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: 7255469
• 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 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7255469
• 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（申请人提供）：蛋白激酶C8（PKC8）在调节心脏收缩、缺血预适应和心脏肥大/衰竭中发挥重要作用。 PKC 激活的传统模型侧重于脂质辅助因子（二酰基甘油，PMA）的作用，其将 PKC 以活性构象锚定到膜上。然而，心肌细胞共表达多种 PKC 同种型，这些同种型可引发不同（有时相反）的细胞作用。信号传导特异性归因于各个 PKC 同工型与其独特的膜相关锚定蛋白 (RACK) 的相互作用； RACK 将单个 PKC 靶向不同的膜子域，靠近其独特的底物。这种机制很容易解释 PKC 同种型在膜中的特异性作用。它没有解释 PKC 对其他位点（例如肌钙蛋白复合物）磷酸化蛋白的众所周知的影响。本申请的初步研究确定了一种在遭受氧化应激的心肌细胞中激活 PKC8 的新模式。我们发现H2O2 促进PKC8 Tyr 磷酸化并诱导PKC8 从膜中释放。 Tyr 磷酸化 PKC8 在 H2O2 处理的心肌细胞的可溶性部分中作为脂质独立酶恢复，准备磷酸化整个细胞（而不仅仅是膜上）的靶蛋白。这些结果表明 PKC8 的作用有所不同，具体取决于激活模式。该提案将测试以下假设：[1] 脂质辅因子激活、膜锚定的 PKC8 和 Tyr 磷酸化 PKCd（在用 H2O2 处理的心肌细胞的膜和细胞质部分中）发挥独特的心脏作用，以及 [2] PKC8 对心脏的有害影响。缺血/梗死的情况是由 Tyr 磷酸化 PKC8 介导的（至少部分）。具体目标是 [I] 识别 PKC 的不同位点？在用去甲肾上腺素、PMA、缺氧或 H202 处理的心肌细胞中，Tyr 磷酸化以及 PKCd 信号传导伴侣和底物的差异，[II] 以确定 PKC8 亚细胞定位的差异（在质膜、细胞膜穴、线粒体和/或细胞核）在用 NE、PMA、缺氧或 H2O2 处理的心肌细胞中，[III] 检验 PKC8 调节的假设通过激酶依赖性和非依赖性机制的生长/凋亡途径。该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 Tyr 磷酸化 PKC8 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演变中起着重要作用。受到氧化应激与生长因子信号传导的心肌细胞中不同的 PKCd 作用对于 PKC8 靶向治疗的设计和评估具有重要意义。