Salomon D, Sacco P A, Roy S G, Simcha I, Johnson K R, Wheelock M J, Ben-Ze'ev A
Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel.
J Cell Biol. 1997 Dec 1;139(5):1325-35. doi: 10.1083/jcb.139.5.1325.
beta-Catenin and plakoglobin (gamma-catenin) are closely related molecules of the armadillo family of proteins. They are localized at the submembrane plaques of cell-cell adherens junctions where they form independent complexes with classical cadherins and alpha-catenin to establish the link with the actin cytoskeleton. Plakoglobin is also found in a complex with desmosomal cadherins and is involved in anchoring intermediate filaments to desmosomal plaques. In addition to their role in junctional assembly, beta-catenin has been shown to play an essential role in signal transduction by the Wnt pathway that results in its translocation into the nucleus. To study the relationship between plakoglobin expression and the level of beta-catenin, and the localization of these proteins in the same cell, we employed two different tumor cell lines that express N-cadherin, and alpha- and beta-catenin, but no plakoglobin or desmosomal components. Individual clones expressing various levels of plakoglobin were established by stable transfection. Plakoglobin overexpression resulted in a dose-dependent decrease in the level of beta-catenin in each clone. Induction of plakoglobin expression increased the turnover of beta-catenin without affecting RNA levels, suggesting posttranslational regulation of beta-catenin. In plakoglobin overexpressing cells, both beta-catenin and plakoglobin were localized at cell-cell junctions. Stable transfection of mutant plakoglobin molecules showed that deletion of the N-cadherin binding domain, but not the alpha-catenin binding domain, abolished beta-catenin downregulation. Inhibition of the ubiquitin-proteasome pathway in plakoglobin overexpressing cells blocked the decrease in beta-catenin levels and resulted in accumulation of both beta-catenin and plakoglobin in the nucleus. These results suggest that (a) plakoglobin substitutes effectively with beta-catenin for association with N-cadherin in adherens junctions, (b) extrajunctional beta-catenin is rapidly degraded by the proteasome-ubiquitin system but, (c) excess beta-catenin and plakoglobin translocate into the nucleus.
β-连环蛋白和桥粒斑珠蛋白（γ-连环蛋白）是犰狳蛋白家族中密切相关的分子。它们定位于细胞间黏附连接的亚膜斑块处，在那里它们与经典钙黏蛋白和α-连环蛋白形成独立的复合物，从而建立与肌动蛋白细胞骨架的联系。桥粒斑珠蛋白也存在于与桥粒钙黏蛋白的复合物中，并参与将中间丝锚定到桥粒斑块上。除了在连接组装中的作用外，β-连环蛋白已被证明在Wnt信号通路的信号转导中起关键作用，该信号通路导致其易位进入细胞核。为了研究桥粒斑珠蛋白表达与β-连环蛋白水平之间的关系，以及这些蛋白在同一细胞中的定位，我们使用了两种表达N-钙黏蛋白、α-连环蛋白和β-连环蛋白，但不表达桥粒斑珠蛋白或桥粒成分的不同肿瘤细胞系。通过稳定转染建立了表达不同水平桥粒斑珠蛋白的单个克隆。桥粒斑珠蛋白的过表达导致每个克隆中β-连环蛋白水平呈剂量依赖性下降。桥粒斑珠蛋白表达的诱导增加了β-连环蛋白的周转，而不影响RNA水平，提示β-连环蛋白的翻译后调控。在桥粒斑珠蛋白过表达的细胞中，β-连环蛋白和桥粒斑珠蛋白都定位于细胞间连接。突变型桥粒斑珠蛋白分子的稳定转染表明，N-钙黏蛋白结合结构域的缺失而非α-连环蛋白结合结构域的缺失消除了β-连环蛋白的下调。在桥粒斑珠蛋白过表达的细胞中抑制泛素-蛋白酶体途径可阻止β-连环蛋白水平的下降，并导致β-连环蛋白和桥粒斑珠蛋白在细胞核中积累。这些结果表明：（a）在黏附连接中，桥粒斑珠蛋白可有效地替代β-连环蛋白与N-钙黏蛋白结合；（b）连接外的β-连环蛋白被蛋白酶体-泛素系统迅速降解；（c）过量的β-连环蛋白和桥粒斑珠蛋白易位进入细胞核。
