Protein kinase C-delta actions in cardiomyocytes

蛋白激酶 C-delta 在心肌细胞中的作用

• 批准号: 7454375
• 负责人: Susan F Steinberg
• 金额: $ 34.88万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-15 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7454375
• 关键词: 1,2-diacylglycerol; Accounting; Agonist; Apoptosis; Binding; Cardiac; Cardiac Myocytes; Caspase; Caveolae; Cell Nucleus; Cell membrane; Cells; Complex; Diglycerides; Docking; Enzymes; Etiology; Evaluation; Evolution; Failure; Family; Gene Transfer; Generations; Goals; Growth; Growth Factor; Heart Hypertrophy; Heart failure; Hydrolysis; Hypoxia; Individual; Infarction; Ischemia; Ischemic Preconditioning; Link; Lipids; Localized; Maps; Mediating; Membrane; Membrane Lipids; Mitochondria; Modeling; Molecular Conformation; Norepinephrine; Nuclear; Oxidants; Oxidative Stress; Pathogenesis; Phorbol Esters; Phosphatidylinositols; Phosphorylation; Phosphotransferases; Physiological; Play; Protein Isoforms; Protein Kinase; Protein-Serine-Threonine Kinases; Proteins; Regulation; Role; Sarcomeres; Signal Transduction; Site; Specificity; Stimulus; Stress; Testing; Tetradecanoylphorbol Acetate; Troponin; adenoviral-mediated; cofactor; design; member; novel; protein kinase C-delta; protein protein interaction; receptor; response; scaffold; therapeutic target; tumor

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.

描述（由申请人提供）：蛋白激酶C8（PKC 8）在心脏收缩、缺血预处理和心脏肥大/衰竭的调节中起重要作用。PKC激活的传统模型集中于脂质辅因子（二酰基甘油，PMA）的作用，其以活性构象将PKC锚于膜。然而，心肌细胞共表达几种PKC亚型，引起不同的（偶尔是相反的）细胞活动。信号传导特异性归因于单个PKC亚型与其独特的膜相关锚定蛋白（RACK）的相互作用; RACK将单个PKC靶向不同的膜亚结构域，靠近其独特的底物。这种机制很容易解释PKC亚型在膜中的特异性作用。它不能解释PKC对其他位点（如肌钙蛋白复合物）磷酸化蛋白的众所周知的作用。本申请的初步研究确定了一种新的模式PKC 8激活心肌细胞受到氧化应激。我们表明，H202促进PKC 8酪氨酸磷酸化，并诱导PKC 8从膜释放。在H2 O2处理的心肌细胞的可溶性级分中作为脂质非依赖性酶回收的Tyr-磷酸化的PKC 8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，PKC 8的行动不同，这取决于激活模式。该提议将测试以下假设：[1]脂质辅因子激活的膜锚定的PKC 8和Tyr-磷酸化的PKCd（在用H2 O2处理的心肌细胞的膜和胞质部分中）发挥不同的心脏作用，[2] PKC 8在缺血/梗死背景下的有害作用（至少部分）由Tyr-磷酸化的PKC 8介导。具体的目的是[I]，以确定不同的网站PKC？酪氨酸磷酸化以及PKCd信号伴侣和底物在去甲肾上腺素、PMA、缺氧或H2 O2处理的心肌细胞中的差异，[II]以鉴定PKC 8亚细胞定位的差异在用NE、PMA、缺氧或H2 O2处理的心肌细胞中，和[III]，以检验PKC 8通过激酶依赖性和非依赖性机制调节生长/凋亡途径的假设。该项目的统一目标是确定PKC 8激活模式的刺激特异性差异（以及Tyr-磷酸化PKC 8作为信号调节支架的激酶独立功能）。氧化应激在心力衰竭的发生发展中起着重要作用。在心肌细胞中受到氧化应激与生长因子信号传导的不同PKCd作用对设计和评价PKC 8靶向治疗具有重要意义。