Two E3 ubiquitin ligases, SCF-Skp2 and DDB1-Cul4, target human Cdt1 for proteolysis.
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.
Nishitani
Hideo
H
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Higashi-ku, Fukuoka, Japan. hideon@molbiol.med.kyushu-u.ac.jp
Sugimoto
Nozomi
N
Roukos
Vassilis
V
Nakanishi
Yohsuke
Y
Saijo
Masafumi
M
Obuse
Chikashi
C
Tsurimoto
Toshiki
T
Nakayama
Keiichi I
KI
Nakayama
Keiko
K
Fujita
Masatoshi
M
Lygerou
Zoi
Z
Nishimoto
Takeharu
T
D000818:Animals; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D002478:Cells, Cultured; D044842:Cullin Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D019925:Cyclin A / Q000378:metabolism; D019927:Cyclin E / Q000378:metabolism; D004249:DNA Damage; D004261:DNA Replication / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D064248:Geminin; D020868:Gene Silencing; D006367:HeLa Cells; D006801:Humans; D007668:Kidney / Q000196:embryology / Q000378:metabolism; D051379:Mice; D010447:Peptide Hydrolases / Q000737:chemistry*; D010766:Phosphorylation; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D044786:S-Phase Kinase-Associated Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D044843:SKP Cullin F-Box Protein Ligases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism; D014466:Ultraviolet Rays
C467199:CDT1 protein, human; C114234:CUL4A protein, human; D018797:Cell Cycle Proteins; D044842:Cullin Proteins; D019925:Cyclin A; D019927:Cyclin E; C091435:DDB1 protein, human; D004268:DNA-Binding Proteins; C577995:GMNN protein, human; D064248:Geminin; D018809:Proliferating Cell Nuclear Antigen; D034741:RNA, Small Interfering; D044786:S-Phase Kinase-Associated Proteins; D025801:Ubiquitin; D044843:SKP Cullin F-Box Protein Ligases; D010447:Peptide Hydrolases