β -Adrenergic Signaling Inhibits G q -Dependent PKD Activation by Preventing PKD Translocation

Rationale: Both β -adrenergic ( β -AR) and G q -coupled agonist (G q R) 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 upon co-activation with G q R agonists. This will test our hypothesis that compartmentalized PKD signaling reconciles disparate findings of protein kinase A (PKA) facilitation and inhibition of PKD activation. Methods and Results: We report on the spatial and temporal profiles of PKD activation using GFP-tagged PKD (wildtype or mutant S427E) and targeted FRET based biosensors (DKARs) in adult cardiomyocytes. We find that  -AR/PKA signaling drives local nuclear activation of PKD, without preceding sarcolemmal translocation. We also discover pronounced interference of  -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. Lastly, we show altered S427 phosphorylation in TAC-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. adult cardiac myocytes. We find that  AR/PKA signaling drives nuclear activation of PKD without requiring a sarcolemmal recruitment phase.  -AR/PKA signaling also prevents G q R-induced translocation, activation and function of PKD and we show that these effects are mediated by PKA-dependent phosphorylation of PKD at S427. Moreover, initial observations in a TAC-induced hypertrophy model indicate altered phosphorylation of this S427 site. Our study provides further evidence of compartmentalized PKD signaling and indicates how S427 could serve as a critical control point for modulation of the spatiotemporal dynamics of PKD activity and function during the pathogenesis of cardiac disease. SS426/427AA, indicating this novel antibody specifically detects phosphorylation at S427. This antibody signal also increased in lysates from ISO- or Forsk-treated myocytes, but not in the presence of PKA inhibitors H89 or 8-Br-Rp-cAMP (Fig6D). Subcellular fractionation experiments found that in response to ISO PKD pS427 signals were increased throughout the cell, but particularly in the sarcolemmal compartment (Supplemental Fig VIII). These findings confirm ISO triggers PKA-dependent phosphorylation of S427. We then probed S427 phosphorylation in cardiac homogenates from 3 week TAC mice vs. their SHAM controls. Echocardiography confirmed banded mice had significant alterations in structural and functional cardiac parameters (Supplemental Fig IX). Fig6E shows that S427 phosphorylation, PKD1 expression and S427-phosphorylated PKD1 were all increased in TAC vs. SHAM. as an important regulatory site on PKD1, controlling the spatiotemporal dynamics of PKD activity and downstream functional effects on cardiac hypertrophy and gene expression. Our finding that PKA signaling renders PKD resistant to G q stimuli provides fundamental insight into how β -AR desensitization in heart failure could contribute to PKD dysregulation and further cardiac dysfunction.