title: Phosphorylation of Cdc6 at serine 74, but not at serine 106, drives translocation of Cdc6 to the cytoplasm.
abstract: Phosphorylation-dependent cytoplasmic translocation of human Cdc6 during S phase is sufficient to control its activity after origin firing. Export from the nucleus also serves as a mechanism for preventing re-replication in mammalian cells. Phosphorylation of the CDK consensus serine residues 54, 74, and 106 has been suggested to be involved in the cytoplasmic translocation of Cdc6. To determine the relative importance of the three phosphorylation sites, we have generated Cdc6 variants by substituting one or more of the three serine residues with alanine or aspartic acid and have assessed their cytoplasmic translocation behavior. Phosphorylation of serine 74 mainly contributes to the cytoplasmic translocation of Cdc6, while serine 54 phosphorylation provides a minor contribution. In contrast, phosphorylation at serine 106 does not affect the nuclear export of Cdc6. Comparative results were found in cells coexpressing the phosphorylation defective mutants of Cdc6 and cyclin A as well as in non-transfected cells synchronized by their release from a double thymidine block. We conclude that Cdk-mediated phosphorylation of Cdc6 at serine 74 is required for the cytoplasmic translocalization of Cdc6 during the cell cycle. Phosphorylation of Cdc6 at serine 54 plays a minor role and phosphorylation of serine 106 plays no role in the cytoplasmic localization of Cdc6. The phosphorylation of S74 in Cdc6 could be important for binding to the nuclear export protein for translocalization.
authors.affiliation: Division of Pharmaceutical Biosciences, Research Institute for Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul, Republic of Korea. hsyim@hanyang.ac.kr
authors.forename: Hyungshin
authors.initials: H
authors.lastname: Yim
authors.forename: Ji-Woong
authors.initials: JW
authors.lastname: Park
authors.forename: Sang Uk
authors.initials: SU
authors.lastname: Woo
authors.forename: Seong-Taek
authors.initials: ST
authors.lastname: Kim
authors.forename: Linhua
authors.initials: L
authors.lastname: Liu
authors.forename: Chul-Hoon
authors.initials: CH
authors.lastname: Lee
authors.forename: Seung Ki
authors.initials: SK
authors.lastname: Lee
mesh_terms: D021581:Active Transport, Cell Nucleus; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D019925:Cyclin A / Q000378:metabolism; D019927:Cyclin E / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012694:Serine; D013997:Time Factors; D014162:Transfection
chemical_list: C103706:CDC6 protein, human; D018797:Cell Cycle Proteins; D019925:Cyclin A; D019927:Cyclin E; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D012694:Serine; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2