PKD Isoforms in Heart

描述(申请人提供)：蛋白激酶D(PKD)是最近出现的一个具有重要心脏作用的酶家族。大多数研究都集中在PKD1(该酶家族的创始成员)上，它可以磷酸化HDAC5，这是一种调节染色质重塑和肥大的II类组蛋白去乙酰基酶。PKD1还可磷酸化CREB和肌节蛋白(如心肌肌钙蛋白I和心肌肌球蛋白结合蛋白C)，但它们在心脏生长反应中的作用尚未被充分考虑。类似地，许多PKD1底物也被PKD2或PKD3磷酸化，但也没有考虑到PKD2或PKD3与PKD1协同调节心脏重构的概念。我们的初步研究表明，PKD1、2和3在体外具有相似(但不完全相同)的底物 在心肌细胞中，PKD1和PKD2以刺激特异性的方式被激活，并且PKD在小鼠胚胎发育和成人心肌肥大中具有重叠和非多余的作用，这为该应用的目的提供了理论基础。AIM#1将使用 最先进的多肽文库筛选以确定PKD亚型底物的特异性。这一目标还将使用生化方法和D-Kinase活性报告分析来确定单个PKD亚型的激活机制和信号谱系。我们将使用基因敲除和过度表达策略(包括对类似物敏感的PKD突变体)来鉴定心肌细胞中PKD异构体特异的底物和效应物。目的研究PKD在心血管发育中的作用。目的：利用PKD 1、2和3单基因敲除小鼠，以及PKD 1/D2、PKD 1/D3和PKD2/D3双基因敲除小鼠，分析PKD在心血管发育中的作用。还将产生可诱导的成人心脏特异性PKD基因敲除小鼠和转基因类似物敏感的PKD1突变小鼠，以检测PKD在成人心肌中的作用。这些研究的首要目标是确定新的调节控制和PKD的心脏作用，可以有针对性地预防或减缓心肌肥厚和心力衰竭表型的演变。 与公共卫生相关：蛋白激酶D(PKD)最近作为一个具有重要心血管作用的信号酶家族出现，但由于我们对PKD家族单个成员的分子控制及其在心脏中的不同作用的理解存在重大差距，考虑将PKD作为治疗靶点的努力已经放缓。在这一应用中的研究将利用创新的生化和分子方法来确定PKD酶的心脏作用。这些研究将对PKD靶向治疗病理性心脏重构药物的设计和临床应用至关重要。

DESCRIPTION (provided by applicant): Protein kinase D (PKD) has recently emerged as a family of enzymes with important cardiac actions. Most studies have focused on PKD1 (the founding member of this enzyme family) which phosphorylates HDAC5, a class II histone deacetylase that regulates chromatin remodeling and hypertrophy. PKD1 also phosphorylates CREB and sarcomeric proteins (such as cardiac troponin I and cardiac myosin-binding protein C), but their role in cardiac growth responses has not adequately been considered. Similarly, many PKD1 substrates also are phosphorylated by PKD2 or PKD3, but the notion that PKD2 or PKD3 cooperate with PKD1 to regulate cardiac remodeling also has not been considered. Our preliminary studies showing that PKD1, 2, and 3 have similar (but not identical) in vitro substrate specificities, PKD1 and PKD2 are activated in a stimulus-specific manner in cardiomyocytes, and PKDs have both overlapping and non-redundant roles in mouse embryonic development and adult heart hypertrophy provide the rationale for the aims of this application. Aim #1 will use state-of-the- art peptide library screens to define PKD isoform substrate specificity. This aim also will use biochemical approaches and D Kinase Activity Reporter assays to identify activation mechanisms and signaling repertoires of individual PKD isoforms. We will use gene knockout and overexpression strategies (including with analog- sensitive PKD mutants) to identify PKD isoform-specific substrates and effectors in cardiomyocytes. Aim #2 will use PKD1, 2, and 3 single global knockout mice and PKD1/D2, PKD1/D3, and PKD2/D3 global and cell type specific double knockout mice to analyze the role of PKDs in cardiovascular development. Inducible adult cardiac specific PKD knockout mice and a transgenic analogue-sensitive PKD1 mutant mouse also will be generated to examine the role of PKDs in the adult myocardium. The overarching goal of these studies is to identify novel regulatory controls and cardiac actions of PKDs that can be targeted to prevent or slow the evolution of cardiac hypertrophy and heart failure phenotypes. PUBLIC HEALTH RELEVANCE: Protein kinase D (PKD) has recently emerged as a family of signaling enzymes with important cardiovascular actions, but efforts to consider PKD as a therapeutic target have been slowed by significant gaps in our understanding of the molecular controls of individual PKD family members and their distinct actions in the heart. Studies in this application will take advantage of innovative biochemical and molecular approaches to define the cardiac actions of PKD enzymes. These studies will be critical for the design and clinical application of PKD-targeted pharmaceuticals for the treatment of pathological cardiac remodeling.