hHBrk1与WAVE1蛋白相互作用位点的鉴定

Interaction between hHBrk1 and WAVE1 protein

目的:鉴定微丝相关蛋白hHBrk1与WAVE1的结合位点。方法:利用PCR的方法克隆人WAVE1及各结构域的基因片段,并将之亚克隆至真核表达载体;免疫共沉淀技术检测hHBrk1与WAVE1结合位点。结果:获得了WAVE1及各结构域基因的真核表达载体;免疫共沉淀发现hHBrk1与WAVE1的Scar同源结构域(SHD)结合,而WAVE1蛋白结合于hHBrk1的羧基端,羧基端七肽重复(HR)结构域点突变后获得的蛋白失去了与WAVE1的结合能力。结论:hHBrk1可能通过HR结构域与WAVE1的SHD区结合,参与WAVE1蛋白的细胞亚定位或维持其稳定性,从而调控肿瘤细胞的迁移。

AIM： To identify the binding sites between hHBrk1 and WAVE1 protein.METHODS： PCR was applied to clone the WAVE1 gene and its truncated domains from human fetal brain library.The gene fragments were sub-cloned into eukaryotic expression vector.Co-immunoprecipitation assay was used to identify the binding sites between hHBrk1 and WAVE1.RESULTS： The sequences of WAVE1 and the truncated domains were successfully amplified and sub-cloned into pCMV-HA vector.An HA tag was added to the fused protein at N-terminal.The interaction between hHBrk1 and the Scar homology domain（SHD） of WAVE1 was identified by co-immunoprecipitation.We also observed that WAVE1 bound to the heptad repeat（HR） domain of hHBrk1,and the site-directed mutations at HR domain abolished the interaction between hHBrk1 and WAVE1.CONCLUSION： hHBrk1 may play an important role in migration of tumor cells through binding to SHD of WAVE1 protein.