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J. Biol. Chem., Vol. 283, Issue 35, 24145-24154, August 29, 2008

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Phosphorylation of the cAMP-dependent Protein Kinase (PKA) Regulatory Subunit Modulates PKA-AKAP Interaction, Substrate Phosphorylation, and Calcium Signaling in Cardiac Cells^*

Sabrina Manni, Joseph H. Mauban, Christopher W. Ward, and Meredith Bond¹

From the Departments of Physiology, School of Medicine, and Organizational System and Adult Health, School of Nursing, University of Maryland Baltimore, Baltimore, Maryland 21201

Subcellular compartmentalization of the cAMP-dependent protein kinase (PKA) by protein kinase A-anchoring proteins (AKAPs) facilitates local protein phosphorylation. However, little is known about how PKA targeting to AKAPs is regulated in the intact cell. PKA binds to an amphipathic helical region of AKAPs via an N-terminal domain of the regulatory subunit. In vitro studies showed that autophosphorylation of type II regulatory subunit (RII) can alter its affinity for AKAPs and the catalytic subunit (PKA_cat). We now investigate whether phosphorylation of serine 96 on RII regulates PKA targeting to AKAPs, downstream substrate phosphorylation and calcium cycling in primary cultured cardiomyocytes. We demonstrated that, whereas there is basal phosphorylation of RII subunits, persistent maximal activation of PKA results in a phosphatase-dependent loss of RII phosphorylation. To investigate the functional effects of RII phosphorylation, we constructed adenoviral vectors incorporating mutants which mimic phosphorylated (RIIS96D), nonphosphorylated (RIIS96A) RII, or wild-type (WT) RII and performed adenoviral infection of neonatal rat cardiomyocytes. Coimmunoprecipitation showed that more AKAP15/18 was pulled down by the phosphomimic, RIIS96D, than RIIS96A. Phosphorylation of phospholamban and ryanodine receptor was significantly increased in cells expressing RIIS96D versus RIIS96A. Expression of recombinant RII constructs showed significant effects on cytosolic calcium transients. We propose a model illustrating a central role of RII phosphorylation in the regulation of local PKA activity. We conclude that RII phosphorylation regulates PKA-dependent substrate phosphorylation and may have significant implications for modulation of cardiac function.

Received for publication, March 21, 2008 , and in revised form, June 9, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants AG16613 and HL79134 (to M. B.). 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 Physiology, School of Medicine, University of Maryland Baltimore, 655 W. Baltimore St, Baltimore, MD 21201. Tel.: 410-706-1922; Fax: 410-706-8341; E-mail: mbond{at}som.umaryland.edu.

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