Project Summary Protein kinase D (PKD) is emerging as a key player in cardiac hypertrophic signaling. It is one of the major histone deacetylase (HDAC) kinases, along with CaMK, and appears to have a pivotal role in the altered gene expression and cardiac remodeling seen in heart failure. Nonetheless, little is known about PKD function and its regulation in adult cardiac myocytes. In recent work with a FRET-based PKD activity reporter (DKAR) and fluorescently tagged PKD and HDAC5, we uncovered uniquely divergent signaling pathways for 2 Gq-coupled receptor agonists, phenylephrine (PE) and endothelin-1 (ET). Although global PKD activity was similar for both, ET caused rapid sustained PKD recruitment to the plasma membrane and only modest nuclear import, while PE triggered transient sarcolemmal localization and more dramatic nuclear import (and activity) of PKD. The more prominent nuclear action of PKD in response to PE was consistent with a more critical role of PKD in PE-induced HDAC5 nuclear export (vs. ET). These studies highlight the context-dependent activation and role of PKD in the heart and illustrate the need to better understand the different levels of structural and spatial regulation of PKD activity. So aim1 focuses on acute control of PKD via the coordinated use of phosphorylation, oxidation and regulatory modules (all structural determinants within PKD). We already have a detailed analysis of the spatiotemporal localization and activity of PKD in response to the neurohumoral stimuli PE and ET, so we will use these contrasting pathways to assess the structural requirements for PKD activation in adult cardiac myocytes. In aim 2, we will address the role of spatial (and temporal) segregation of PKD in achieving signal specificity. We will measure the magnitude and duration of PKD activity at defined intracellular regions (e.g. nucleus, mitochondria). We will also determine whether PKD1 (the predominant cardiac isoform) has a critical role in these compartments. The proposed work should provide great insight into when, where and what PKD is doing in the heart. Most experiments will be done in isolated, adult ventricular myocytes, using our innovative fluorescence methods (FRET, TIRF, FRAP measurements) complemented by molecular and biochemistry approaches. These experiments interweave both fundamental mechanistic studies of PKD activation and specific determination of the regulation and role of PKD in adult cardiomyocytes (and add to our understanding of both). Moreover we will gain greater insight into the potential of PKD as a therapeutic target for cardiac dysfunction. PUBLIC HEALTH RELEVANCE: Project Narrative In response to stress the heart undergoes a remodeling process that ultimately leads to increased risk of heart failure and arrhythmias. Protein kinase D has been identified as a key signal transducer in this process but its function and regulation in the heart are poorly understood. This project is aimed at gaining insight in the molecular mechanisms that regulate Protein kinase D in cardiac cells and further defining the functions of PKD in the heart. In the long term these studies could provide clues to novel therapeutic strategies in the treatment of cardiac disease such as heart failure.