Piepenhagen P A, Nelson W J
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, CA 94305-5426.
J Cell Sci. 1993 Mar;104 ( Pt 3):751-62. doi: 10.1242/jcs.104.3.751.
Ca(2+)-dependent cell adhesion is mediated by a family of proteins termed cadherins, and is modulated by cytosolic proteins that include alpha-, beta-, and gamma-catenin and other cytoskeletal proteins that bind to the cytoplasmic domain of cadherins. Recent studies have suggested that either beta- or gamma-catenin may be identical to plakoglobin, a protein associated with adherens junctions. However, the relationship between these proteins, and their interaction with cadherins, are not well understood. In this study, we have further defined the relationship between plakoglobin and the catenins in complexes with E-cadherin in Madin-Darby canine kidney (MDCK) cells. Specific immunoprecipitations revealed that plakoglobin (86 kDa) and beta-catenin (92 kDa) have different detergent extractabilities and apparent molecular weights in these cells; however, plakoglobin has an apparent molecular weight similar to that of gamma-catenin (86 kDa). Immunoblotting of E-cadherin immunoprecipitates demonstrated that both plakoglobin and beta-catenin co-immunoprecipitate with E-cadherin. Laser-scanning confocal microscopy demonstrated temporally and spatially co-ordinate redistribution of plakoglobin and E-cadherin following induction of cell-cell contact in MDCK cells. Although plakoglobin comigrated with gamma-catenin on SDS-PAGE, quantitative analysis of E-cadherin and plakoglobin immunoprecipitates revealed that plakoglobin accounted for < 50% of the gamma-catenin signal. Two-dimensional gel electrophoresis resolved the gamma-catenin protein band into two proteins. One protein was identified as plakoglobin, based upon apparent molecular weight, immunoreactivity and isoelectric point (pI approximately 6.1). The other protein comigrated with gamma-catenin on SDS-PAGE, did not react with plakoglobin antibodies and had a pI of approximately 4.25; we refer to this protein as gamma-catenin to distinguish it from plakoglobin. Two-dimensional gel electrophoresis further revealed that plakoglobin comprised multiple isoelectric variants, but that, within the newly synthesized pool of plakoglobin, only the most basic of these variants co-immunoprecipitated with E-cadherin; phosphorylation did not account for the plakoglobin isoelectric variants seen by two-dimensional gel electrophoresis. These results demonstrate directly that plakoglobin associates and co-localizes with the E-cadherin in MDCK epithelial cells in a complex that contains alpha-, beta-, and gamma-catenin. Although plakoglobin shares sequence similarity with beta-catenin, and comigrates with gamma-catenin in SDS-PAGE, plakoglobin is distinct from the catenins. The association of plakoglobin with E-cadherin may be regulated by post-translational modifications of plakoglobin.
依赖钙离子的细胞黏附由一类称为钙黏蛋白的蛋白质介导，并受胞质蛋白调节，这些胞质蛋白包括α -、β - 和γ - 连环蛋白以及其他与钙黏蛋白胞质结构域结合的细胞骨架蛋白。最近的研究表明，β - 连环蛋白或γ - 连环蛋白可能与桥粒芯蛋白相同，桥粒芯蛋白是一种与黏附连接相关的蛋白质。然而，这些蛋白质之间的关系以及它们与钙黏蛋白的相互作用尚未完全清楚。在本研究中，我们进一步明确了在Madin - Darby犬肾（MDCK）细胞中，桥粒芯蛋白与连环蛋白在与E - 钙黏蛋白形成的复合物中的关系。特异性免疫沉淀显示，在这些细胞中，桥粒芯蛋白（86 kDa）和β - 连环蛋白（92 kDa）具有不同的去污剂提取能力和表观分子量；然而，桥粒芯蛋白的表观分子量与γ - 连环蛋白（86 kDa）相似。对E - 钙黏蛋白免疫沉淀物的免疫印迹表明，桥粒芯蛋白和β - 连环蛋白都能与E - 钙黏蛋白共免疫沉淀。激光扫描共聚焦显微镜显示，在MDCK细胞中诱导细胞 - 细胞接触后，桥粒芯蛋白和E - 钙黏蛋白在时间和空间上协调重新分布。尽管桥粒芯蛋白在SDS - PAGE上与γ - 连环蛋白迁移一致，但对E - 钙黏蛋白和桥粒芯蛋白免疫沉淀物的定量分析表明，桥粒芯蛋白占γ - 连环蛋白信号的不到50%。二维凝胶电泳将γ - 连环蛋白蛋白条带解析为两种蛋白质。根据表观分子量、免疫反应性和等电点（pI约为6.1），其中一种蛋白质被鉴定为桥粒芯蛋白。另一种蛋白质在SDS - PAGE上与γ - 连环蛋白迁移一致，不与桥粒芯蛋白抗体反应，pI约为4.25；我们将这种蛋白质称为γ - 连环蛋白以区别于桥粒芯蛋白。二维凝胶电泳进一步显示，桥粒芯蛋白包含多个等电变体，但在新合成的桥粒芯蛋白池中，只有这些变体中最碱性的与E - 钙黏蛋白共免疫沉淀；磷酸化不能解释二维凝胶电泳中看到的桥粒芯蛋白等电变体。这些结果直接表明，在MDCK上皮细胞中，桥粒芯蛋白与E - 钙黏蛋白结合并共定位在一个包含α -、β - 和γ - 连环蛋白的复合物中。尽管桥粒芯蛋白与β - 连环蛋白具有序列相似性，并且在SDS - PAGE上与γ - 连环蛋白迁移一致，但桥粒芯蛋白与连环蛋白不同。桥粒芯蛋白与E - 钙黏蛋白的结合可能受桥粒芯蛋白翻译后修饰的调节。
