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J. Biol. Chem., Vol. 282, Issue 7, 4884-4893, February 16, 2007

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ROCK1 Phosphorylates and Activates Zipper-interacting Protein Kinase^*

Laura Hagerty¹, Douglas H. Weitzel¹, Jenica Chambers, Christopher N. Fortner, Matthew H. Brush, David Loiselle, Hiroshi Hosoya, and Timothy A. J. Haystead²

From the Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710 and Department of Biological Sciences, Graduate School of Science, Hiroshima University, Hiroshima 739 8526, Japan

Zipper-interacting protein kinase (ZIPK) regulates Ca²⁺-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.

Received for publication, October 24, 2006 , and in revised form, December 6, 2006.

^* This work was supported by National Institutes of Health Grant R01 HL07895. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental data.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Pharmacology and Cancer Biology, Duke University Medical Center, C119 LSRC Research Dr., Durham, NC 27710. Tel.: 919-613-8606; Fax: 919-668-0977; E-mail: hayst001{at}mc.duke.edu.

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