FIG. 6.

Mutations in the major CK1 phosphorylation residue of E-cadherin affect adhesiveness of cell-cell contacts in L fibroblast cells stably expressing E-cadherin mutants. L fibroblast clones stably expressing the Myc-tagged wild type and nonphosphorylatable (S846A) and pseudophosphorylated (S846D) mutants of E-cadherin were obtained. More than five independent clones were analyzed for each of the different types of E-cadherin, and analogous data were obtained between clones expressing the same type of E-cadherin. The data using representative clones (WT2, A5, and D13 for wild-type, S846A, and S846D E-cadherin, respectively) are shown. (a) Expression level of E-cadherin mutants in the L fibroblast clones. Cell lysates (20 μg of proteins) from the indicated clones were analyzed by Western blotting (WB) with anti-Myc antibody (Ab). (b) Effect of mutations of E-cadherin on cell-cell contact formation. Nontransfected cells or the indicated clones were cultured at high density and analyzed by phase-contrast microscopy. Scale bar, 40 μm. (c) Effect of mutations of E-cadherin on formation of cell aggregates. Nontransfected cells or the indicated clones were cultured in suspension and examined by phase-contrast microscopy. Scale bar, 40 μm. (d) Effect of mutations on subcellular localization of E-cadherin. The indicated clones were cultured at low density and analyzed by immunostaining with anti-Myc antibody. Scale bar, 20 μm. IF, immunofluorescence. (e) Effect of mutations of E-cadherin on IC261-induced stabilization of cell-cell contacts. The indicated clones were cultured at a low density in the presence or absence of 10 μM IC261 for 4 h and analyzed by phase-contrast microscopy. Scale bar, 20 μm.