Abstract
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.
Footnotes
↵*Present address: Lancaster University, Faculty of Health and Medicine, Biomedical and Life Sciences, Lancaster LA1 4YQ, UK.
↵‡Present address: The University of Oxford, Wellcome Trust Centre for Human Genetics, Roosevelt Drive, Oxford OX3 7BN, UK.
Competing interests
The authors declare no competing or financial interests.
Author contributions
N.A.C. and D.C. designed experiments. N.A.C., H.E.S. and R.H.C.W. performed experiments. N.A.C. analysed data. N.A.C. and D.C. wrote the manuscript.
Funding
This work was supported by Yorkshire Cancer Research [grant number Y252 to D.C]; and the North West Cancer Research [grant numbers CR879 and CR891 to N.A.C]. D.C. is supported by the Higher Education Funding Council; and R.W. and H.S. studentships were supported by the Biotechnology and Biological Sciences Research Council.
Supplementary material available online at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.161919/‐/DC1
- Received September 9, 2014.
- Accepted February 23, 2015.
- © 2015. Published by The Company of Biologists Ltd