β-Adrenergic Signaling Inhibits Gq-Dependent PKD Activation by Preventing PKD Translocation
Rationale: Both β-adrenergic (β-AR) and Gq-coupled agonist (GqR) 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 GqR 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 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 GqR-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 GqR-induced PKD translocation and activation. In neonatal myocytes, S427E inhibits GqR-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 GqR-mediated PKD activation by preventing its intracellular translocation. PKA-dependent phosphorylation of PKD S427 fine-tunes the PKD responsiveness to GqR-agonists, serving as a key integration point for β-adrenergic and Gq-coupled stimuli.
- protein kinase D
- beta-adrenergic signaling
- cardiac myocytes
- protein kinase A phosphorylation
- adrenergic receptor
- G proteins
- gene expression/regulation
- Received March 4, 2014.
- Revision received March 13, 2014.
- Accepted March 18, 2014.