HEK 293T细胞中TRAF6多聚泛素化修饰KLF5的方式及其修饰位点的鉴定

The pattern and site identification of KLF5 polyubiquitination by TRAF6 in HEK 293T cells

目的:研究人胚肾293T(HEK 293T,简称293T)细胞中外源性肿瘤坏死因子受体相关因子(tumor necrosis factor receptor⁃associated factor 6,TRAF6)与Krüppel样因子5(Krüppel⁃like factor 5,KLF5)的结合及TRAF6多聚泛素化修饰KLF5的方式和修饰的位点。方法:将构建的Flag⁃TRAF6、HA⁃KLF5、泛素过表达质粒、shTRAF6小干扰质粒和TRAF6 C70A位点突变质粒行不同组合转染293T细胞48 h。用免疫沉淀(immunoprecipitation,IP)和免疫印迹(immunoblotting,IB)实验检查TRAF6与KLF5的结合以及KLF5 K63或K48多聚泛素化水平。此外,构建KLF5全部赖氨酸突变的质粒,分别与TRAF6质粒共转染293T细胞。用前述IP/IB检测KLF5 K63连接的多聚泛素化修饰,并确定KLF5 K63泛素化修饰的位点。结果:293T细胞中TRAF6能与KLF5结合;TRAF6过表达和基因沉默或TRA6酶活性缺失能相应上调或下调KLF5 K63的多聚泛素化;KLF5被TRAF6 K63多聚泛素化修饰的位点是其第99位和第100位的赖氨酸。结论:TRAF6能与KLF5相互作用,并对KLF5⁃K99和K100进行K63多聚泛素化修饰。

Objective:To study the binding of exogenous tumor necrosis factor receptor⁃associated factor 6(TRAF6)to Krüppel⁃like factors 5(KLF5)as well as the pattern and site of KLF5 polyubiquitination by TRAF6 in HEK 293T(i.e.293 T)cells.Methods:The 293T cells were co⁃transfected with Flag⁃TRAF6,HA⁃KLF5 and ubiquitin(Ub)expression plasmids,or shTRAF6 and TRAF6 C70A plasmids in different combinations for 48 h.Then,the binding of TRAF6 to KLF5 and KLF5 K48/K63⁃linked polyubiquitination by TRAF6 were detected using immunoprecipitation(IP)and immunoblotting(IB)assays.Moreover,the plasmids with all lysine mutation of KLF5 were constructed,and co⁃transfected with TRAF6 overexpression plasmids into 293T cells.Thereafter,the level of KLF5 K63⁃linked polyubiquitination and the lysine(site)of KLF5 K63⁃linked polyubiquitination were measured or identified by IP and IB.Results:TRAF6 and KLF5 in 293T cells could bind with each other.The overexpression of TRAF6 up⁃regulated while the knockdown or activity deficiency of TRAF6 down⁃regulated the level of KLF5 K63⁃linked polyubiquitination.The site of KLF5 K63⁃linked polyubiquitination was its K99 or K100 lysine.Conclusion:TRAF6 can interact with KLF5 and modify the K99 and K100 of KLF5 via K63⁃linked polyubiquitination.