摘要
为鉴定微丝相关蛋白HSPC300/hHBRK1在肝脏组织的功能,采用GSTpull-down结合质谱技术,检测该蛋白在肝脏中的结合蛋白,结果提示,肌球蛋白Ⅵ与HSPC300/hHBRK1共沉降,Western印迹杂交证实了质谱的结果.构建HSPC300/hHBRK1原核表达载体,诱导并获得了His-hHBRK1融合蛋白.利用免疫共沉淀证实hHBRK1与肌球蛋白Ⅵ存在相互作用,激光共聚焦检测显示hHBRK1与肌球蛋白Ⅵ在肺癌95D细胞的胞浆共定位,提示其相互作用可能是直接结合.肌球蛋白Ⅵ参与细胞迁移、高尔基分泌泡的运输和维持高尔基体稳定性等作用.hHBRK1与肌球蛋白Ⅵ相互作用,为微丝相关蛋白HSPC300/hHBRK1参与细胞迁移和胞内物质运输提供了进一步佐证.
The Scar/Wave protein, a major activator of Arp2/3 complex, is required for aetin polymerization during cell morphogenesis. The mammalian Wave/Scar complex is composed of five proteins： Sra1/PIR121, Nap1/Nap125, Abi-1/Abi-2, Brk1/HSPC300 and Scar/Wave. Knockout of HSPC300 in the Dorsophila central nervous system recapitulates various aspects of neuralogocal defects refected by Scar, CYFIP and Kette mutants. It has been generally accepted that the Wave complex subunits are critical for Scar stabilization, cellular localization and its activity, Whereas HSPC300 is unique for its lack of association with the complex and primarily remained in a cytoplasmic pool. Whether HSPC300/hHBRK1 has additional cytoplasmic targets is till unclear. To identify binding partners of HSPC300/hHBRK1, GST pull-down assay following peptide finger mass spectrometry were applied to discover the associated protein in the mouse liver. The interaction between hHBRK1 and myosin Ⅵ was found by GST pull-down and co-immunoprecipitation. The co-localization of hHBRK1 and myosin Ⅵ in the cytoplasm was observed in 95D cells using confoeal laser scanning microscopy. Since myosin Ⅵ is involved in a wide variety of intracellular processes such as endocytosis, cargo transport and cell migration, our results suggested that it binding to HSPC300/hHBRK1 might play an important role in cell motility and cargo transport.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2009年第2期146-151,共6页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金(No.30570550)
北京市自然科学基金(No.5042023,No.7062052)资助~~
