 (32). The available evidence indicates that c-Src phosphorylates the EGF-R and thereby contributes to mitogenesis and transformation (33). Mitogenesis induced by PDGF is also positively regulated by c-Src-mediated phosphorylation of the PDGF-R (34). Other substrates of c-Src that include focal adhesion kinase, p130Cas, and cortactin have functional associations with the actin cytoskeleton (32). These findings have collectively provided support for the involvement of c-Src in the integration of mitogenic, cell adhesion, and cytoskeletal responses. The present studies extend these findings by demonstrating that the MUC1 carcinoma-associated antigen is also a substrate for c-Src and that interaction of MUC1 with GSK3beta and beta -catenin are regulated by c-Src-dependent signals.


    ACKNOWLEDGEMENTS

We thank Joan Brugge for GST-Src-SH3 and GST-Src-SH3De90/92, Ricky Rickles for pCMV-c-Src, and John Hilkens for pCMV-MUC1 constructs.


    FOOTNOTES

* This work was supported by NCI, National Institutes of Health Grant CA87421.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 617-632-3141; Fax: 617-632-2934; E-mail: donald_kufe@dfci.harvard.edu.

Published, JBC Papers in Press, January 10, 2001, DOI 10.1074/jbc.C000754200


    ABBREVIATIONS

The abbreviations used are: APC, adenomatous polyposis coli; GSK3beta , glycogen synthase kinase 3beta ; CD, cytoplasmic domain; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; MAb, monoclonal antibody; EGF-R, epidermal growth factor receptor; PDGF-R, platelet-derived growth factor receptor; P-Tyr, phosphotyrosine.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES


1. Takeichi, M. (1990) Annu. Rev. Biochem. 59, 237-252[CrossRef][Medline] [Order article via Infotrieve]
2. Jou, T., Stewart, D., Stappert, J., Nelson, W., and Marrs, J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 5067-5071[Abstract]
3. Kawanishi, J., Kato, J., Sasaki, K., Fujii, S., Watanabe, N., and Niitsu, Y. (1995) Mol. Cell. Biol. 15, 1175-1181[Abstract]
4. Rubinfield, B., Souza, B., Albert, I., Muller, O., Chamberlain, S., Masiarz, S., Munemitsu, S., and Polakis, P. (1993) Science 262, 1731-1734[Medline] [Order article via Infotrieve]
5. Rubinfield, B., Souza, B., Albert, I., Muller, O., Munemitsu, S., and Polakis, P. (1995) J. Biol. Chem. 270, 5549-5555[Abstract/Free Full Text]
6. Su, L.-K., Vogelstein, B., and Kinzler, K. W. (1993) Science 262, 1734-1737[Medline] [Order article via Infotrieve]
7. Rubinfield, B., Albert, I., Porfiri, E., Fiol, C., Munemitsu, S., and Polakis, P. (1996) Science 272, 1023-1026[Abstract]
8. Aberie, H., Bauer, A., Stappert, J., Kispert, A., and Kemler, R. (1997) EMBO J. 16, 3797-3804[Abstract/Free Full Text]
9. Orford, K., Crockett, C., Jensen, J., Weissman, A., and Byers, S. (1997) J. Biol. Chem. 272, 24735-24738[Abstract/Free Full Text]
10. Solomon, D., Sacco, P., Roy, S., Simcha, I., Johnson, K., Wheelock, M., and Ben-Ze'ev, A. (1997) J. Cell Biol. 139, 1325-1335[Abstract/Free Full Text]
11. Munemitsu, S., Albert, I., Souza, B., Rubinfeld, B., and Polakis, P. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 3046-3050[Abstract]
12. Behrens, J., von Kries, J. P., Kühl, M., Bruhn, L., Wedlich, D., Grosschedi, R., and Birchmeier, W. (1996) Nature 382, 638-642[CrossRef][Medline] [Order article via Infotrieve]
13. Huber, O., Korn, R., McLaughlin, J., Ohsugi, M., Hermann, B. G., and Kemler, R. (1996) Mech. Dev. 59, 3-10[CrossRef][Medline] [Order article via Infotrieve]
14. Molenaar, M., van de Wetering, M., Oosterwegel, M., Peterson-Maduro, J., Godsave, S., Korinek, V., Roose, J., Destrée, O., and Clevers, H. (1996) Cell 86, 391-399[Medline] [Order article via Infotrieve]
15. Brunner, E., Peter, O., Schweizer, L., and Basler, K. (1997) Nature 385, 829-833[CrossRef][Medline] [Order article via Infotrieve]
16. Yamamoto, M., Bharti, A., Li, Y., and Kufe, D. (1997) J. Biol. Chem. 272, 12492-12494[Abstract/Free Full Text]
17. Li, Y., Bharti, A., Chen, D., Gong, J., and Kufe, D. (1998) Mol. Cell. Biol. 18, 7216-7224[Abstract/Free Full Text]
18. Kufe, D., Inghirami, G., Abe, M., Hayes, D., Justi-Wheeler, H., and Schlom, J. (1984) Hybridoma 3, 223-232[Medline] [Order article via Infotrieve]
19. Friedman, E. L., Hayes, D. F., and Kufe, D. W. (1986) Cancer Res. 46, 5189-5194[Abstract]
20. Perey, L., Hayes, D. F., Maimonis, P., Abe, M., O'Hara, C., and Kufe, D. W. (1992) Cancer Res. 52, 2563-3568[Abstract]
21. Gendler, S., Taylor-Papadimitriou, J., Duhig, T., Rothbard, J., and Burchell, J. A. (1988) J. Biol. Chem. 263, 12820-12823[Abstract/Free Full Text]
22. Siddiqui, J., Abe, M., Hayes, D., Shani, E., Yunis, E., and Kufe, D. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 2320-2323[Abstract]
23. Ligtenberg, M., Buijs, F., Vos, H., and Hilkens, J. (1992) Cancer Res. 52, 223-232
24. Kamps, M. P., and Sefton, B. M. (1986) Mol. Cell. Biol. 6, 751-757[Medline] [Order article via Infotrieve]
25. Shiue, L., Zoller, M., and Brugge, J. (1995) J. Biol. Chem. 270, 10498-10502[Abstract/Free Full Text]
26. Weng, Z., Rickles, R., Feng, S., Richard, S., Shaw, A., Schreiber, S., and Brugge, J. (1995) Mol. Cell. Biol. 15, 5627-5634[Abstract]
27. Songyang, 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., and Cantley, L. C. (1993) Cell 72, 767-778[Medline] [Order article via Infotrieve]
28. Malarkey, K., Belham, C., Paul, A., Graham, A., Mclees, A., Scott, P., and Plevin, R. (1995) Biochem. J. 309, 361-375[Medline] [Order article via Infotrieve]
29. Holmes, T., Fadool, D., and Levitan, I. (1996) J. Neurosci. 16, 581-590
30. Yu, S., Yeh, C., Sensi, S., Gwag, B., Canzoniero, L., Farhangrazi, Z., Ying, H., Tian, M., Dugan, L., and Choi, D. (1997) Science 278, 114-117[Abstract/Free Full Text]
31. van Hoek, M., Allen, C., and Parsons, S. (1997) Biochem. J. 326, 271-277[Medline] [Order article via Infotrieve]
32. Biscardi, J., Tice, D., and Parsons, S. (1999) Adv. Cancer Res. 76, 61-119[Medline] [Order article via Infotrieve]
33. Maa, M., Leu, T., McCarley, D., Schatzman, R., and Parsons, S. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 6981-6985[Abstract]
34. Hansen, K., Johnell, M., Siegbahn, A., Rorsman, C., Engstrom, U., Wernstedt, C., Heldin, C., and Ronnstrand, L. (1996) EMBO J. 15, 5299-5313[Abstract]


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