Tat蛋白与Plk1相互作用位点的鉴定

Identification of the interaction site of Tat and Plk1

目的确定Tat蛋白与Plk1的相互作用及作用位点。方法构建tat和plk1基因的不同表达载体,通过GST-pull down、免疫共沉淀检测它们表达产物的相互作用位点,激光共聚焦确定其在细胞内的定位。结果 GST-pull down和免疫共沉淀实验发现Tat蛋白能结合在Plk1 N端的激酶区域,激光共聚焦发现Tat蛋白和Plk1在细胞核内有相同的亚细胞定位。结论 Tat蛋白能结合在Plk1 N端的激酶区域引起Plk1 T210的磷酸化水平提高,从而激活Plk1的活性,导致细胞周期的紊乱。

ObjectiveTo identify the interaction site of Tat with Plk1.MethodsDifferent expression vectors of tat and plk1 were constructed.The interaction of tat and plk1 was determined by GST-pull down and immunoprecipitation assay.The localization of Tat and Plk1 was observed by the confocal laser scanning microscopy.ResultsTat protein could interact with the amino-terminal kinase domain of Plk1 as shown in GST-pull down and immunoprecipitation assay.The co-localiation of Tat and Plk1 in the nuclei of HeLa and 293 cells was observed by the confocal laser scanning microscopy.ConclusionTat protein can bind to Plk1 and activate the kinase activity by up-regulating the level of phosphorylation of Plk1,eventually resulting in the disorder of cell cycle.