Identification of Novel Human Cdt1-binding Proteins by a Proteomics Approach: Proteolytic Regulation by APC/CCdh1
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
DOI:
10.1091/mbc.e07-09-0859
Publication Date:
2007-12-28T01:34:19Z
AUTHORS (9)
ABSTRACT
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.
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