Identification of novel human Cdt1-binding proteins by a proteomics approach:: Proteolytic regulation by APC/CCdh1

In mammalian cells, Cdt1 activity is strictly controlled by multiple independent mechanisms, implying that it is central to the regulation of DNA replication during the cell cycle. In fact, unscheduled Cdt1 hyperfunction results in rereplication and/or chromosomal damage. Thus, it is important to understand its function and regulations precisely. We sought to comprehensively identify human Cdt1-binding proteins by a combination of Cdt1 affinity chromatography and liquid chromatography and tandem mass spectrometry analysis. Through this approach, we could newly identify 11 proteins, including subunits of anaphase-promoting complex/cyclosome (APC/C), SNF2H and WSTF, topoisomerase I and II alpha, GRWD1/WDR28, nucleophosmin/nucleoplasmin, and importins. In vivo interactions of Cdt1 with APC/C-Cdh1, SNF2H, topoisomerase I and II alpha, and GRWD1/WDR28 were confirmed by coimmunoprecipitation assays. A further focus on APC/C-Cdh1 indicated that this ubiquitin ligase controls the levels of Cdt1 during the cell cycle via three destruction boxes in the Cdt1 N-terminus. Notably, elimination of these destruction boxes resulted in induction of strong rereplication and chromosomal damage. Thus, in addition to SCFSkp2 and cullin4-based ubiquitin ligases, APC/C-Cdh1 is a third ubiquitin ligase that plays a crucial role in proteolytic regulation of Cdt1 in mammalian cells.