title: ROCK1 phosphorylates and activates zipper-interacting protein kinase.
abstract: Zipper-interacting protein kinase (ZIPK) regulates Ca(2+)-independent phosphorylation of both smooth muscle (to regulate contraction) and non-muscle myosin (to regulate non-apoptotic cell death) through either phosphorylation and inhibition of myosin phosphatase, the myosin phosphatase inhibitor CPI17, or direct phosphorylation of myosin light chain. ZIPK is regulated by multisite phosphorylation. Phosphorylation at least three sites Thr-180, Thr-225, and Thr-265 has been shown to be essential for full activity, whereas phosphorylation at Thr-299 regulates its intracellular localization. Herein we utilized an unbiased proteomics screen of smooth muscle extracts with synthetic peptides derived from the sequence of the regulatory phosphorylation sites of the enzyme to identify the protein kinases that might regulate ZIPK activity in vivo. Discrete kinase activities toward Thr-265 and Thr-299 were defined and identified by mass spectrometry as Rho kinase 1 (ROCK1). In vitro, ROCK1 showed a high degree of substrate specificity toward native ZIPK, both stoichiometrically phosphorylating the enzyme at Thr-265 and Thr-299 as well as bringing about activation. In HeLa cells, coexpression of ZIPK with ROCK1 altered the ROCK-induced phenotype of focused stress fiber pattern to a Rho-like phenotype of parallel stress fiber pattern. This effect was also dependent upon phosphorylation at Thr-265. Our findings provide a new regulatory pathway in smooth muscle and non-muscle cells whereby ROCK1 phosphorylates and regulates ZIP kinase.
authors.affiliation: Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710 and.
authors.forename: Laura
authors.initials: L
authors.lastname: Hagerty
authors.affiliation: Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710 and.
authors.forename: Douglas H
authors.initials: DH
authors.lastname: Weitzel
authors.affiliation: Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710 and.
authors.forename: Jenica
authors.initials: J
authors.lastname: Chambers
authors.affiliation: Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710 and.
authors.forename: Christopher N
authors.initials: CN
authors.lastname: Fortner
authors.affiliation: Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710 and.
authors.forename: Matthew H
authors.initials: MH
authors.lastname: Brush
authors.affiliation: Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710 and.
authors.forename: David
authors.initials: D
authors.lastname: Loiselle
authors.affiliation: Department of Biological Sciences, Graduate School of Science, Hiroshima University, Hiroshima 739 8526, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Hosoya
authors.affiliation: Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710 and. Electronic address: hayst001@mc.duke.edu.
authors.forename: Timothy A J
authors.initials: TAJ
authors.lastname: Haystead
mesh_terms: D000818:Animals; D051017:Apoptosis Regulatory Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D016923:Cell Death / Q000502:physiology; D064550:Death-Associated Protein Kinases; D004789:Enzyme Activation / Q000502:physiology; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008297:Male; D009130:Muscle, Smooth / Q000201:enzymology*; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism; D009218:Myosins / Q000378:metabolism; D010455:Peptides / Q000494:pharmacology; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D040901:Proteomics; D015398:Signal Transduction / Q000502:physiology*; D022502:Stress Fibers / Q000378:metabolism; D013552:Swine; D054460:rho-Associated Kinases
chemical_list: D051017:Apoptosis Regulatory Proteins; D047908:Intracellular Signaling Peptides and Proteins; D010455:Peptides; D064550:Death-Associated Protein Kinases; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D009219:Myosin-Light-Chain Kinase; D043263:Myosin-Light-Chain Phosphatase; D009218:Myosins