Ozawa M, Kemler R
Department of Biochemistry, Faculty of Medicine, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan.
J Biol Chem. 1998 Mar 13;273(11):6166-70. doi: 10.1074/jbc.273.11.6166.
Leukemia cells (K562) that grow as non-adhesive single cells and have no endogenous cadherin were transfected with an E-cadherin expression vector, and cell clones stably expressing E-cadherin on their surface were established. The expression of E-cadherin induced the up-regulation of catenins, and E-cadherin became associated with catenins. The transfected cells grew as floating aggregates. Cell aggregation was Ca2+-dependent and was inhibited by E-cadherin antibodies. The aggregates dissociated into single cells on the addition of pervanadate. Pervanadate caused a dramatic augmentation of the phosphorylation of E-cadherin, beta-catenin, and gamma-catenin (plakoglobin), but alpha-catenin was not detectably phosphorylated. After pervanadate treatment, beta-catenin and gamma-catenin migrated more slowly on gel electrophoresis, suggesting changes in their conformations due to eventual changes in their phosphorylation levels. In the treated cells, a significant amount of alpha-catenin was dissociated from the E-cadherin.catenin complex. Aggregates of cells expressing an E-cadherin chimeric molecule covalently linked with alpha-catenin were not dissociated on pervanadate treatment, supporting the idea that the dissociation of alpha-catenin from the complex underlies the observed E-cadherin dysfunction.
作为非贴壁单细胞生长且无内源性钙黏蛋白的白血病细胞（K562）用E-钙黏蛋白表达载体进行转染，并建立在其表面稳定表达E-钙黏蛋白的细胞克隆。E-钙黏蛋白的表达诱导连环蛋白上调，且E-钙黏蛋白与连环蛋白结合。转染后的细胞以漂浮聚集体形式生长。细胞聚集依赖于Ca2+，并被E-钙黏蛋白抗体抑制。加入过氧钒酸盐后，聚集体解离为单细胞。过氧钒酸盐导致E-钙黏蛋白、β-连环蛋白和γ-连环蛋白（桥粒斑蛋白）的磷酸化显著增强，但α-连环蛋白未检测到磷酸化。过氧钒酸盐处理后，β-连环蛋白和γ-连环蛋白在凝胶电泳上迁移更慢，表明由于其磷酸化水平的最终变化导致其构象改变。在处理后的细胞中，大量α-连环蛋白从E-钙黏蛋白-连环蛋白复合物中解离。表达与α-连环蛋白共价连接的E-钙黏蛋白嵌合分子的细胞聚集体在过氧钒酸盐处理后未解离，支持α-连环蛋白从复合物中解离是观察到的E-钙黏蛋白功能障碍基础的观点。
