[Isolation and characterization of a BRCA1-interacting protein].
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 DH5 alpha. The purified fusion proteins were obtained by GST-Sepharose 4B affinity chromatography. The purified fusion proteins were incubated with in vitro translated 35S-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 pHABRCT1/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.
Beijing Institute of Biotechnology, Beijing 100850, China.
Jing-Hua
JH
Yan
Qi-Nong
QN
Ye
Jian-Hua
JH
Zhu
Hong-Jun
HJ
Zhong
Hui-Yong
HY
Zheng
Cui-Fen
CF
Huang
D019313:BRCA1 Protein / Q000737:chemistry* / Q000378:metabolism; D005260:Female; D006801:Humans; D020798:Two-Hybrid System Techniques; D015003:Yeasts / Q000235:genetics
D019313:BRCA1 Protein