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 IIalpha, GRWD1/WDR28, nucleophosmin/nucleoplasmin, and importins. In vivo interactions of Cdt1 with APC/C(Cdh1), SNF2H, topoisomerase I and IIalpha, 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 SCF(Skp2) 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.
Sugimoto
Nozomi
N
Virology Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuohku, Tokyo 104-0045, Japan.
Kitabayashi
Issay
I
Osano
Satoko
S
Tatsumi
Yasutoshi
Y
Yugawa
Takashi
T
Narisawa-Saito
Mako
M
Matsukage
Akio
A
Kiyono
Tohru
T
Fujita
Masatoshi
M
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D064173:Anaphase-Promoting Complex-Cyclosome; D002352:Carrier Proteins / Q000032:analysis*; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D002846:Chromatography, Affinity; D002877:Chromosomes, Human / Q000378:metabolism; D004249:DNA Damage; D004261:DNA Replication; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D040901:Proteomics; D034741:RNA, Small Interfering / Q000378:metabolism; D016192:Resting Phase, Cell Cycle; D044786:S-Phase Kinase-Associated Proteins / Q000378:metabolism; D013816:Thermodynamics; D043743:Ubiquitin-Protein Ligase Complexes / Q000378:metabolism*; D054875:Ubiquitination
C467199:CDT1 protein, human; D002352:Carrier Proteins; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins; D050505:Mutant Proteins; D034741:RNA, Small Interfering; D044786:S-Phase Kinase-Associated Proteins; C577488:CDC20 protein, human; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome