Interaction of the DF3/MUC1 Breast Carcinoma-associated Antigen and beta -Catenin in Cell Adhesion*

(Received for publication, March 5, 1997)

Makiko Yamamoto , Ajit Bharti , Yongqing Li and Donald Kufe

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

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

The DF3/MUC1 mucin-like glycoprotein is aberrantly overexpressed in human breast carcinomas. The functional role of DF3 is unknown. The present studies demonstrate that DF3 associates with beta -catenin. Similar findings have been obtained for gamma -catenin but not alpha -catenin. DF3, like E-cadherin and the adenomatous polyposis coli gene product, contains an SXXXXXSSL site that is responsible for direct binding to beta -catenin. The results further demonstrate that interaction of DF3 and beta -catenin is dependent on cell adhesion. These findings and the role of beta -catenin in cell signaling support a role for DF3 in the adhesion of epithelial cells.


INTRODUCTION

The human DF3 (MUC1, episialin, PEM) gene encodes a high molecular mass membrane-associated glycoprotein with a mucin-like external domain. The DF3 glycoprotein is expressed on the apical borders of secretory mammary epithelial cells and aberrantly expressed over the entire surface of carcinoma cells (1). The ectodomain consists of varying numbers of 20-amino acid tandem repeats that are subject to O-glycosylation and that contribute to the expression of a polymorphic protein (2-4). The N-terminal region contains hydrophobic signal sequences that vary as a consequence of alternate splicing (5-7). The C-terminal region includes a transmembrane domain and a 72-amino acid cytoplasmic tail that contains tyrosine phosphorylation sites (8, 9). The function of DF3 is unclear. However, high levels found on carcinoma cells reduce cell-cell and cell-extracellular matrix adhesion in a nonspecific manner (10-12). These studies have suggested that DF3 interferes with cellular adhesion by steric hindrance from the rigid ectodomain (11).

Cadherin cell adhesion molecules form complexes with the cytoplasmic alpha -, beta -, and gamma -catenin proteins (13). alpha -Catenin is required for cadherin-mediated cell adhesion and links cadherins to the actin cytoskeleton (14, 15). beta -Catenin links alpha -catenin to the cadherins and is highly related to plakoglobin (gamma -catenin) (16-18). beta -Catenin is homologous to the Drosophila segment polarity gene product Armadillo (19) that acts downstream of Wingless (20). Armadillo forms complexes with Drosophila E-cadherin and alpha -catenin (21, 22). These findings have supported a role for beta -catenin in morphogenetic signals. Other studies have demonstrated that beta -catenin binds directly to the adenomatous polyposis coli (APC)1 gene product (23-25). The APC protein and E-cadherin form independent complexes with beta -catenin (25). gamma -Catenin mediates similar interactions among APC, alpha -catenin, and the cytoskeleton (16).

The present results demonstrate that DF3 interacts directly with beta -catenin. An SXXXXXSSL motif in the DF3 cytoplasmic domain is responsible for binding to beta -catenin. We also demonstrate that cell adhesion induces the interaction between DF3 and beta -catenin.


MATERIALS AND METHODS

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. Cells were grown in suspension (0.3 × 108/100 ml) with gentle rocking or as a monolayer on polystyrene culture dishes.

Cell Lysate

Cells (~70% confluent) were lysed in ice-cold lysis buffer (150 mM NaCl, 50 mM Tris, pH 7.6, 0.5% Brij 97, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol) for 30 min on ice. Lysates were cleared by centrifugation at 14,000 × g for 15 min.

Immunoprecipitation and Immunoblotting

Lysates were incubated with monoclonal antibody (mAb) DF3 (1), anti-alpha -catenin (Zymed Laboratories, Inc., San Francisco, CA), anti-beta -catenin (Zymed), anti-gamma -catenin (Zymed), or anti-E-cadherin (Transduction Laboratories, Lexington, KY) for 2 h at 4 °C. Immunoprecipitates were prepared by incubation with rabbit anti-mouse IgG (Upstate Biotechnology, Inc., Lake Placid, NY) and protein A-Sepharose (Pharmacia Biotech Inc.) for 1 h at 4 °C. The precipitates were subjected to electrophoresis in 7.5% or 6% SDS-polyacrylamide gels. Proteins were transferred to nitrocellulose membranes by dry transfer. The membranes were blocked in 5% nonfat dry milk in phosphate-buffered saline containing 0.05% Tween 20 and then incubated with an appropriate antibody for immunoblot analysis. Reactivity was detected by horseradish peroxidase-conjugated second antibodies and chemiluminescence (ECL, Amersham Corp.).

Direct Binding Studies

The GST fusion construct expressing the DF3 cytoplasmic domain (CD) was prepared by polymerase chain reaction cloning and ligation into the pGEX2T vector. GST or GST-DF3/CD was affinity-purified with glutathione-Sepharose 4B beads and suspended in elution buffer (50 mM Tris-HCl, pH 8.0, 5 mM glutathione). Nitrocellulose filters were incubated with GST or GST-DF3/CD for 1.5 h at room temperature. Reactivity was detected with an anti-GST antibody (Santa Cruz Biotechnology).


RESULTS AND DISCUSSION

To identify proteins that associate with DF3, we analyzed mAb DF3 immunoprecipitates by SDS-PAGE and silver staining. The detection of a coprecipitated protein of 92 kDa was confirmed by reactivity with an antibody against beta -catenin (Fig. 1A). Since E-cadherin forms complexes with alpha -, beta -, and gamma -catenins (26), we analyzed anti-DF3 immunoprecipitates for an association with alpha - and gamma -catenins. While there was no detectable alpha -catenin in the precipitates, the results indicate that DF3 forms complexes with gamma -catenin (Fig. 1, B and C). In the reciprocal experiments, anti-catenin immunoprecipitates were analyzed by immunoblotting with anti-DF3. The findings confirm binding of DF3 to beta - and gamma -catenins (Fig. 1D). As previously shown (26), E-cadherin formed complexes with all three of the catenins (Fig. 1D).


Fig. 1. Association of DF3 with beta -catenin (beta -cat) and gamma -catenin. Lysates from adherent ZR-75-1 cells were subjected to immunoprecipitation with mAb DF3. The immunoprecipitates were analyzed for reactivity with anti-beta -catenin (A), anti-alpha -catenin (B), and anti-gamma -catenin (C). Lysates were directly analyzed by immunoblotting as controls. D, lysates were subjected to immunoprecipitation with the indicated antibodies. The precipitates were analyzed by immunoblotting with mAb DF3 (upper panel) or anti-E-cadherin (E-cad, lower panel).
[View Larger Version of this Image (29K GIF file)]

To determine if binding to DF3 is direct, we subjected anti-beta - catenin immunoprecipitates to SDS-PAGE and then transferred the separated proteins to filters. Incubation of the filters with a GST fusion protein that contains the DF3 cytoplasmic domain (GST-DF3/CD) demonstrated binding to beta -catenin (Fig. 2A). By contrast, there was no detectable binding to GST (Fig. 2A). Similar results were obtained for gamma -catenin (Fig. 2B).


Fig. 2. Direct binding of DF3 to beta -catenin (beta -cat) and gamma -catenin. Lysates were subjected to immunoprecipitation with anti-beta -catenin (A) or anti-gamma -catenin (B). The immunoprecipitates were separated by SDS-PAGE, and the proteins were transferred to nitrocellulose filters. The filters were incubated with GST or GST-DF3/CD and then washed and analyzed for reactivity with anti-GST.
[View Larger Version of this Image (25K GIF file)]

Previous studies have demonstrated that beta -catenin binds to SXXXXXSSL sites in E-cadherin (amino acids 840-848) and APC (seven motifs) (23, 24, 27) (Fig. 3A). beta -Catenin also associates with the epidermal growth factor receptor, which contains a SRTPLLSSLS (amino acids 1030-1039) site (28). A similar site is present at amino acids 1239-1243 in DF3 (Fig. 3A). To assess whether beta -catenin binds to the SXXXXXSSL site in DF3, we subjected cell lysates to immunoprecipitation with mAb DF3 in the presence of the synthetic peptide GGSSLSY. The results demonstrate that the peptide inhibits binding of beta -catenin and DF3 (Fig. 3B). By contrast, there was no detectable effect on this interaction when using an irrelevant peptide (Fig. 3B). The GGSSLSY peptide also blocked interaction of DF3 and gamma -catenin (Fig. 3B). These findings suggested that beta - and gamma -catenin bind to DF3 at similar sites.


Fig. 3. DF3 binds to catenins at an SXXXXXSSL site. A, SXXXXXSSL sites in E-cadherin, APC, and DF3. B, lysates were subjected to immunoprecipitation with mAb DF3 in the presence of no added peptide, a control peptide (50 µM; EAPPPKIPDKQ), or a 50 µM GGSSLSY peptide. The immunoprecipitates were analyzed by immunoblotting with anti-beta -catenin (beta -cat) or anti-gamma -catenin.
[View Larger Version of this Image (18K GIF file)]

The functional role of the association between DF3 and beta -catenin was studied in cells grown in suspension and then grown as a monolayer. There was no detectable beta -catenin in the mAb DF3 immunoprecipitates prepared from the suspension cells. By contrast, binding of DF3 to beta -catenin was detectable at 1 and 3 h of adherence (Fig. 4A). Cell adhesion was also associated with formation of a complex with DF3 and gamma -catenin (Fig. 4B), but not alpha -catenin (data not shown). A similar analysis of E-cadherin immunoprecipitates demonstrated little if any difference in binding to beta - or gamma -catenin in suspension as compared with adherent cells (Fig. 4C).


Fig. 4. Cell adhesion induces binding of DF3 with beta -catenin (beta -cat) and gamma -catenin. Cells were trypsinized and grown in suspension by gentle agitation for 6 h. Suspension cells were allowed to adhere to culture dishes for 1 and 3 h. Lysates were subjected to immunoprecipitation with mAb DF3 (A and B) or anti-E-cadherin (anti-E-cad, C). The immunoprecipitates were analyzed for reactivity with anti-beta -catenin, anti-gamma -catenin, or mAb DF3. Lysates were directly analyzed by immunoblotting as controls.
[View Larger Version of this Image (25K GIF file)]

beta -Catenin is involved in the formation of adherens junctions of epithelial cells. The cell adhesion E-cadherin protein and the APC tumor suppressor gene product compete for binding to the arm repeats of beta -catenin (16) that are also found in Armadillo, gamma -catenin, and certain other junctional proteins (29). The present studies demonstrate that DF3 also binds directly to beta -catenin and that the SXXXXXSSL motif in DF3 is responsible for this interaction. Similar results were obtained with the highly related gamma -catenin. Whereas the cytoplasmic domain of DF3/MUC1 is phosphorylated on tyrosine (8, 9), it is not known if tyrosine sites influence binding of catenins to the serine-rich motif. The formation of a complex between DF3 and beta -catenin (or gamma -catenin) may differ from those found in other beta -catenin complexes. The interaction of E-cadherin or APC complexes to the cytoskeleton is mediated by binding of beta -catenin to alpha -catenin (16). By contrast, there was little if any alpha -catenin in the complex of DF3 and beta -catenin. Moreover, while E-cadherin forms a stable complex with beta -catenin in suspension and adherent cells, the interaction of DF3 with beta -catenin is detectable following cell adhesion. Similar findings were obtained for the interaction of DF3 and gamma -catenin. These findings support a role for DF3 in the adhesion of cells and provide support for a novel interaction of DF3 with catenins.


FOOTNOTES

*   This work was supported by Department of the Army Grant DAMD 17-94-J-4394.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.
1   The abbreviations used are: APC, adenomatous polyposis coli; mAb, monoclonal antibody; CD, cytoplasmic domain; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis.

REFERENCES

  1. Kufe, D., Inghirami, G., Abe, M., Hayes, D., Justi-Wheeler, H., and Schlom, J. (1984) Hybridoma 3, 223-232 [Medline] [Order article via Infotrieve]
  2. 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]
  3. Gendler, S. J., Burchell, J. M., Duhig, T., Lamport, D., White, R., Parker, M., and Taylor-Papadimitriou, J. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 6060-6064 [Abstract]
  4. Gendler, S., Taylor-Papadimitriou, J., Duhig, T., Rothbard, J., and Burchell, J. A. (1988) J. Biol. Chem. 263, 12820-12823 [Abstract/Free Full Text]
  5. Abe, M., and Kufe, D. (1989) Biochem. Biophys. Res. Commun. 165, 644-649 [Medline] [Order article via Infotrieve]
  6. Ligtenberg, M. J. L., Vos, H. L., Gennissen, A. M. C., and Hilkens, J. (1990) J. Biol. Chem. 265, 5573-5578 [Abstract/Free Full Text]
  7. Wreschner, D. H., Hareuveni, M., Tsarfaty, I., Smorodinsky, N., Horev, J., Zaretsky, J., Kotkes, P., Weiss, M., Lathe, R., Dion, A., and Keydar, I. (1990) Eur. J. Biochem. 189, 463-473 [Abstract]
  8. Pandey, P., Kharbanda, S., and Kufe, D. (1995) Cancer Res. 55, 4000-4003 [Abstract]
  9. Zrihan-Licht, S., Baruch, A., Keydar, I., and Wreschner, D. H. (1994) FEBS Lett. 356, 130-137 [CrossRef][Medline] [Order article via Infotrieve]
  10. Ligtenberg, M. J. L., Buijs, F., Vos, H. L., and Hilkens, J. (1992) Cancer Res. 52, 2318-2324 [Abstract]
  11. Wesseling, J., van der Valk, S. W., and Hilkens, J. (1996) Mol. Biol. Cell 7, 565-577 [Abstract]
  12. van de Wiel-van Kemenade, E., Ligtenberg, M. J. L., de Boer, A. J., Buijs, F., Vos, H. L., Melief, C. J. M., Hilkens, J., and Figdor, C. G. (1993) J. Immunol. 151, 767-776 [Abstract/Free Full Text]
  13. Ozawa, M., Baribault, H., and Kemler, R. (1989) EMBO J. 8, 1711-1717 [Abstract]
  14. Ozawa, M., Ringwald, M., and Kemler, R. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4246-4250 [Abstract]
  15. Rimm, D. L., Koslov, E. R., Kebriaei, P., Cianci, C. D., and Morrow, J. S. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8813-8817 [Abstract]
  16. Hulsken, J., Birchmeier, W., and Behrens, J. (1994) J. Cell Biol. 127, 2061-2069 [Abstract]
  17. Knudsen, K. A., and Wheelock, M. (1992) J. Cell Biol. 118, 671-679 [Abstract]
  18. Peifer, M., McCrea, P. D., Green, K. J., Wieschaus, E., and Gumbiner, B. M. (1992) J. Cell Biol. 118, 681-689 [Abstract]
  19. McCrea, P. D., Turck, C. W., and Gumbiner, B. (1991) Science 254, 1359-1361 [Medline] [Order article via Infotrieve]
  20. Noordermeer, J., Klingensmith, J., Perrimon, N., and Nusse, R. (1994) Nature 367, 80-83 [CrossRef][Medline] [Order article via Infotrieve]
  21. Oda, H., Uemura, T., Shiomi, K., Nagafuchi, A., Tsukita, S., and Takeichi, M. (1993) J. Cell Biol. 121, 1133-1140 [Abstract]
  22. Oda, H., Uemura, T., Harada, Y., Iwai, Y., and Takeichi, M. (1994) Dev. Biol. 165, 716-726 [CrossRef][Medline] [Order article via Infotrieve]
  23. Su, L. K., Vogelstein, B., and Kinzler, K. W. (1993) Science 262, 1734-1737 [Medline] [Order article via Infotrieve]
  24. Rubinfeld, B., Souza, B., Albert, I., Muller, O., Chamberlain, S. H., Mastarz, F. R., Munemitsu, S., and Polakis, P. (1993) Science 262, 1731-1734 [Medline] [Order article via Infotrieve]
  25. Rubinfeld, B., Souza, B., Albert, I., Munemitsu, S., and Polakis, P. (1995) J. Biol. Chem. 270, 5549-5555 [Abstract/Free Full Text]
  26. Kintner, C. (1992) Cell 69, 225-236 [Medline] [Order article via Infotrieve]
  27. Stappert, J., and Kemler, R. (1994) Cell Adhes. Commun. 2, 319-327 [Medline] [Order article via Infotrieve]
  28. Hoschuetzky, H., Aberle, H., and Kemler, R. (1994) J. Cell Biol. 127, 1375-1380 [Abstract]
  29. Peifer, M., Berg, S., and Reynolds, A. B. (1994) Cell 76, 789-791 [Medline] [Order article via Infotrieve]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.