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Zipper-interacting protein kinase (ZIPK) is a widely expressed serine/threonine kinase implicated in cell death and smooth muscle contractility, but its mechanism of regulation is unknown. We have identified six phosphorylation sites in ZIPK that regulate both its enzyme activity and localization, including Thr180, Thr225, Thr265, Thr299, Thr306, and Ser311. Mutational analysis showed that phosphorylation of Thr180 in the kinase activation T-loop, Thr225 in the substrate-binding groove, and Thr265 in kinase subdomain X is essential for full ZIPK autophosphorylation and activity toward exogenous substrates. Abrogation of phosphorylation of Thr299, Thr306, and Ser311 had little effect on enzyme activity, but mutation of Thr299 and Thr300 to alanine resulted in redistribution of ZIPK from the cytosol to the nucleus. Mutation of Thr299 alone to alanine caused ZIPK to assume a diffuse cellular localization, whereas T299D redistributed the enzyme to the cytoplasm. C-terminal truncations of ZIPK at amino acid 273 or 342 or mutation of the leucine zipper motif increased ZIPK activity toward exogenous substrates by severalfold, suggesting a phosphorylation-independent autoinhibitory role for the C-terminal domain. Additionally, mutation of the leucine zipper reduced the ability of ZIPK to oligomerize and also caused ZIPK to relocalize from the cytoplasm to the nucleus in vivo. Together, our findings show that ZIPK is positively regulated by phosphorylation within its kinase domain and that it contains an inhibitory C-terminal domain that controls enzyme activity, localization, and oligomerization.

Chromatid catenation is actively monitored in human cells, with progression from G(2) to mitosis being inhibited when chromatids are insufficiently decatenated. Mitotic delay was quantified in normal and checkpoint-deficient human cells during treatment with ICRF-193, a topoisomerase II catalytic inhibitor that prevents chromatid decatenation without producing topoisomerase-associated DNA strand breaks. Ataxia telangiectasia (A-T) cells, defective in DNA damage checkpoints, showed normal mitotic delay when treated with ICRF-193. The mitotic delay in response to ICRF-193 was ablated in human fibroblasts expressing an ataxia telangiectasia mutated- and rad3-related (ATR) kinase-inactive ATR allele (ATR(ki)). BRCA1-mutant HCC1937 cells also displayed a defect in ICRF-193-induced mitotic delay, which was corrected by expression of wild-type BRCA1. Phosphorylations of hCds1 or Chk1 and inhibition of Cdk1 kinase activity, which are elements of checkpoints associated with DNA damage or replication, did not occur during ICRF-193-induced mitotic delay. Over-expression of cyclin B1 containing a dominant nuclear localization signal, and inhibition of Crm1-mediated nuclear export, reversed ICRF-193-induced mitotic delay. In combination, these results imply that ATR and BRCA1 enforce the decatenation G(2) checkpoint, which may act to exclude cyclin B1/Cdk1 complexes from the nucleus. Moreover, induction of ATR(ki) produced a 10-fold increase in chromosomal aberrations, further emphasizing the vital role for ATR in genetic stability.

Cdc25C is a dual-specificity protein kinase that controls entry into mitosis by dephosphorylating Cdc2 on both threonine 14 and tyrosine 15. Cdc25C is phosphorylated on serine 216 throughout interphase but not during mitosis. Serine 216 phosphorylation mediates the binding of 14-3-3 protein to Cdc25C, and Cdc25C/14-3-3 complexes are present throughout interphase but not during mitosis. Here we report the cloning of a human kinase denoted C-TAK1 (for Cdc twenty-five C associated protein kinase) that phosphorylates Cdc25C on serine 216 in vitro. C-TAK1 is ubiquitously expressed in human tissues and cell lines and is distinct from the DNA damage checkpoint kinase Chk1, shown previously to phosphorylate Cdc25C on serine 216. Cotransfection of Cdc25C with C-TAK1 resulted in enhanced phosphorylation of Cdc25C on serine 216. In addition, a physical interaction between C-TAK1 and Cdc25C was observed upon transient overexpression in COS-7 cells. Finally, coproduction of Cdc25C and C-TAK1 in bacteria resulted in the stoichiometric phosphorylation of Cdc25C on serine 216 and facilitated 14-3-3 protein binding in vitro. Taken together, these results suggest that one function of C-TAK1 may be to regulate the interactions between Cdc25C and 14-3-3 in vivo by phosphorylating Cdc25C on serine 216.