Identification of FANCA as a protein interacting with centromere-associated protein E.
This study sought to isolate and identify proteins that interact with centromere-associated protein E (CENPE), 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 coimmunoprecipitation 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.
Du
Jian
J
Department of Biochemistry and Molecular Biology, Anhui Medical University, Hefei, China. dudu@mail.ustc.edu.cn
Chen
Lijian
L
Shen
Jilong
J
D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics*; D005199:Fanconi Anemia / Q000235:genetics*; D020022:Genetic Predisposition to Disease; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D054730:Protein Interaction Domains and Motifs; D014407:Tumor Cells, Cultured; D020798:Two-Hybrid System Techniques
D002868:Chromosomal Proteins, Non-Histone; C068688:centromere protein E; D005982:Glutathione Transferase