Identification of Novel Human Cdt1-binding Proteins by a Proteomics Approach: Proteolytic Regulation by APC/CCdh1

• DOI: 10.1091/mbc.e07-09-0859
• Publication Date: 2007-12-28T01:34:19Z
• Authors: Nozomi Sugimoto; Issay Kitabayashi; Satoko Osano; Yasutoshi Tatsumi; Takashi Yugawa; Mako Narisawa-Saito; Akio Matsukage; Tohru Kiyono; Masatoshi Fujita
• Keywords: Immunoprecipitation

In mammalian cells, Cdt1 activity is strictly controlled by multiple independent mechanisms, implying that it central to the regulation of DNA replication during cell cycle. fact, unscheduled hyperfunction results in rereplication and/or chromosomal damage. Thus, important understand its function and regulations precisely. We sought comprehensively identify human Cdt1-binding proteins a combination affinity chromatography liquid tandem mass spectrometry analysis. Through this approach, we could newly 11 proteins, including subunits anaphase-promoting complex/cyclosome (APC/C), SNF2H WSTF, topoisomerase I IIalpha, GRWD1/WDR28, nucleophosmin/nucleoplasmin, importins. vivo interactions with APC/C(Cdh1), SNF2H, GRWD1/WDR28 were confirmed coimmunoprecipitation assays. A further focus on APC/C(Cdh1) indicated ubiquitin ligase controls levels cycle via three destruction boxes N-terminus. Notably, elimination these resulted induction strong addition SCF(Skp2) cullin4-based ligases, third plays crucial role proteolytic cells.