Induction of tyrosine phosphorylation and association of β-catenin with EGF receptor upon tryptic digestion of quiescent cells at confluence

Abstract

Normal human breast epithelial (HBE) cells which reached confluence ceased growth and tightly adhered to each other, forming a monolayer. In quiescent cells thus arrested by density, E-cadherin colocalized and coimmunoprecipitated with α- and β-catenins in the boundary region between adjacent cells. By contrast, immunocytostaining and Western blot analyses revealed that E-cadherin colocalized and coprecipitated with β-catenin but not with α-catenin in exponentially growing cells at low density. As a comparable amount of α-catenin was detected in the total cell lysate of cells at different densities, it is suggested that α-catenin is present but dissociates from the E-cadherin-β-catenin complex in growing cells. β-Catenin was tyrosine phosphorylated in growing cells at low density but not in quiescent cells at confluence. Tyrosine phosphorylation of β-catenin was concomitantly induced with association of β-catenin with EGF receptor (EGFR) when quiescent cells at confluence were dissociated into single cells by tryptic digestion, being accompanied by dissociation of α-catenin from E-cadherin. Both tyrosine phosphorylation and association of β-catenin with EGFR were inhibited by tyrphostin, a specific inhibitor of the EGFR tyrosine kinase, whereas dissociation of α-catenin from E-cadherin was not. The results suggest that tyrosine phosphorylation of β-catenin is achieved by EGFR upon tryptic digestion of cells and concurrent with but independent of dissociation of α-catenin from E-cadherin. β-Catenin thus phosphorylated at tyrosine is suggested to play the role in preventing α-catenin once dissociated from reassociating with E-cadherin until cells reach confluence.