Protein kinase C-delta actions in cardiomyocytes

描述(申请人提供)：蛋白激酶C4(PKC4)是一种丝氨酸/苏氨酸激酶，与心脏重塑和再灌注损伤的发病机制有关。PKC4的激活通常归因于受体驱动的、脂类辅助因子依赖的机制，这些机制将PKC4的活性构象锚定在膜上。这个变构模型假设PKC4活性是酶的一种固有的/不可变的性质，不会被激活过程改变。然而，在HL77680中的研究表明，PKC4自动磷酸化和PKC4酪氨酸磷酸化是翻译后修饰，导致生理相关靶底物的PKC4磷酸化发生功能上的重要变化。我们还表明，酪氨酸磷酸化形式的PKC4积累在氧化应激下心肌细胞的胞浆部分，作为一种脂质无关的酶，它准备在整个细胞内磷酸化底物，而不仅仅是在脂膜上。其他研究表明，PKC4激活导致一系列效应蛋白的磷酸化，对细胞重塑有不同的(和潜在相反的)影响。此次更新的研究将集中在PKC4自动磷酸化和酪氨酸磷酸化机制上，这些机制决定了PKC4‘S的细胞作用，并可以作为靶向治疗，以防止不利的心脏重构。特定目的I将使用生化方法来鉴定由Src引起的PKC4自磷酸化和酪氨酸磷酸化的位置和后果。我们的目标是确定调控PKC4催化活性的翻译后修饰，特别是其底物特异性。特殊目的II将使用RNAi沉默和腺病毒介导的过度表达策略来确定以刺激特异性方式积累的不同分子形式的PKC4是否调节不同的效应器反应。在特定目标III中的研究将集中在PKC4易位的多肽调节剂的心脏保护作用的潜在机制。研究的重点是这些化合物是否发挥了“非靶标”作用，以防止酶上调节磷酸化所需的对接相互作用。具体目的IV将研究基因工程小鼠体内依赖PKC4的心脏重塑的分子要求。长期目标是[1]区分因生长因子受体激活而积聚的变构激活形式的PKC4和在氧化应激期间积聚的酪氨酸磷酸化形式的PKC4的心脏作用，以及[2]检验只有某些分子形式的PKC4才能招募促进不利心脏重塑和缺血/再灌注损伤的效应物的假设。这些信息将为开发新的药物提供基础，这些药物旨在通过选择性地抑制特定分子形式的PKC4(或对酶的翻译后修饰)的细胞作用来防止不利的心脏重构。

DESCRIPTION (provided by applicant): Protein kinase C4 (PKC4) is a serine/threonine kinase implicated in the pathogenesis of cardiac remodeling and reperfusion injury. PKC4 activation is generally attributed to receptor-driven, lipid cofactor-dependent mechanisms that anchor PKC4 in its active conformation to membranes. This allosteric model assumes that PKC4 activity is an inherent/immutable property of the enzyme that is not altered by the activation process. However, studies in HL77680 implicate PKC4 autophosphorylation and PKC4 tyrosine phosphorylation by Src as post-translational modifications that lead to functionally important changes in PKC4 phosphorylation of physiologically relevant target substrates. We also show that a tyrosine phosphorylated form of PKC4 accumulates in the cytosolic fraction of cardiomyocytes subjected to oxidative stress as a lipid-independent enzyme that is poised to phosphorylate substrates throughout the cell, not just on lipid membranes. Other studies show that PKC4 activation leads to the phosphorylation of a range of effector proteins with varied (and potentially opposing) effects on cellular remodeling. Studies in this renewal will focus on the PKC4 autophosphorylation and tyrosine phosphorylation mechanisms that dictate PKC4's cellular actions and can be targeted therapeutically to prevent adverse cardiac remodeling. Specific Aim I will use biochemical methods to identify the sites and consequences of PKC4 autophosphorylation and tyrosine phosphorylation by Src. The goal is to identify post-translational modifications that regulate PKC4 catalytic activity, and particularly its substrate specificity. Specific aim II will use RNAi silencing and adenoviral-mediated overexpression strategies to determine whether distinct molecular forms of PKC4, that accumulate in a stimulus-specific manner, regulate different effector responses. Studies in Specific Aim III will focus on the mechanism underlying the cardioprotective actions of peptide modulators of PKC4 translocation. The focus is on whether these compounds exert 'off-target' actions to prevent docking interactions required for regulatory phosphorylations on the enzyme. Specific aim IV will examine the molecular requirements for PKC4- dependent cardiac remodeling in vivo in genetically-engineered mice. The long-term goals are [1] to distinguish the cardiac actions of the allosterically-activated form of PKC4 that accumulates in response to growth factor receptor activation versus the tyrosine phosphorylated form of PKC4 that accumulates during oxidative stress and [2] to test the hypothesis that only certain molecular forms of PKC4 recruit effectors that promote adverse cardiac remodeling and ischemia/reperfusion injury. This information would provide the basis for the development of novel pharmaceuticals designed to prevent adverse cardiac remodeling by selectively inhibiting the cellular actions of specific molecular forms of PKC4 (or post-translational modifications on the enzyme).