β-Adrenergic Signaling Inhibits G q -Dependent Protein Kinase D Activation by Preventing Protein Kinase D Translocation

Abstract

Rationale: Both β-adrenergic receptor (β-AR) and G q -coupled receptor (G q R) agonist–driven signaling play key roles in the events, leading up to and during cardiac dysfunction. How these stimuli interact at the level of protein kinase D (PKD), a nodal point in cardiac hypertrophic signaling, remains unclear. Objective: To assess the spatiotemporal dynamics of PKD activation in response to β-AR signaling alone and on coactivation with G q R-agonists. This will test our hypothesis that compartmentalized PKD signaling reconciles disparate findings of PKA facilitation and inhibition of PKD activation. Methods and Results: We report on the spatial and temporal profiles of PKD activation using green fluorescent protein-tagged PKD (wildtype or mutant S427E) and targeted fluorescence resonance energy transfer–based biosensors (D-kinase activity reporters) in adult cardiomyocytes. We find that β-AR/PKA signaling drives local nuclear activation of PKD, without preceding sarcolemmal translocation. We also discover pronounced interference of β-AR/cAMP/PKA signaling on G q R-induced translocation and activation of PKD throughout the cardiomyocyte. We attribute these effects to direct, PKA-dependent phosphorylation of PKD-S427. We also show that phosphomimetic substitution of S427 likewise impedes G q R-induced PKD translocation and activation. In neonatal myocytes, S427E inhibits G q R-evoked cell growth and expression of hypertrophic markers. Finally, we show altered S427 phosphorylation in transverse aortic constriction–induced hypertrophy. Conclusions: β-AR signaling triggers local nuclear signaling and inhibits G q R-mediated PKD activation by preventing its intracellular translocation. PKA-dependent phosphorylation of PKD-S427 fine-tunes the PKD responsiveness to G q R-agonists, serving as a key integration point for β-adrenergic and G q -coupled stimuli.