泛素连接酶Smurf1促进PDK1的多聚泛素化修饰  

Ubiquitin ligase Smurf1 promotes poly-ubiquitination of PDK1

作　　者：魏军 时慧森 党智笙 张令强 张学利 WEI Jun;SHI Hui-sen;DANG Zhi-sheng;ZHAGN Ling-qiang;ZHAGN Xue-li(Fengxian Hospital Graduate Training Base, Jinzhou Medical University, Shanghai 201499, China;Graduate School, Jinzhou Medical University, Jinzhou, Liaoning 121000, China;State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences Beijing, Beijing Institute of Lifeomics, Beijing 102206, China)

机构地区：[1]锦州医科大学奉贤中心医院研究生培训基地,上海201499 [2]锦州医科大学研究生院,锦州121000 [3]蛋白质组学国家重点实验室,北京蛋白质组研究中心,国家蛋白质科学中心(北京),北京生命组学研究所,北京102206

出　　处：《军事医学》2017年第12期947-951,共5页Military Medical Sciences

基　　金：国家自然科学基金资助项目(81772959);上海市科委实验动物研究领域项目(16140902000)

摘　　要：目的探讨3-磷酸肌醇依赖性蛋白激酶1(PDK1)发生多聚泛素化修饰的机制。方法免疫共沉淀(COIP)和免疫印迹(WB)分析PDK1的多聚泛素化修饰,MEF细胞内印证泛素连接酶Smad泛素化修饰调节因子1(Smurf1)增强PDK1多聚泛素化修饰,定点突变和CO-IP及WB确定PDK1多聚泛素化修饰位点。结果 PDK1可发生多聚泛素化修饰,泛素连接酶Smurf1增强PDK1多聚泛素化修饰,且依赖HECT类泛素连接酶Smurf1 K699位点活性;K304是PDK1多聚泛素化修饰位点。结论泛素连接酶Smurf1增强PDK1的多聚泛素化修饰。Objective To investigate the mechanism of 3-phosphoinositide-dependent protein kinase 1 （PDK1） polyubiquitination. Methods Co-immunopreeipitation （Co-IP） and Western blot （WB） were used to analyze poly ubiquitination of PDK1. It was confirmed that ubiquitin ligase smad ubiquitylation regulatory factor 1 （ Smurfl ） inprove PDK1 poly-ubiquitination within MEF cells, site-directed mutagenesis and WB before PDK1 poly-ubiquitination sites were determined. Results We found that PDK1 could undergoes poly-ubiquitination, ubiqufiin ligase Smurfl was found to be a direct E3 ligase for PDK1 poly-ubiquitination and might rely on the ubiquitin ligase Smurfl K699 site activity. K304 was PDK1 poly-ubiquitination modification site point. Conclusion The ubiquitin ligase Smurfl can promate poly-ubiquitination of PDK1.

关 键 词：泛素连接酶 泛素化修饰 免疫共沉淀 免疫印迹法 Smurf1 PDK1 PDPK1 细胞增殖 质粒 突变 

分 类 号：R3411[医药卫生—基础医学]