Protein kinase C-delta actions in cardiomyocytes

描述（由申请人提供）：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激活通常归因于受体驱动的脂质辅助因子依赖机制，该机制将PKC4固定在其活性构象中。该变构模型假设PKC4活性是酶的固有/不可变特性，不受激活过程的改变。然而，对HL77680的研究表明，Src介导的PKC4自磷酸化和PKC4酪氨酸磷酸化是翻译后修饰，导致PKC4磷酸化生理相关靶底物在功能上发生重要变化。我们还发现，酪氨酸磷酸化形式的PKC4在氧化应激下的心肌细胞的细胞质部分中积累，作为一种脂质无关的酶，它可以在整个细胞中磷酸化底物，而不仅仅是在脂质膜上。其他研究表明，PKC4激活导致一系列效应蛋白的磷酸化，这些效应蛋白对细胞重塑具有不同（可能相反）的影响。这一更新的研究将集中在PKC4的自磷酸化和酪氨酸磷酸化机制上，这些机制决定PKC4的细胞作用，可以靶向治疗以防止不良的心脏重构。我将使用生化方法来确定Src对PKC4自磷酸化和酪氨酸磷酸化的位点和后果。目的是确定调节PKC4催化活性的翻译后修饰，特别是其底物特异性。Specific aim II将使用RNAi沉默和腺病毒介导的过表达策略来确定以刺激特异性方式积累的PKC4的不同分子形式是否调节不同的效应反应。Specific Aim III的研究将重点关注PKC4易位的肽调节剂的心脏保护作用的机制。重点是这些化合物是否发挥“脱靶”作用，以防止对酶进行调节磷酸化所需的对接相互作用。特异性目的IV将研究基因工程小鼠体内PKC4依赖性心脏重构的分子需求。长期目标是[1]区分变构激活形式的PKC4在响应生长因子受体激活时积累的心脏作用与酪氨酸磷酸化形式的PKC4在氧化应激期间积累的心脏作用，[2]是为了验证PKC4只有某些分子形式招募效应物促进不利的心脏重构和缺血/再灌注损伤的假设。这一信息将为开发新型药物提供基础，这些药物旨在通过选择性抑制PKC4特定分子形式的细胞作用（或酶的翻译后修饰）来预防不良的心脏重构。

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).