The regulation and role of PKD in the heart

描述（由申请人提供）： 蛋白激酶D（PKD）是心脏肥大信号传导中的关键参与者。它是一种主要的组蛋白去乙酰化酶（HDAC）激酶，沿着CaMK，似乎在心力衰竭中观察到的基因表达改变和心脏重塑中具有关键作用。然而，对PKD功能及其在成年心肌细胞中的调节知之甚少。在最近的工作与FRET为基础的PKD活性报告（DKAR）和荧光标记的PKD和HDAC 5，我们发现了独特的分歧信号通路的2 Gq偶联受体激动剂，苯肾上腺素（PE）和内皮素-1（ET）。虽然全球PKD活动是相似的，ET引起快速持续的PKD招聘质膜和只有适度的核输入，而PE触发短暂的肌膜定位和更显着的核输入（和活动）的PKD。PKD对PE的反应中更突出的核作用与PKD在PE诱导的HDAC 5核输出（与ET相比）中更关键的作用一致。这些研究强调了PKD在心脏中的背景依赖性激活和作用，并说明了需要更好地了解PKD活性的不同水平的结构和空间调节。因此，aim 1的重点是通过协调使用磷酸化，氧化和调节模块（PKD中的所有结构决定因素）来急性控制PKD。我们已经对PKD在神经体液刺激PE和ET下的时空定位和活性进行了详细的分析，因此我们将使用这些对比的途径来评估成人心肌细胞中PKD激活的结构要求。在目标2中，我们将讨论PKD的空间（和时间）分离在实现信号特异性中的作用。我们将测量在确定的细胞内区域（例如细胞核、线粒体）PKD活性的幅度和持续时间。我们还将确定PKD 1（主要的心脏亚型）是否在这些隔室中起关键作用。拟议的工作应该提供很大的洞察力，何时，何地和PKD在做什么的心。大多数实验将在分离的成年心室肌细胞中进行，使用我们的创新荧光方法（FRET，TIRF，FRAP测量），辅以分子和生物化学方法。这些实验交织了PKD激活的基本机制研究和PKD在成人心肌细胞中的调节和作用的具体确定（并增加了我们对两者的理解）。此外，我们将更深入地了解PKD作为心功能障碍治疗靶点的潜力。

DESCRIPTION (provided by applicant): 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.