ERK2的CC区域在HeLa细胞中的定位作用

The focal adhesion location of ERK2 is dependent on CC domain of ERK2 in HeLa after EGF stimulation

目的探索细胞外调节蛋白激酶2(ERK2)的coiled-coil(CC)区域在表皮生长因子(EGF)所诱导的HeLa细胞中的定位作用。方法用定点突变试剂盒删除ERK2的盘绕结构[ERK2(del CC)],免疫共沉淀的方法检测ERK2(del CC)在EGF刺激的条件下和G蛋白耦联受体激酶相互作用分子1(GIT1)的相互关系,免疫荧光染色检测ERK2(del CC)在细胞内的位置以及和GIT1的相互关系。结果成功删除ERK2的CC区域;免疫共沉淀结果显示,删除ERK2的CC区域显著抑制了ERK2和GIT1在EGF刺激条件下的相互结合;免疫荧光染色结果显示,删除ERK2的CC区域显著抑制了磷酸化的ERK2在细胞局部黏附内的定位。结论在EGF的刺激条件下,增加了ERK2和GIT1的相互结合,ERK2的CC区域是与GIT1相互结合的重要区域。删除ERK2的CC区域不仅显著抑制了ERK2和GIT1的相互结合,而且明显降低ERK2在细胞局部黏附内的定位。

Objective To investigate the function of coiled-coil（CC） domain of ERK2 in HeLa cell localization.Methods Quik change site-directed mutagenesis kit was used to delete the CC domian of ERK2[ERK2（del CC）].The ERK2（del CC） associated with G-protein coulped recepor kinase interacting protein 1（GIT1） was analysed by co-immunoprecipitation.The localization of phosphorylation ERK2 was detected by immunofluorescence.Results We successfully constructed the ERK2（del CC）.The result of co-immunoprecipotation showed that ERK2 could not associate with the GIT1 after deletion of the CC domain.The localization of phosphorylation ERK2 in focal adhesion was dramatically inhibited after deletion of the CC domain.Conclusion Under EGF stimulation,the CC domain of ERK2 is very important for the association of ERK2 with GIT1.The deletion of CC domain can inhibit the association of ERK2 with GIT1 and decrease ERK2 localization in cell focal adhesions.