Protein kinase C-delta actions in cardiomyocytes

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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.