Hu Peiqi, Berkowitz Paula, O'Keefe Edward J, Rubenstein David S
Department of Dermatology, University of North Carolina--Chapel Hill, School of Medicine, Chapel Hill, North Carolina 27599-7287, USA.
J Invest Dermatol. 2003 Aug;121(2):242-51. doi: 10.1046/j.1523-1747.2003.12376.x.
Because changes in cell-cell adhesion have profound effects on cellular behavior, we hypothesized a link between the adhesion and signaling functions of plakoglobin and beta-catenin. To investigate the existence of adherens-junction-mediated signaling, we used peroxovanadate to tyrosine phosphorylate plakoglobin and beta-catenin and to dissociate adherens junctions. The distribution of plakoglobin and beta-catenin was determined by immunofluorescence, western blot analysis, pulse-chase radiolabeling, and biochemical subcellular fractionation. Coimmunoprecipitation studies from nuclear fractions, gel-shift assays, and transient transfections with T cell factor (TCF)/lymphoid enhancer factor (LEF) optimized promoter reporter constructs were used to investigate the ability of plakoglobin and beta-catenin that had redistributed from the membrane to the nucleus to form functional transcriptional regulatory complexes with TCF/LEF family member transcription factors. Tyrosine phosphorylation of plakoglobin and beta-catenin resulted in their rapid translocation from the cell membrane to the nucleus. Nuclear translocation was associated with increased plakoglobin and decreased beta-catenin binding to nuclear TCF/LEF and downregulation of gene transcription from TCF/LEF reporter constructs. These results are consistent with a signaling pathway initiated by structural changes in the adherens junction in which adherens-junction-derived plakoglobin regulates nuclear transcription by antagonizing the binding of beta-catenin to TCF/LEF proteins.
由于细胞间黏附的变化对细胞行为具有深远影响，我们推测桥粒斑珠蛋白和β-连环蛋白的黏附功能与信号传导功能之间存在联系。为了研究黏附连接介导的信号传导的存在，我们用过氧钒酸盐使桥粒斑珠蛋白和β-连环蛋白酪氨酸磷酸化，并解离黏附连接。通过免疫荧光、蛋白质印迹分析、脉冲追踪放射性标记和生化亚细胞分级分离来确定桥粒斑珠蛋白和β-连环蛋白的分布。利用来自核级分的共免疫沉淀研究、凝胶迁移分析以及用T细胞因子（TCF）/淋巴细胞增强因子（LEF）优化的启动子报告构建体进行瞬时转染，来研究已从膜重新分布到核的桥粒斑珠蛋白和β-连环蛋白与TCF/LEF家族成员转录因子形成功能性转录调节复合物的能力。桥粒斑珠蛋白和β-连环蛋白的酪氨酸磷酸化导致它们迅速从细胞膜转运至细胞核。核转位与桥粒斑珠蛋白增加、β-连环蛋白与核TCF/LEF的结合减少以及TCF/LEF报告构建体的基因转录下调有关。这些结果与由黏附连接的结构变化引发的信号通路一致，其中源自黏附连接的桥粒斑珠蛋白通过拮抗β-连环蛋白与TCF/LEF蛋白的结合来调节核转录。
