Protein kinase C-delta actions in cardiomyocytes

描述（由申请人提供）：蛋白激酶C8 （PKC8）在心脏收缩、缺血预处理和心脏肥厚/衰竭的调节中起重要作用。PKC活化的传统模型侧重于脂质辅助因子（二酰基甘油，PMA）的作用，它以活性构象将PKC锚定在膜上。然而，心肌细胞共表达几种PKC亚型，引起不同的（偶尔相反的）细胞作用。信号特异性归因于单个PKC异构体与其独特的膜相关锚定蛋白（rack）的相互作用；rack将单个PKCs靶向到不同的膜亚结构域，靠近它们独特的底物。这一机制很容易解释PKC异构体在膜中的特异性作用。它不能解释PKC对其他位点（如肌钙蛋白复合体）磷酸化蛋白的众所周知的作用。该应用的初步研究确定了氧化应激下心肌细胞中PKC8激活的新模式。我们发现H202促进PKC8 tyrr磷酸化并诱导PKC8从膜中释放。酪氨酸磷酸化的PKC8，作为一种脂质无关的酶，在h2处理的心肌细胞的可溶部分中被恢复，准备在整个细胞中磷酸化靶蛋白（不仅仅是在膜上）。这些结果表明PKC8的作用是不同的，取决于激活的模式。本研究将验证以下假设：[1]脂质辅助因子激活的、膜锚定的PKC8和酪氨酸磷酸化的PKCd（在H202处理的心肌细胞的膜和细胞质部分中）发挥不同的心脏作用；[2]PKC8在缺血/梗死背景下的有害作用是由（至少部分）酪氨酸磷酸化的PKC8介导的。具体目的是[1]确定PKC的不同位点？在去甲肾上腺素、PMA、缺氧或H202处理的心肌细胞中，酪氨酸磷酸化以及PKCd信号伙伴和底物的差异，[II]以确定NE、PMA、缺氧或H202处理的心肌细胞中PKC8亚细胞定位（在质膜、小泡、线粒体和/或细胞核中）的差异，[III]以检验PKC8通过激酶依赖和激酶独立机制调节生长/凋亡途径的假设。该项目的统一目标是确定PKC8激活模式的刺激特异性差异（以及酪氨酸磷酸化PKC8作为信号调节支架的激酶无关功能）。氧化应激在心力衰竭的发展过程中起着重要作用。在氧化应激与生长因子信号传导的心肌细胞中，PKCd的独特作用对pkc8靶向治疗的设计和评估具有重要意义。

DESCRIPTION (provided by applicant): Protein kinase C8 (PKC8) plays an important role in the regulation of cardiac contraction, ischemic preconditioning, and cardiac hypertrophy/failure. The traditional model of PKC activation focuses on the role of lipid cofactors (diacylglycerol, PMA) that anchor PKC, in an active conformation, to membranes. However, cardiomyocytes co-express several PKC isoforms that elicit distinct (and occasionally opposing) cellular actions. Signaling specificity has been attributed to individual PKC isoform interactions with their unique membrane-associated anchoring proteins (RACKs); RACKs target individual PKCs to distinct membrane subdomains, in close proximity to their unique substrates. This mechanism readily accounts for PKC isoform specific actions in membranes. It does not explain the well-known effects of PKC to phosphorylation proteins at other sites (such as in the troponin complex). Preliminary studies in this application identify a novel mode for PKC8 activation in cardiomyocytes subjected to oxidative stress. We show that H202 promotes PKC8 Tyr phosphorylation and induces PKC8 release from membranes. The Tyr-phosphorylated PKC8, recovered as a lipid-independent enzyme in the soluble fraction of H202-treated cardiomyocytes, is poised to phosphorylate target proteins throughout the cell (not just on membranes). These results suggest that PKC8 actions differ, depending upon the mode of activation. This proposal will test the hypotheses that [1] lipid cofactor-activated, membrane-anchored PKC8 and Tyr-phosphorylated PKCd (in membrane and cytosolic fractions of cardiomyocytes treated with H202) exert distinct cardiac actions and [2] the deleterious effects of PKC8 in the context of ischemia/infaction are mediated (at least in part) by Tyr-phosphorylated PKC8. The specific aims are [I] to identify the distinct sites for PKC? Tyr-phosphorylation as well as differences in PKCd signaling partners and substrates in cardiomyocytes treated with norepinephrine, PMA, hypoxia, or H202, [II] to identify differences in the subcellular localization of PKC8 (in plasma membrane, caveolae, mitochondria, and/or nuclei) in cardiomyocytes treated with NE,PMA, hypoxia, or H202, and [III] to test the hypothesis that PKC8 regulates growth/apoptosis pathways via both kinase-dependent and -independent mechanisms. The unifying goal of this project is to identify stimulus-specific differences in the mode of PKC8 activation (as well as kinase-independent functions for Tyr-phosphorylated PKC8 as a signal-regulated scaffold). Oxidant stress plays an important role in the evolution of cardiac failure. The distinct PKCd actions in cardiomyocytes subjected to oxidative stress vs. growth factor signaling have important implications for the design and evaluation of PKC8-targeted therapeutics.