Article
- The EMBO Journal (2006) 25, 1126 - 1136
- doi:10.1038/sj.emboj.7601002
Published online: 16 February 2006
Subject Categories:
Two E3 ubiquitin ligases, SCF-Skp2 and DDB1-Cul4, target human Cdt1 for proteolysis
Hideo Nishitani1, Nozomi Sugimoto2, Vassilis Roukos3, Yohsuke Nakanishi1, Masafumi Saijo4, Chikashi Obuse5, Toshiki Tsurimoto6, Keiichi I Nakayama7, Keiko Nakayama7, Masatoshi Fujita2, Zoi Lygerou3 and Takeharu Nishimoto1
- Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Higashi-ku, Fukuoka, Japan
- Virology Division, National Cancer Center Research Institute, Chuoh-ku, Tokyo, Japan
- Laboratory of General Biology, School of Medicine, University of Patras, Rio, Patras, Greece
- Graduate School of FrontierBioscience, Osaka University, Japan
- Department of Gene Mechanisms, Graduate School of Biostudies, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto, Japan
- Department of Biology, School of Sciences, Kyushu University, Higashi-ku, Fukuoka, Japan
- Medical Institute of Bioregulation, Kyushu University, Higashi-ku, Fukuoka, Japan
Correspondence to:
Hideo Nishitani, Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan. Tel.: +81 92 642 6177; Fax: +81 92 642 6183; E-mail: hideon@molbiol.med.kyushu-u.ac.jp
Received 21 October 2005; Accepted 23 January 2006
Abstract
Replication licensing is carefully regulated to restrict replication to once in a cell cycle. In higher eukaryotes, regulation of the licensing factor Cdt1 by proteolysis and Geminin is essential to prevent re-replication. We show here that the N-terminal 100 amino acids of human Cdt1 are recognized for proteolysis by two distinct E3 ubiquitin ligases during S–G2 phases. Six highly conserved amino acids within the 10 first amino acids of Cdt1 are essential for DDB1-Cul4-mediated proteolysis. This region is also involved in proteolysis following DNA damage. The second E3 is SCF-Skp2, which recognizes the Cy-motif-mediated Cyclin E/A-cyclin-dependent kinase-phosphorylated region. Consistently, in HeLa cells cosilenced of Skp2 and Cul4, Cdt1 remained stable in S–G2 phases. The Cul4-containing E3 is active during ongoing replication, while SCF-Skp2 operates both in S and G2 phases. PCNA binds to Cdt1 through the six conserved N-terminal amino acids. PCNA is essential for Cul4- but not Skp2-directed degradation during DNA replication and following ultraviolet-irradiation. Our data unravel multiple distinct pathways regulating Cdt1 to block re-replication.
Keywords:
- cell cycle,
- Cdt1,
- Geminin,
- proteolysis,
- replication
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