Final published version
Research output: Contribution to Journal/Magazine › Journal article › peer-review
Research output: Contribution to Journal/Magazine › Journal article › peer-review
}
TY - JOUR
T1 - Cyclin A/CDK2 mediated phosphorylation of CIZ1 blocks replisome formation and initiation of mammalian DNA replication
AU - Copeland, Nikki
AU - Sercombe, Heather E
AU - Wilson, Rosemary H. C.
AU - Coverley, Dawn
PY - 2015/4/15
Y1 - 2015/4/15
N2 - 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.
AB - 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.
KW - CDK
KW - replisome
KW - DNA replication
KW - cell cycle
KW - cancer
U2 - 10.1242/jcs.161919
DO - 10.1242/jcs.161919
M3 - Journal article
VL - 128
SP - 1518
EP - 1527
JO - Journal of Cell Science
JF - Journal of Cell Science
SN - 0021-9533
IS - 8
ER -