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: DNA Replication; Proteomics; 0303 health sciences; Cdc20 Proteins; Amino Acid Motifs; Molecular Sequence Data; Cell Cycle Proteins; Resting Phase, Cell Cycle; Anaphase-Promoting Complex-Cyclosome; Chromatography, Affinity; Mass Spectrometry; Cell Line; 03 medical and health sciences; Chromosomes, Human; Humans; Mutant Proteins; Amino Acid Sequence; RNA, Small Interfering; Carrier Proteins; Protein Processing, Post-Translational; DNA Damage; Protein Binding

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 IIα, GRWD1/WDR28, nucleophosmin/nucleoplasmin, and importins. In vivo interactions of Cdt1 with APC/CCdh1, SNF2H, topoisomerase I and IIα, and GRWD1/WDR28 were confirmed by coimmunoprecipitation assays. A further focus on APC/CCdh1indicated 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 SCFSkp2and cullin4-based ubiquitin ligases, APC/CCdh1is a third ubiquitin ligase that plays a crucial role in proteolytic regulation of Cdt1 in mammalian cells.