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 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.
Graves
Paul R
PR
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Winkfield
Karen M
KM
Haystead
Timothy A J
TA
D000595:Amino Acid Sequence; D002460:Cell Line; D017931:DNA Primers; D006367:HeLa Cells; D006801:Humans; D007668:Kidney; D020930:MAP Kinase Kinase Kinases; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D010710:Phosphates; D011994:Recombinant Proteins; D010769:Phosphothreonine; D010768:Phosphoserine; D017346:Protein Serine-Threonine Kinases; D020930:MAP Kinase Kinase Kinases; C090757:mitogen-activated protein kinase kinase kinase 12