Takahashi K, Suzuki K, Tsukatani Y
Department of Biochemistry, Kanagawa Cancer Center Research Institute, Yokohama, Japan.
Oncogene. 1997 Jul 3;15(1):71-8. doi: 10.1038/sj.onc.1201160.
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 alpha- and beta-catenins in the boundary region between adjacent cells. By contrast, immunocytostaining and Western blot analyses revealed that E-cadherin colocalized and coprecipitated with beta-catenin but not with alpha-catenin in exponentially growing cells at low density. As a comparable amount of alpha-catenin was detected in the total cell lysate of cells at different densities, it is suggested that alpha-catenin is present but dissociates from the E-cadherin-beta-catenin complex in growing cells. beta-Catenin was tyrosine phosphorylated in growing cells at low density but not in quiescent cells at confluence. Tyrosine phosphorylation of beta-catenin was concomitantly induced with association of beta-catenin with EGF receptor (EGFR) when quiescent cells at confluence were dissociated into single cells by tryptic digestion, being accompanied by dissociation of alpha-catenin from E-cadherin. Both tyrosine phosphorylation and association of beta-catenin with EGFR were inhibited by tyrphostin, a specific inhibitor of the EGFR tyrosine kinase, whereas dissociation of alpha-catenin from E-cadherin was not. The results suggest that tyrosine phosphorylation of beta-catenin is achieved by EGFR upon tryptic digestion of cells and concurrent with but independent of dissociation of alpha-catenin from E-cadherin. beta-Catenin thus phosphorylated at tyrosine is suggested to play the role in preventing alpha-catenin once dissociated from reassociating with E-cadherin until cells reach confluence.
正常人类乳腺上皮（HBE）细胞达到汇合状态后停止生长，并彼此紧密粘附，形成单层。在因密度而停滞的静止细胞中，E-钙粘蛋白在相邻细胞之间的边界区域与α-连环蛋白和β-连环蛋白共定位并共免疫沉淀。相比之下，免疫细胞化学染色和蛋白质印迹分析显示，在低密度指数生长的细胞中，E-钙粘蛋白与β-连环蛋白共定位并共沉淀，但不与α-连环蛋白共沉淀。由于在不同密度细胞的总细胞裂解物中检测到相当数量的α-连环蛋白，提示α-连环蛋白存在，但在生长细胞中从E-钙粘蛋白-β-连环蛋白复合物中解离。β-连环蛋白在低密度生长细胞中发生酪氨酸磷酸化，但在汇合的静止细胞中不发生。当汇合的静止细胞通过胰蛋白酶消化解离为单细胞时，β-连环蛋白的酪氨酸磷酸化与β-连环蛋白与表皮生长因子受体（EGFR）的结合同时被诱导，同时伴有α-连环蛋白从E-钙粘蛋白上解离。EGFR酪氨酸激酶的特异性抑制剂 tyrphostin 可抑制β-连环蛋白的酪氨酸磷酸化以及β-连环蛋白与EGFR的结合，而α-连环蛋白从E-钙粘蛋白上的解离不受影响。结果提示，细胞经胰蛋白酶消化后，β-连环蛋白的酪氨酸磷酸化由EGFR实现，且与α-连环蛋白从E-钙粘蛋白上的解离同时发生但相互独立。如此磷酸化的酪氨酸β-连环蛋白被认为在防止α-连环蛋白一旦解离后重新与E-钙粘蛋白结合直至细胞达到汇合状态中发挥作用。
