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 language | English |
|---|---|
| Pages (from-to) | 1518-1527 |
| Number of pages | 10 |
| Journal | Journal of Cell Science |
| Volume | 128 |
| Issue number | 8 |
| Early online date | 3 Mar 2015 |
| DOIs | |
| Publication status | Published - 15 Apr 2015 |
Keywords
- CIZ1
- Cyclin A
- Cyclin dependent kinase 2
- DNA replication
- Phosphyorylation
- Replisome