Protein kinase C-delta actions in cardiomyocytes

描述(申请人提供)：蛋白激酶C8(PKC8)在心脏收缩、缺血预适应和心肌肥大/衰竭的调节中发挥重要作用。传统的PKC激活模型侧重于将PKC以活性构象锚定在膜上的脂类辅助因子(二酰甘油，PMA)的作用。然而，心肌细胞共表达几种PKC亚型，从而引发不同的(有时是相反的)细胞行为。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(RAKS)的相互作用；RAKS将单个PKC靶向不同的膜亚结构域，靠近其独特的底物。这一机制很容易解释PKC异构体在膜上的特定作用。它不能解释众所周知的PKC对其他部位(如肌钙蛋白复合体)的磷酸化蛋白质的影响。这项应用的初步研究确定了氧化应激下心肌细胞中PKC8的一种新的激活模式。我们发现，H202促进了PKC8酪氨酸的磷酸化，并诱导PKC8从膜上释放。酪氨酸磷酸化的PKC8在H202处理的心肌细胞的可溶性部分中恢复为一种脂类无关的酶，准备在整个细胞内(不仅仅是在膜上)磷酸化目标蛋白。这些结果表明，根据激活模式的不同，PKC8的作用也不同。这项提议将检验以下假设：[1]脂质辅因子激活的膜锚定的PKC8和酪氨酸磷酸化的PKCd(在经H202处理的心肌细胞的膜和胞浆部分中)发挥不同的心脏作用；[2]PKC8在缺血/梗塞背景下的有害作用(至少部分)是由酪氨酸磷酸化的PKC8介导的。具体目标是[i]确定PKC的不同位置？目的：比较去甲肾上腺素、PMA、低氧或H202处理的心肌细胞中PKC8的酪氨酸磷酸化以及PKCd信号伙伴和底物的差异，[ii]以确定去甲肾上腺素、PMA、低氧或H202处理的心肌细胞中PKC8的亚细胞定位(位于质膜、小窝、线粒体和/或细胞核)的差异，以及[iii]以验证PKC8通过依赖和非依赖的机制调控生长/凋亡通路的假设。这个项目的统一目标是确定PKC8激活模式中刺激特异性的差异(以及作为信号调节支架的酪氨酸磷酸化PKC8的不依赖于激酶的功能)。氧化应激在心力衰竭的发生发展中起着重要作用。在氧化应激和生长因子信号作用下的心肌细胞中PKCd的不同作用对于PKC8靶向治疗的设计和评估具有重要的意义。

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.