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: 7987695
• 负责人: Susan F Steinberg
• 金额: $ 40.25万
• 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 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7987695
• 关键词: Agonist; Apoptosis; Arts; Biochemical; CREB1 gene; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cell Fraction; Cells; Cytosol; Development; Docking; Enzymatic Biochemistry; Enzymes; Etiology; Event; Evolution; Funding; Future; GTP-Binding Proteins; Generations; Genetically Engineered Mouse; Goals; Growth Factor; Growth Factor Receptors; HDAC5 gene; Heart failure; Hypertrophy; In Vitro; Ischemia; Lead; Link; Lipids; Membrane; Membrane Lipids; Methods; Modeling; Molecular; Molecular Conformation; Mutagenesis; Oxidative Stress; Pathogenesis; Peptide Library; Peptides; Pharmacologic Substance; Phosphorylation; Phosphotransferases; Post-Translational Protein Processing; Process; Property; Protein Kinase; Protein-Serine-Threonine Kinases; Proteins; RNA Interference; Reaction; Reactive Oxygen Species; Receptor Activation; Recruitment Activity; Reperfusion Injury; Reperfusion Therapy; Role; Signal Transduction; Site; Specificity; Stimulus; Substrate Specificity; Testing; Treatment Efficacy; Troponin I; Tyrosine; Tyrosine Phosphorylation; Tyrosine Phosphorylation Site; adapter protein; adenoviral-mediated; base; cofactor; delta protein; design; improved; in vivo; inhibitor/antagonist; novel; overexpression; p66(ShcA) protein; prevent; protein kinase C-delta; protein kinase D; protein kinase modulator; public health relevance; receptor; response; small molecule; therapeutic target

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). PUBLIC HEALTH RELEVANCE: Protein kinase C-delta (PKC4) is viewed as an important therapeutic target that contributes to adverse cardiac remodeling and reperfusion injury. Studies funded by HL77680 identify significant gaps in our understanding of the molecular mechanisms that regulate PKC4 activation and the consequences of PKC4 activation in cardiomyocytes. This renewal focuses on novel PKC4 activation mechanisms and cellular actions that can provide the basis for future efforts to design small-molecule PKC4 inhibitors with improved specificity and efficacy for the treatment of pathological cardiac remodeling.

描述（由申请人提供）：蛋白激酶C4（PKC 4）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。PKC 4活化通常归因于受体驱动的脂质辅因子依赖性机制，其将活性构象的PKC 4锚于膜。该变构模型假设PKC 4活性是酶的固有/不变性质，其不被活化过程改变。然而，在HL 77680中的研究表明，Src导致的PKC 4自磷酸化和PKC 4酪氨酸磷酸化是翻译后修饰，导致生理相关靶底物的PKC 4磷酸化发生功能性重要变化。我们还表明，酪氨酸磷酸化形式的PKC 4积累在心肌细胞的胞质部分进行氧化应激作为脂质非依赖性酶，准备磷酸化底物在整个细胞，而不仅仅是在脂质膜。其他研究表明，PKC 4激活导致一系列效应蛋白的磷酸化，对细胞重塑具有不同的（和潜在的相反）影响。在这个更新的研究将集中在PKC 4的自磷酸化和酪氨酸磷酸化机制，决定PKC 4的细胞作用，并可以有针对性的治疗，以防止不良的心脏重塑。具体目的我将使用生物化学方法来确定的网站和后果的PKC 4自磷酸化和酪氨酸磷酸化的Src。我们的目标是确定翻译后修饰，调节PKC 4的催化活性，特别是其底物特异性。具体目标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不同分子形式的PKC 4是否调节不同的效应器应答。特定目标III的研究将集中在PKC 4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，以防止调节磷酸化酶所需的对接相互作用。具体目标IV将检查基因工程小鼠体内PKC 4依赖性心脏重塑的分子要求。长期目标是[1]区分响应于生长因子受体活化而蓄积的变构活化形式的PKC 4与在氧化应激期间蓄积的酪氨酸磷酸化形式的PKC 4的心脏作用[2]，并检验只有某些分子形式的PKC 4招募促进不良心脏重塑和缺血/再灌注损伤的效应子的假设。这些信息将为开发新药物提供基础，这些药物旨在通过选择性抑制PKC 4的特定分子形式（或酶的翻译后修饰）的细胞作用来预防不良心脏重塑。 公共卫生相关性：蛋白激酶C-δ（PKC 4）被认为是一个重要的治疗靶点，有助于不利的心脏重塑和再灌注损伤。由HL 77680资助的研究发现，我们对心肌细胞中调节PKC 4活化的分子机制和PKC 4活化的后果的理解存在重大差距。此次更新的重点是新型PKC 4激活机制和细胞作用，这些机制和细胞作用可以为未来设计具有更高特异性和疗效的小分子PKC 4抑制剂以治疗病理性心脏重塑提供基础。