摘要
乳腺癌易感基因 (breastcancersusceptibilitygene 1,BRCA1)在DNA损伤修复、细胞周期调控、染色质的稳定、基因转录激活以及细胞凋亡等方面起着重要作用。BRCA1C 末端是富含酸性氨基酸的转录激活结构域 (AD) ,AD核心结构为两个串联的BRCT结构域 (BRCT1和BRCT2 )。应用酵母双杂交技术 ,以BRCT2为诱饵蛋白 ,从卵巢文库中筛选到了与BRCT2结构域相互作用蛋白FHL2 (fourandhalfLIMdomains)。利用酵母交配的方法证明FHL2与BRCA1BRCT2特异结合 ,而不与BRCA1BRCT1、Rap1BRCT结构域结合。GST沉淀实验表明 ,FHL2在体外特异地与BRCT2结构域相结合 ;免疫共沉淀实验表明 ,FHL2在体内特异地与BRCT2结构域结合 ;FHL2可与全长BRCA1结合。BRCA1与FHL2相互作用的发现为研究BRCA1以及FHL2在肿瘤发生、发展中的作用打下了坚实的基础。
BRCA1 (breast cancer susceptibility gene 1) plays important roles in DNA damage repair,cell checkpoint regulation,gene transcription,chromosome stability,and apoptosis.At the C terminus of BRCA1 is the activation domain with a number of acidic amino acid residues that includes two tandem repeats of BRCT(BRCT1 and BRCT2).In this study,to identify proteins that interact with the BRCT2 domain of BRCA1,the standard yeast two hybird screen was performed.FHL2 was isolated from a human ovary library,with the BRCT2 domain of BRCA1 as bait.Furthermore,the specific interaction of FHL2 with the BRCT2 domain of BRCA1,but not with the BRCT1 domain of BRCA1 and the BRCT domain of Rap1,was verified by yeast mating.To confirm the interaction between BRCA1 and FHL2 in vitro ,the GST pull down assay was performed,the coding sequences of BRCT1 and BRCT2 domains were fused in frame with the coding region of GST in the pGEX 2T vector,generating the pGST BRCT1 and pGST BRCT2 recombinant plasmids the fusion proteins GST BRCT1 and GST BRCT2 were expressed in E.coli DH5α.The purified fusion proteins were obtained by GST Sepharose 4B affinity chromatography.The purified fusion proteins were incubated with in vitro translated 35 S methinine labeled FHL2.Consistent with the two hybird results,FHL2 could specifically bind to the BRCT2 domain,but not BRCT1 in vitro. To further assess the binding specificity of FHL2 to the BRCT2 domain of BRCA1 in vivo ,pFLAG FHL2 and pHA BRCT1/ pHA BRCT2 recombinant plasmids were cotransfected into 293T cells.Then the coimmunoprecipitation assay were performed.The results also showed that FHL2 specifically interacted with the BRCT2 domain in vivo. Furthermore,the coimmunoprecipitation assay demonstrated that FHL2 could interact with endogenous BRCA1 in vivo. These findings lay solid foundations for study on the function of BRCA1 and FHL2 in cancer development and progression.
