PKD Isoforms in Heart

描述（由申请人提供）：蛋白激酶D（PKD）最近作为具有重要心脏作用的酶家族出现。大多数研究都集中在PKD 1（该酶家族的创始成员），它磷酸化HDAC 5，一种II类组蛋白脱乙酰酶，调节染色质重塑和肥大。PKD 1也磷酸化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得到充分考虑。类似地，许多PKD 1底物也被PKD 2或PKD 3磷酸化，但PKD 2或PKD 3与PKD 1协同调节心脏重塑的概念也未被考虑。我们的初步研究表明PKD 1、2和3具有相似（但不相同）的体外底物 PKD 1和PKD 2在心肌细胞中以刺激特异性的方式被激活，并且PKD在小鼠胚胎发育和成年心脏肥大中具有重叠和非冗余的作用，这为本申请的目的提供了理论基础。目标1将使用 最先进的肽库筛选以确定PKD同种型底物特异性。这一目标也将使用生化方法和D激酶活性报告分析，以确定激活机制和信号库的个别PKD亚型。我们将使用基因敲除和过表达策略（包括类似物敏感的PKD突变体）来鉴定心肌细胞中PKD亚型特异性底物和效应物。目的#2将使用PKD 1、2和3单次整体敲除小鼠和PKD 1/D2、PKD 1/D3和PKD 2/D3整体和细胞类型特异性双敲除小鼠来分析PKD在心血管发育中的作用。诱导成年心脏特异性PKD基因敲除小鼠和转基因类似物敏感的PKD 1突变小鼠也将被产生，以检查PKD在成年心肌中的作用。这些研究的总体目标是确定PKD的新的调节控制和心脏作用，可以有针对性地预防或减缓心脏肥大和心力衰竭表型的演变。 公共卫生相关性：蛋白激酶D（PKD）最近已成为一个家庭的信号传导酶与重要的心血管活动，但努力考虑PKD作为一个治疗目标已经放缓了显着的差距，我们的理解个别PKD家族成员的分子控制和他们的独特的行动在心脏。这项应用的研究将利用创新的生物化学和分子方法来定义PKD酶的心脏作用。这些研究对于PKD靶向药物治疗病理性心脏重构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.