Cyclin-dependent kinases phosphorylate human Cdt1 and induce its degradation.
Eukaryotic cells tightly control DNA replication so that replication origins fire only once during S phase within the same cell cycle. Cell cycle-regulated degradation of the replication licensing factor Cdt1 plays important roles in preventing more than one round of DNA replication per cell cycle. We have previously shown that the SCF(Skp2)-mediated ubiquitination pathway plays an important role in Cdt1 degradation. In this study, we demonstrate that human Cdt1 is a substrate of Cdk2 and Cdk4 both in vivo and in vitro. Overexpression of cyclin-dependent kinase inhibitors such as p21 and p27 dramatically suppresses the phosphorylation of Cdt1, disrupts the interaction of Cdt1 with the F-box protein Skp2, and blocks the degradation of Cdt1. Further analysis reveals that Cdt1 interacts with cyclin/cyclin-dependent kinase (Cdk) complexes through a cyclin/Cdk binding consensus site, located at the N terminus of Cdt1. A Cdt1 mutant carrying four amino acid substitutions at the Cdk binding site dramatically reduces associations with cyclin/Cdk complexes. This mutant is not phosphorylated, fails to bind Skp2 and is more stable than wild-type Cdt1. These data suggest that cyclin/Cdk-mediated Cdt1 phosphorylation is required for the association of Cdt1 with the SCF(Skp2) ubiquitin ligase and thus is important for the cell cycle dependent degradation of Cdt1 in mammalian cells.
Liu
Enbo
E
Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA.
Li
Xianghong
X
Yan
Feng
F
Zhao
Qiping
Q
Wu
Xiaohua
X
D001665:Binding Sites; D015153:Blotting, Western; D042846:CDC2-CDC28 Kinases / Q000378:metabolism*; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism / Q000502:physiology*; D002478:Cells, Cultured; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins; D012190:Retroviridae / Q000235:genetics; D016196:S Phase; D044786:S-Phase Kinase-Associated Proteins / Q000378:metabolism / Q000502:physiology*; D013997:Time Factors; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism
C493956:CDKN1A protein, human; C467199:CDT1 protein, human; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D011518:Proto-Oncogene Proteins; D044786:S-Phase Kinase-Associated Proteins; D025521:Tumor Suppressor Proteins; D025801:Ubiquitin; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004247:DNA; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495900:CDK4 protein, human; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases