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J. Biol. Chem., Vol. 282, Issue 42, 30691-30698, October 19, 2007

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Protein Kinase C

A NOVEL REGULATOR OF BOTH PHOSPHORYLATION AND DE-PHOSPHORYLATION OF CARDIAC SARCOMERIC PROTEINS^*

From the Center for Cardiovascular Research, Department of Physiology and Biophysics, University of Illinois at Chicago College of Medicine, Chicago, Illinois 60612

Our experiments investigated associations of specific isoforms of protein kinase C (PKC) with individual proteins in the cardiac troponin complex. Troponin I (cTnI) associated with PKC and and troponin T (cTnT) associated with PKC , , and . Based on its association with cTnI, we hypothesized that PKC is a major regulator of myofilament protein phosphorylation. To test this, we infected adult cardiac myocytes with adenoviral constructs containing DsRed monomer-tagged wild type (WT) and the following constitutively active forms of PKC: the pseudo-substrate region (A119E), 3'-phospho-inositide-dependent kinase-1 (T410E), and auto-phosphorylation (T560E). The A119E and T410E mutants displayed increased localization to the Z-discs compared with WT and T560E. Immunoprecipitations were performed in myocytes expressing PKC using PKC phospho-motif antibodies to determine the phosphophorylation of cTnI, cTnT, tropomyosin, myosin-binding protein C, and desmin. We did not find serine (Ser) phosphorylation of cTnI or cTnT. However, we observed a significant decrease in threonine (Thr) phosphorylation of cTnI and cTnT notably by PKC T560E. Ser phosphorylation of tropomyosin was increased by all three active mutants of PKC. Ser/Thr phosphorylation of myosin-binding protein C increased primarily by PKC A119E. Both PKC A119E and T410E mutants increased desmin Ser/Thr phosphorylation. To explain the apparent Thr dephosphorylation of cTnI and cTnT, we hypothesized that PKC exists as a complex with p21-activated kinase-1 (Pak1) and protein phosphatase 2A (PP2A), and this was confirmed by immunoprecipitation Western blot. Our data demonstrate that PKC is a novel regulator of myofilament protein phosphorylation.

Received for publication, May 3, 2007 , and in revised form, August 16, 2007.

^* This work was supported by an American Heart Association pre-doctoral fellowship and National Institutes of Health Training Grants T32 HL07692-17 (to S. C. W.) and R01 HL64035 and PO1 HL62426 (to R. J. S.). 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 Fig. S1.

¹ To whom correspondence should be addressed: Dept. of Physiology and Biophysics (M/C 901), University of Illinois at Chicago College of Medicine, 835 South Wolcott Ave., Chicago, IL 60612-7342, Tel.: 312-996-7620; Fax: 312-996-1414; E-mail: solarorj{at}uic.edu.

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