Interaction of the DF3/MUC1 breast carcinoma-associated antigen and beta-catenin in cell adhesion.

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.

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 ␤-catenin. Similar findings have been obtained for ␥-catenin but not ␣-catenin. DF3, like E-cadherin and the adenomatous polyposis coli gene product, contains an SXXXXXSSL site that is responsible for direct binding to ␤-catenin. The results further demonstrate that interaction of DF3 and ␤-catenin is dependent on cell adhesion. These findings and the role of ␤-catenin in cell signaling support a role for DF3 in the adhesion of epithelial cells.
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)(3)(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).
The present results demonstrate that DF3 interacts directly with ␤-catenin. An SXXXXXSSL motif in the DF3 cytoplasmic domain is responsible for binding to ␤-catenin. We also demonstrate that cell adhesion induces the interaction between DF3 and ␤-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 g/ml streptomycin, 100 units/ml penicillin, and 2 mM Lglutamine. Cells were grown in suspension (0.3 ϫ 10 8 /100 ml) with gentle rocking or as a monolayer on polystyrene culture dishes.
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 ␤-catenin (Fig. 1A). Since E-cadherin forms complexes with ␣-, ␤-, and ␥-catenins (26), we analyzed anti-DF3 immunoprecipitates for an association with ␣and ␥-catenins. While there was no detectable ␣-catenin in the precipitates, the results indicate that DF3 forms complexes with ␥-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 ␤and ␥-catenins (Fig. 1D). As previously shown (26), E-cadherin formed complexes with all three of the catenins (Fig. 1D).
To determine if binding to DF3 is direct, we subjected anti-␤-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 ␤-catenin ( Fig.  2A). By contrast, there was no detectable binding to GST ( Fig.  2A). Similar results were obtained for ␥-catenin (Fig. 2B).
Previous studies have demonstrated that ␤-catenin binds to SXXXXXSSL sites in E-cadherin (amino acids 840 -848) and APC (seven motifs) (23,24,27) (Fig. 3A). ␤-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 ␤-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 ␤-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 ␥-catenin (Fig. 3B). These findings suggested that ␤and ␥-catenin bind to DF3 at similar sites.
The functional role of the association between DF3 and ␤-catenin was studied in cells grown in suspension and then grown as a monolayer. There was no detectable ␤-catenin in the mAb DF3 immunoprecipitates prepared from the suspension cells. By contrast, binding of DF3 to ␤-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 ␥-catenin (Fig. 4B), but not ␣-catenin (data not shown). A similar analysis of E-cadherin immunoprecipitates demonstrated little if any difference in binding to ␤or ␥-catenin in suspension as compared with adherent cells (Fig. 4C).
␤-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 ␤-catenin (16) that are also found in Armadillo, ␥-catenin, and certain other junctional proteins (29). The present studies demonstrate that DF3 also binds directly to ␤-catenin and that the SXXXXXSSL motif in DF3 is responsible for this interaction. Similar results were obtained with the highly related ␥-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 ␤-catenin (or ␥-catenin) may differ from those found in other ␤-catenin complexes. The interaction of E-cadherin or APC complexes to the cytoskeleton is mediated by binding of ␤-catenin to ␣-catenin (16). By contrast, there was little if any ␣-catenin in the complex of DF3 and ␤-catenin. Moreover, while E-cadherin forms a stable complex with ␤-catenin in suspension and adherent cells, the interaction of DF3 with ␤-catenin is detectable following cell adhesion. Similar findings were obtained for the interaction of DF3 and ␥-catenin. These findings support a role for DF3 in the adhesion of cells and provide support for a novel interaction of DF3 with catenins.