Identification of FANCA as a protein interacting with centromere-associated protein E
Identification of FANCA as a protein interacting with centromere-associated protein E
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DOI：
10.1093/abbs/gmp074
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发表时间：
2009-10-01
影响因子：
3.7
通讯作者：
Shen, Jilong
中科院分区：
文献类型：
--
作者：
Du, Jian;Chen, Lijian;Shen, Jilong
本研究旨在分离鉴定与着丝粒相关蛋白E（CENP-E）相互作用的蛋白，为探索CENP-E在细胞周期调控和肿瘤发病机制中的功能提供新线索。采用酵母二杂交筛选和常规分子生物学技术筛选具有CENP-E动粒结合域的人HeLa cDNA文库。 CENP-E C 末端的诱饵是通过亚克隆方法创建的，以找出与 CENP-E 着丝粒结合域相互作用的最佳候选蛋白。获得了八种新型 CENP-E 相互作用蛋白，包括智人范可尼贫血补充 A 组 (FANCA)。在酵母双杂交测定中，发现FANCA的N端260个氨基酸对于与CENP-E的C端相互作用是必要且充分的。这种相互作用通过体外谷胱甘肽S-转移酶下拉试验和体内免疫共沉淀试验得到证实。我们对 CENP-E 与 FANCA 相互作用的发现表明 CENP-E 和 FANCA 可能在有丝分裂检查点信号通路的功能调节中发挥重要作用。
This study sought to isolate and identify proteins that interact with centromere-associated protein E (CENP-E), provide new clues for exploring the function of CENP-E in cell cycle control and the pathogenesis of tumor. Yeast two-hybrid screen and regular molecular biologic techniques were undertaken to screen human HeLa cDNA library with the kinetochore binding domain of CENP-E. The bait from the C-terminus of CENP-E was created by subcloning methods to find out optimal candidate proteins that interact with the kinetochore binding domain of CENP-E. Eight novel CENP-E interacting proteins including Homo sapiens Fanconi anemia complementation group A (FANCA) were obtained. In yeast two-hybrid assay, the N-terminal 260 amino acids of FANCA were found to be necessary and sufficient for the interaction with the C-terminus of CENP-E. The interaction was confirmed by in vitro glutathione S-transferase pull-down assay and in vivo co-immunoprecipitation assay. Our finding of the interaction of CENP-E with FANCA demonstrates that CENP-E and FANCA may play important roles in the functional regulation of the mitotic checkpoint signal pathway.