Regulation of zipper-interacting protein kinase activity in vitro and in vivo by multisite phosphorylation

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 Thr(180), Thr(225), Thr(265), Thr(299), Thr(306), and Ser(311). Mutational analysis showed that phosphorylation of Thr(180) in the kinase activation T-loop, Thr(225) in the substrate-binding groove, and Thr(265) in kinase subdomain X is essential for full ZIPK autophosphorylation and activity toward exogenous substrates. Abrogation of phosphorylation of Thr(299), Thr(306), and Ser(311) had little effect on enzyme activity, but mutation of Thr(299) and Thr(300) to alanine resulted in redistribution of ZIPK from the cytosol to the nucleus. Mutation of Thr(299) 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.