Phosphorylation of mammalian CDC6 by cyclin A/CDK2 regulates its subcellular localization.
Cyclin-dependent kinases (CDKs) are essential for regulating key transitions in the cell cycle, including initiation of DNA replication, mitosis and prevention of re-replication. Here we demonstrate that mammalian CDC6, an essential regulator of initiation of DNA replication, is phosphorylated by CDKs. CDC6 interacts specifically with the active Cyclin A/CDK2 complex in vitro and in vivo, but not with Cyclin E or Cyclin B kinase complexes. The cyclin binding domain of CDC6 was mapped to an N-terminal Cy-motif that is similar to the cyclin binding regions in p21(WAF1/SDI1) and E2F-1. The in vivo phosphorylation of CDC6 was dependent on three N-terminal CDK consensus sites, and the phosphorylation of these sites was shown to regulate the subcellular localization of CDC6. Consistent with this notion, we found that the subcellular localization of CDC6 is cell cycle regulated. In G1, CDC6 is nuclear and it relocalizes to the cytoplasm when Cyclin A/CDK2 is activated. In agreement with CDC6 phosphorylation being specifically mediated by Cyclin A/CDK2, we show that ectopic expression of Cyclin A, but not of Cyclin E, leads to rapid relocalization of CDC6 from the nucleus to the cytoplasm. Based on our data we suggest that the phosphorylation of CDC6 by Cyclin A/CDK2 is a negative regulatory event that could be implicated in preventing re-replication during S phase and G2.
Petersen
B O
BO
European Institute of Oncology, Department of Experimental Oncology, Via Ripamonti 435, 20141 Milan, Italy.
Lukas
J
J
Sørensen
C S
CS
Bartek
J
J
Helin
K
K
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D042846:CDC2-CDC28 Kinases; D019556:COS Cells; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D019925:Cyclin A / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016196:S Phase; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection
C103706:CDC6 protein, human; D018797:Cell Cycle Proteins; D019925:Cyclin A; D017931:DNA Primers; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D011494:Protein Kinases; C102333:histone H1 kinase; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495892:Cdk2 protein, rat; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases