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J. Biol. Chem., Vol. 281, Issue 34, 24322-24335, August 25, 2006

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Phosphorylation of Protein Phosphatase Inhibitor-1 by Protein Kinase C^*

Bogachan Sahin, Hongjun Shu, Joseph Fernandez^¶, Ali El-Armouche^||, Jeffery D. Molkentin^**, Angus C. Nairn, and James A. Bibb¹

From the Department of Psychiatry, Protein Chemistry Laboratory, Alliance for Cellular Signaling, The University of Texas Southwestern Medical Center, Dallas, Texas 75390, the ^¶Protein/DNA Technology Center and Laboratory of Cellular and Molecular Neuroscience, Rockefeller University, New York, New York 10021, ^||Institute of Experimental and Clinical Pharmacology, University Hospital Eppendorf, 20246 Hamburg, Germany, the ^**Department of Pediatrics, University of Cincinnati, Children's Hospital Medical Center, Cincinnati, Ohio 45229, and Yale University School of Medicine, New Haven, Connecticut 06519

Inhibitor-1 becomes a potent inhibitor of protein phosphatase 1 when phosphorylated by cAMP-dependent protein kinase at Thr³⁵. Moreover, Ser⁶⁷ of inhibitor-1 serves as a substrate for cyclin-dependent kinase 5 in the brain. Here, we report that dephosphoinhibitor-1 but not phospho-Ser⁶⁷ inhibitor-1 was efficiently phosphorylated by protein kinase C at Ser⁶⁵ in vitro. In contrast, Ser⁶⁷ phosphorylation by cyclin-dependent kinase 5 was unaffected by phospho-Ser⁶⁵. Protein kinase C activation in striatal tissue resulted in the concomitant phosphorylation of inhibitor-1 at Ser⁶⁵ and Ser⁶⁷, but not Ser⁶⁵ alone. Selective pharmacological inhibition of protein phosphatase activity suggested that phospho-Ser⁶⁵ inhibitor-1 is dephosphorylated by protein phosphatase 1 in the striatum. In vitro studies confirmed these findings and suggested that phospho-Ser⁶⁷ protects phospho-Ser⁶⁵ inhibitor-1 from dephosphorylation by protein phosphatase 1 in vivo. Activation of group I metabotropic glutamate receptors resulted in the up-regulation of diphospho-Ser⁶⁵/Ser⁶⁷ inhibitor-1 in this tissue. In contrast, the activation of N-methyl-D-aspartate-type ionotropic glutamate receptors opposed increases in striatal diphospho-Ser⁶⁵/Ser⁶⁷ inhibitor-1 levels. Phosphomimetic mutation of Ser⁶⁵ and/or Ser⁶⁷ did not convert inhibitor-1 into a protein phosphatase 1 inhibitor. On the other hand, in vitro and in vivo studies suggested that diphospho-Ser⁶⁵/Ser⁶⁷ inhibitor-1 is a poor substrate for cAMP-dependent protein kinase. These observations extend earlier studies regarding the function of phospho-Ser⁶⁷ and underscore the possibility that phosphorylation in this region of inhibitor-1 by multiple protein kinases may serve as an integrative signaling mechanism that governs the responsiveness of inhibitor-1 to cAMP-dependent protein kinase activation.

Received for publication, April 6, 2006 , and in revised form, May 30, 2006.

^* This work was supported by National Institute on Drug Abuse Grants DA16672 (to J. A. B.) and DA10044 (to A. C. N.), National Institute of Mental Health Grant MH67777 (to J. A. B.), NHLBI, National Institutes of Health, Grant SCCoR P50 HL077101 (to J. D. M. and J. A. B.), a grant from the National Alliance for Research on Schizophrenia and Depression (to J. A. B.), a grant from the Ella McFadden Charitable Trust Fund at the Southwestern Medical Foundation (to J. A. B.), and United States Army Medical Research and Materiel Command Neurotoxin Exposure Treatment Research Program Grant W81XWH-04-2-0009 (to Intracellular Therapies, Inc.). 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 Figs. S1 and S2.

¹ To whom correspondence should be addressed: Dept. of Psychiatry, The University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9070. Tel.: 214-648-4168; Fax: 214-648-1293; E-mail: james.bibb{at}utsouthwestern.edu.

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