Phosphorylation of mammalian CDC6 by Cyclin A/CDK2 regulates its subcellular localization

• Published in: The EMBO journal Vol. 18; no. 2; pp. 396 - 410
• Main Authors: Petersen, Birgit Otzen, Lukas, Jiri, Sørensen, Claus Storgaard, Bartek, Jiri, Helin, Kristian
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 15.01.1999; Blackwell Publishing Ltd
• Subjects: Animals; Base Sequence; Binding Sites; CDC2-CDC28 Kinases; CDC6; Cell Cycle; Cell Cycle Proteins - chemistry; Cell Cycle Proteins - genetics; Cell Cycle Proteins - metabolism; Cell Line; COS Cells; Cyclin A; Cyclin A - metabolism; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinases - metabolism; Deoxyribonucleic acid; DNA; DNA Primers - genetics; DNA replication; Humans; In Vitro Techniques; Mammals; Nuclear Proteins - chemistry; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Phosphorylation; Protein Kinases - metabolism; Protein-Serine-Threonine Kinases - metabolism; Rats; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; S Phase; Subcellular Fractions - metabolism; Transfection
• ISSN: 0261-4189; 1460-2075; 1460-2075
• DOI: 10.1093/emboj/18.2.396

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 p21WAF1/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.