Romer L H, McLean N, Turner C E, Burridge K
Department of Pediatrics, University of North Carolina at Chapel Hill 27599.
Mol Biol Cell. 1994 Mar;5(3):349-61. doi: 10.1091/mbc.5.3.349.
Tyrosine phosphorylation of cytoskeletal proteins occurs during integrin-mediated cell adhesion to extracellular matrix proteins. We have investigated the role of tyrosine phosphorylation in the migration and initial spreading of human umbilical vein endothelial cells (HUVEC). Elevated phosphotyrosine concentrations were noted in the focal adhesions of HUVEC migrating into wounds. Anti-phosphotyrosine Western blots of extracts of wounded HUVEC monolayers demonstrated increased phosphorylation at 120-130 kDa when compared with extracts of intact monolayers. The pp125FAK immunoprecipitated from wounded monolayers exhibited increased kinase activity as compared to pp125FAK from intact monolayers. The time to wound closure in HUVEC monolayers was doubled by tyrphostin AG 213 treatment. The same concentration of AG 213 interfered with HUVEC focal adhesion and stress fiber formation. AG 213 inhibited adhesion-associated tyrosine phosphorylation of pp125FAK in HUVEC. Tyrphostins AG 213 and AG 808 inhibited pp125FAK activity in in vitro kinase assays. pp125FAK immunoprecipitates from HUVEC treated with both of these inhibitors also had kinase activity in vitro that was below levels seen in untreated HUVEC. These findings suggest that tyrosine phosphorylation of cytoskeletal proteins may be important in HUVEC spreading and migration and that pp125FAK may mediate phosphotyrosine formation during these processes.
细胞骨架蛋白的酪氨酸磷酸化发生在整合素介导的细胞与细胞外基质蛋白的黏附过程中。我们研究了酪氨酸磷酸化在人脐静脉内皮细胞（HUVEC）迁移和初始铺展中的作用。在迁移到伤口处的HUVEC的黏着斑中，磷酸酪氨酸浓度升高。与完整单层细胞提取物相比，受伤的HUVEC单层细胞提取物的抗磷酸酪氨酸免疫印迹显示，120 - 130 kDa处的磷酸化增加。与完整单层细胞的pp125FAK相比，从受伤单层细胞中免疫沉淀的pp125FAK表现出增加的激酶活性。酪氨酸磷酸化抑制剂AG 213处理使HUVEC单层细胞伤口闭合时间加倍。相同浓度的AG 213干扰HUVEC的黏着斑和应力纤维形成。AG 213抑制HUVEC中pp125FAK的黏附相关酪氨酸磷酸化。在体外激酶测定中，酪氨酸磷酸化抑制剂AG 213和AG 808抑制pp125FAK活性。用这两种抑制剂处理的HUVEC的pp125FAK免疫沉淀物在体外也具有低于未处理HUVEC的激酶活性。这些发现表明，细胞骨架蛋白的酪氨酸磷酸化可能在HUVEC铺展和迁移中起重要作用，并且pp125FAK可能在这些过程中介导磷酸酪氨酸的形成。
