The regulation and role of PKD in the heart

PKD在心脏中的调节及作用

• 批准号: 8458117
• 负责人: Julie B Bossuyt
• 金额: $ 36.17万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-15 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8458117
• 关键词: Ablation; Acute; Address; Adult; Affect; Agonist; Arrhythmia; Binding; Biochemical; Biochemistry; Calcium/calmodulin-dependent protein kinase; Cardiac; Cardiac Myocytes; Cell Nucleus; Cell membrane; Cells; Complement; DAG/PE-Binding Domain; Employee Strikes; Endothelin-1; Fluorescence; Fluorescence Resonance Energy Transfer; Functional disorder; Gene Expression; Genetic Transcription; Golgi Apparatus; HDAC5 gene; Heart; Heart Diseases; Heart failure; Histone Deacetylase; Hydrogen Peroxide; Knockout Mice; Location; Longitudinal Studies; Measurement; Measures; Membrane; Methionine; Methods; Mitochondria; Molecular; Muscle Cells; Nuclear; Nuclear Export; Nuclear Import; Oxidative Stress; PH Domain; PKC Phosphorylation Site; Pathway interactions; Phenylephrine; Phosphorylation; Phosphotransferases; Positioning Attribute; Process; Protein Isoforms; Regulation; Relative (related person); Reporter; Risk; Role; Signal Pathway; Signal Transduction; Specificity; Stimulus; Stress; Testing; Total Internal Reflection Fluorescent; Transducers; Ventricular; Work; base; calmodulin-dependent protein kinase II; innovation; insight; member; mutant; novel therapeutics; oxidation; protein kinase D; public health relevance; receptor coupling; research study; response; scaffold; segregation; spatiotemporal; therapeutic target; trafficking

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.

描述（由申请人提供）： 项目摘要蛋白激酶D（PKD）正在成为心脏肥厚信号的关键参与者。它是主要的组蛋白脱乙酰基酶（HDAC）激酶之一，与CAMK一起，似乎在基因表达改变和心力衰竭中看到的心脏衰竭中具有关键作用。尽管如此，关于PKD功能及其在成人心脏肌细胞中的调节知之甚少。在最新的基于FRET的PKD活性报告基因（DKAR）和荧光标记的PKD和HDAC5的工作中，我们发现了2个GQ耦合受体激动剂，苯肾上腺素（PE）和Endophelin-1（ET）（ET）的2 GQ耦合受体激动剂（ET）。尽管两者的全局PKD活性都相似，但ET引起了质膜的快速持续募集，而PE又触发了PE的瞬时肌膜定位和PKD的急剧核进口（和活动）。 PKD响应PE的更为突出的核作用与PKD在PE诱导的HDAC5核出口（vs.ET）中的更关键作用一致。这些研究强调了PKD在心脏中的上下文依赖性激活和作用，并说明了需要更好地了解PKD活性的结构和空间调节的不同水平。因此，AIM1通过协调的磷酸化，氧化和调节模块（PKD中的所有结构决定因素）的协调使用，重点介绍了PKD的急性控制。我们已经对PKD的时空定位和响应神经性刺激PE和ET的活性进行了详细的分析，因此我们将使用这些对比途径来评估成人心脏心肌细胞中PKD激活的结构要求。在AIM 2中，我们将解决PKD在达到信号特异性方面的空间（和时间）分离的作用。我们将测量定义的细胞内区域（例如核，线粒体）上PKD活性的幅度和持续时间。我们还将确定PKD1（主要的心脏同工型）是否在这些隔室中起关键作用。拟议的工作应提供有关PKD在心脏中何时，何地，何处和在何地做什么的洞察力。大多数实验将使用我们的创新荧光方法（FRET，TIRF，FRAP测量）进行孤立的成年心室肌细胞进行，并通过分子和生物化学方法补充。这些实验既相互交织的PKD激活的基本机理研究，又是PKD在成人心肌细胞中的调节和作用的特定确定（并增加了我们对两者的理解）。此外，我们将更深入地了解PKD作为心脏功能障碍的治疗靶点的潜力。