Phosphorylation of the cAMP-dependent protein kinase (PKA) regulatory subunit modulates PKA-AKAP interaction, substrate phosphorylation, and calcium signaling in cardiac cells.

Subcellular compartmentalization of the cAMP-dependent protein kinase by protein kinase A-anchoring proteins (AKAPs) facilitates local protein phosphorylation. However, little is known about how targeting to AKAPs is regulated in the intact cell. duanyu1529 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 We now investigate whether phosphorylation of serine 96 on RII regulates duanyu1529 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 duanyu1529 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 duanyu1529 activity. We conclude that RII phosphorylation regulates substrate phosphorylation and may have significant implications for modulation of cardiac function.

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