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Vol. 19, Issue 3, 1007-1021, March 2008

This Article Full Text Full Text (PDF) Supplemental Materials All Versions of this Article: E07-09-0859v1 19/3/1007    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Google Scholar Articles by Sugimoto, N. Articles by Fujita, M. PubMed PubMed Citation Articles by Sugimoto, N. Articles by Fujita, M.

Identification of Novel Human Cdt1-binding Proteins by a Proteomics Approach: Proteolytic Regulation by APC/C^Cdh1

Nozomi Sugimoto^*,, Issay Kitabayashi, Satoko Osano^*,, Yasutoshi Tatsumi^*, Takashi Yugawa^*, Mako Narisawa-Saito^*, Akio Matsukage, Tohru Kiyono^*, and Masatoshi Fujita^*

*Virology Division and Molecular Oncology Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuohku, Tokyo 104-0045, Japan; and Faculty of Science, Japan Women's University, 2-8-1 Mejirodai, Bunkyouku, Tokyo 112-8679, Japan

Submitted September 4, 2007; Revised November 27, 2007; Accepted December 19, 2007
Monitoring Editor: Orna Cohen-Fix

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, GRWD1/WDR28, nucleophosmin/nucleoplasmin, and importins. In vivo interactions of Cdt1 with APC/C^Cdh1, SNF2H, topoisomerase I and II, 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.

Address correspondence to: Masatoshi Fujita (mafujita{at}gan2.res.ncc.go.jp)

Abbreviations used: pre-RC, prereplication complex; APC/C, anaphase-promoting complex/cyclosome.

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