Cyclin-A-CDK2-mediated phosphorylation of CIZ1 blocks replisome formation and initiation of mammalian DNA replication.
CIZ1 is a nuclear matrix protein that cooperates with cyclin A2 (encoded by CCNA2) and CDK2 to promote mammalian DNA replication. We show here that cyclin-A-CDK2 also negatively regulates CIZ1 activity by phosphorylation at threonines 144, 192 and 293. Phosphomimetic mutants do not promote DNA replication in cell-free and cell-based assays, and also have a dominant-negative effect on replisome formation at the level of PCNA recruitment. Phosphorylation blocks direct interaction with cyclin-A-CDK2 and recruitment of endogenous cyclin A to the nuclear matrix. In contrast, phosphomimetic CIZ1 retains the ability to bind to the nuclear matrix, and its interaction with CDC6 is not affected. Phospho-T192-specific antibodies confirm that CIZ1 is phosphorylated during S phase and G2, and show that phosphorylation at this site occurs at post-initiation concentrations of cyclin-A-CDK2. Taken together, the data suggest that CIZ1 is a kinase sensor that promotes initiation of DNA replication at low kinase levels, when in a hypophosphorylated state that is permissive for cyclin-A-CDK2 interaction and delivery to licensed origins, but blocks delivery at higher kinase levels when it is phosphorylated.
University of York, Department of Biology, Heslington, York YO10 5DD, UK.
Nikki A
NA
Copeland
University of York, Department of Biology, Heslington, York YO10 5DD, UK.
Heather E
HE
Sercombe
University of York, Department of Biology, Heslington, York YO10 5DD, UK.
Rosemary H C
RH
Wilson
University of York, Department of Biology, Heslington, York YO10 5DD, UK dawn.coverley@york.ac.uk.
Dawn
D
Coverley
D016475:3T3 Cells; D000818:Animals; D002478:Cells, Cultured; D002843:Chromatin / Q000235:genetics; D056751:Cyclin A2 / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism*; D004261:DNA Replication; D016195:G2 Phase; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D016196:S Phase
C503721:CCNA2 protein, human; C508184:CCNA2 protein, mouse; D002843:Chromatin; C400423:Ciz1 protein, human; C540084:Ciz1 protein, mouse; D056751:Cyclin A2; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; C495894:CDK2 protein, human; C495893:Cdk2 protein, mouse; D051357:Cyclin-Dependent Kinase 2
CIZ1; Cyclin A; Cyclin dependent kinase 2; DNA replication; Phosphorylation; Replisome