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.
| Gong, Jianli; Holewinski, Ronald J; Van Eyk, Jennifer E et al. (2016) A novel phosphorylation site at Ser130 adjacent to the pseudosubstrate domain contributes to the activation of protein kinase C-?. Biochem J 473:311-20 |
| Gong, Jianli; Yao, Yongneng; Zhang, Pingbo et al. (2015) The C2 Domain and Altered ATP-Binding Loop Phosphorylation at Ser³?? Mediate the Redox-Dependent Increase in Protein Kinase C-? Activity. Mol Cell Biol 35:1727-40 |
| Steinberg, Susan F (2013) Oxidative stress and sarcomeric proteins. Circ Res 112:393-405 |
| Steinberg, Susan F (2012) Regulation of protein kinase D1 activity. Mol Pharmacol 81:284-91 |
| Steinberg, Susan F (2012) Cardiac actions of protein kinase C isoforms. Physiology (Bethesda) 27:130-9 |
| Rybin, Vitalyi O; Guo, Jianfen; Harleton, Erin et al. (2012) Regulatory domain determinants that control PKD1 activity. J Biol Chem 287:22609-15 |
| Drosatos, Konstantinos; Bharadwaj, Kalyani G; Lymperopoulos, Anastasios et al. (2011) Cardiomyocyte lipids impair ?-adrenergic receptor function via PKC activation. Am J Physiol Endocrinol Metab 300:E489-99 |
| Sumandea, Marius P; Steinberg, Susan F (2011) Redox signaling and cardiac sarcomeres. J Biol Chem 286:9921-7 |
| Guo, Jianfen; Gertsberg, Zoya; Ozgen, Nazira et al. (2011) Protein kinase D isoforms are activated in an agonist-specific manner in cardiomyocytes. J Biol Chem 286:6500-9 |
| Guo, Jianfen; Cong, Lin; Rybin, Vitalyi O et al. (2010) Protein kinase C-{delta} regulates the subcellular localization of Shc in H2O2-treated cardiomyocytes. Am J Physiol Cell Physiol 299:C770-8 |
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