The regulation and role of PKD in the heart

描述(由申请人提供)： 项目摘要蛋白激酶D(PKD)在心肌肥大信号转导中发挥着重要作用。它是组蛋白脱乙酰酶(HDAC)的主要激酶之一，与CaMK一起，在心力衰竭的基因表达改变和心脏重构中发挥关键作用。然而，对PKD在成人心肌细胞中的功能及其调控知之甚少。在最近对基于FRET的PKD活性报告(DKAR)和荧光标记的PKD和HDAC5的研究中，我们发现了两种GQ偶联受体激动剂苯肾上腺素(PE)和内皮素-1(ET)独特的不同信号通路。尽管两者的PKD活性相似，但ET导致PKD快速持续地向质膜募集，仅有少量的核输入，而PE则触发了短暂的肌膜定位和更剧烈的核输入(和活动)。在PE诱导的HDAC5核输出(vs.ET)中，PKD的核作用更为显著，这与PKD在PE诱导的HDAC5核输出(vs.ET)中的重要作用是一致的。这些研究强调了PKD在心脏中的上下文依赖的激活和作用，并说明了更好地理解PKD活动的不同结构和空间调节水平的必要性。因此，AIM 1专注于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。我们已经详细分析了神经体液刺激PE和ET对PKD的时空定位和活性的影响，因此我们将使用这些不同的途径来评估成年心肌细胞对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方面的作用。我们将测量特定细胞内区域(如细胞核、线粒体)的PKD活性的大小和持续时间。我们还将确定PKD1(主要的心脏亚型)是否在这些间隔中起关键作用。拟议的工作应该提供对PKD在何时、何地和什么情况下在心脏中发挥作用的巨大洞察力。大多数实验将在分离的成人心室肌细胞中进行，使用我们创新的荧光方法(FRET、TIRF、FRAP测量)，辅之以分子和生物化学方法。这些实验交织了PKD激活的基础机制研究和PKD在成年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，我们还将更深入地了解PKD作为心脏功能障碍治疗靶点的潜力。 公共卫生相关性： 应对压力的项目叙述心脏经历了一个重塑过程，最终导致心力衰竭和心律失常的风险增加。蛋白激酶D被认为是这一过程中的关键信号转导，但对其在心脏中的功能和调节知之甚少。该项目旨在深入了解调节心肌细胞中蛋白激酶D的分子机制，并进一步确定PKD在心脏中的功能。从长远来看，这些研究可能为心力衰竭等心脏病的治疗提供新的治疗策略的线索。

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. 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.