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Acta Biochimica et Biophysica Sinica

Year: 2009  |  Volume: 41  |  Issue: 10  |  Page No.: 816 - 821

Identification of FANCA as a protein interacting with centromere-associated protein E

J Du, L Chen and J. Shen

Abstract

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.

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