Cyclin A/CDK2 phosphorylation of CIZ1 blocks replisome formation and initiation of mammalian DNA replication

Nikki A. Copeland, Heather E. Sercombe, Rosemary H. C. Wilson, Dawn Coverley

Research output: Contribution to journalArticlepeer-review

Abstract

CIZ1 is a nuclear matrix protein that cooperates with cyclin A/CDK2 to promote mammalian DNA replication. We show here that cyclin A/CDK2 also negatively regulates CIZ1 activity via 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 nuclear matrix binding capability, and interaction with CDC6 is not affected. Phospho-threonine 192-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. 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 itself phosphorylated.

Original languageEnglish
Pages (from-to)1518-1527
Number of pages10
JournalJournal of Cell Science
Volume128
Issue number8
Early online date3 Mar 2015
DOIs
Publication statusPublished - 15 Apr 2015

Keywords

  • CIZ1
  • Cyclin A
  • Cyclin dependent kinase 2
  • DNA replication
  • Phosphyorylation
  • Replisome

Cite this