14-3-3/HIP-55复合体增强HIP-55蛋白稳定性  

14-3-3 / HIP-55 complex increases the stability of HIP-55

作　　者：田爱炬[1] 李子健[1] 

机构地区：[1]北京大学第三医院心内科血管医学研究所,卫生部心血管分子生物学与调节肽重点实验室,分子心血管学教育部重点实验室,心血管受体研究北京市重点实验室,北京100191

出　　处：《北京大学学报（医学版）》2015年第6期893-897,共5页Journal of Peking University:Health Sciences

基　　金：国家自然科学基金(81471893,81070078,81270157);北京市自然科学基金(7102158)资助~~

摘　　要：目的:用双分子荧光互补及免疫共沉淀技术验证HIP-55与14-3-3在HEK293细胞中存在相互作用,并进一步研究其生物学意义。方法:利用GATEWAY系统构建PDEST-N-Venus-HIP-55WT(野生型),PDEST-N-VenusHIP-55AA(突变体S269A/T291A),PDEST-GST-HIP-55WT及PDEST-C-Venus-14-3-3τ重组质粒,利用双分子荧光互补及免疫共沉淀技术检测两者的相互作用,同时应用14-3-3蛋白相互作用抑制肽R18和HIP-55蛋白突变体(HIP-55AA突变体S269A/T291A不能与14-3-3相互作用)作为工具研究两者结合后对嘌呤霉素诱导的HIP-55蛋白表达的影响。结果:外源转入的Venus-HIP-55WT、Venus-HIP-55AA及Venus-14-3-3蛋白能够在HEK293细胞中表达;双分子荧光互补实验结果表明HIP-55与14-3-3存在相互作用,HIP-55蛋白的S269/T291位点介导HIP-55与14-3-3的相互作用;免疫共沉淀技术表明内源性HIP-55与14-3-3存在相互作用;进一步研究发现HIP-55与14-3-3复合体增强HIP-55蛋白的稳定性,保护HIP-55不被降解。结论:14-3-3与HIP-55存在相互作用,14-3-3/HIP-55复合体可以促进HIP-55蛋白的稳定性。Objective： To further demonstrate the interaction of a new 14-3-3 interaction protein hematopoietic progenitor kinase 1[HPK1]-interacting protein（ HIP-55） and 14-3-3 proteins and its potential biological function in HEK293 cells. Methods： PDEST-N-Venus-HIP-55WT（ wild type）,PDEST-N-Venus-HIP-55AA（ mutants,S269 A / T291 A,abolishing the binding of HIP-55 to 14-3-3）,PDEST-GSTHIP-55 WT and PDEST-C-Venus-14-3-3τ plasmids were constructed by gateway system. Their expressions were demonstrated by Western blotting method. Then we used Bimolecular Fluorescence Complementation（ Bi FC） and co-immunoprecipitation（ co-IP） methods to demonstrate the interaction of HIP-55 and 14-3-3 in HEK293 cells. Moreover,the 14-3-3 antagonist peptide,R18 and HIP-55 protein mutant plasmid HIP-55 AA were used to detect the protein synthesis of HIP-55 at different time points induced by puromycin,an inhibitor of protein production. Results： The HEK293 cells expressed HIP-55 protein respectively,after being transected with PDEST-N-Venus-HIP-55 WT,PDEST-N-Venus-HIP-55 AA,PDEST-GSTHIP-55 WT plasmids and expressed 14-3-3 protein after being transected with PDEST-C-Venus-14-3-3τplasmids. We could detect venus fluorescence of venus-HIP-55 protein via confocal microscopy in HEK293 cells transfected with N-Venus-HIP-55 and C-14-3-3τ plasmids by Bi FC,butnot in HEK 293 cells transfected with N-Venus-HIP-55 AA（ mutants S269 A / T291A） and C-14-3-3τ plasmids. The results of Bi FC suggested that 14-3-3 interacted with HIP-55 through HIP-55 S269 / T291 sites. At the same time,the data of co-IP showed that there were endogenous interactions between 14-3-3 and HIP-55. Furthermore,puromycin had no influence in HIP-55 protein synthesis at hours 0,4,or 8 in HEK 293 cells expressing GST-HIP-55 WT and 14-3-3 plasmids,while puromycin blocked HIP-55 protein synthesis in HEK 293 cells transfected with N-Venus-HIP-55AA（ mutants S269 A / T291A） and C-14-3-3τ plasmids.The results indicated that the 14-3-3 / HIP-55 complex could co

关 键 词：HIP-55蛋白 人 14-3-3蛋白质类 蛋白质相互作用 

分 类 号：R34[医药卫生—基础医学]