Protein kinase C-delta (PKCd) is a signal-regulated enzyme that plays pleotropic roles in the control of cardiac contraction, ventricular remodeling, ischemia-reperfusion injury and cardioprotection. PKCd is traditionally viewed as an allosterically-activated enzyme that exerts membrane-delimited actions at lipid membranes. This conventional model of PKCd activation does not adequately explain PKCd's actions in the heart, where PKCd phosphorylates proteins in non-membrane compartments and exerts diverse (and in some cases opposing) actions in both ischemic injury and cardioprotection. Our previous studies began to address this longstanding dilemma by showing that PKCd is activated in a stimulus-specific manner in cardiomyocytes. We showed that PKCd is phosphorylated at Y311 in cardiomyocytes subjected to oxidative stress (but not G protein-coupled receptor agonists) and that Y311 phosphorylation alters PKCd activity toward the sarcomeric regulatory proteins cardiac troponin I and cardiac troponin T. New data in this application expose the mechanism underlying the Y311-phosphorylation dependent change in PKCd's enzymology. We show that Y311 phosphorylation generates a docking site for PKCd's phospho-Tyr (pY) binding C2 domain. The C2 domain-pY311 interaction in turn controls PKCd activity indirectly by regulating phosphorylation at a novel site (S357) in the catalytic pocket of the kinase domain. The redox-dependent decrease in PKCd-S357 phosphorylation leads to a high level of lipid-independent activity (allowing for the phosphorylation of substrates throughout the cell, not just on lipid membranes) and a change in PKCd's substrate phosphoacceptor site (P-site) specificity. A mechanism to dynamically alter P-site specificity (through a change in kinase domain phosphorylation) is both novel for PKCd and unprecedented for any other kinase. Studies in this application will consider changes in S357 phosphorylation as a mechanism to explain PKCd's distinctive cellular actions during oxidative stress.
Aim #1 will use in vitro biochemical approaches to identify the role of the C2 domain and S357 phosphorylation in the control of PKCd signaling to pathways that regulate cardiac growth and apoptosis responses. We will use biochemical approaches to identify growth factor- and ROS-dependent mechanisms that regulate PKCd-S357 phosphorylation and take advantage of genetic approaches and overexpression strategies (including with analogue-sensitive forms of PKCd) to identify substrates/effectors that are uniquely activated by distinct molecular forms of PKCd and (in conjunction with studies Aim 2) examine their role in cardiac injury responses.
Aim #2 will use mouse models engineered to express mutant PKCdS357A or PKCdS357E alleles, in place of the WT-PKCd allele, to determine the role of PKCd- S357 phosphorylation/dephosphorylation in cardiac function and cardiac pathogenesis following ischemia- reperfusion injury in vivo. The overarching goal of these studies is to identify novel molecular determinant of PKCd that can be targeted to prevent or mitigate ischemia-reperfusion injury and pathologic cardiac remodeling.
Protein kinase C-delta (PKCd) is a signaling enzyme that has been implicated in both ischemia/reperfusion injury and pathways that contribute to ischemic preconditioning and cardiomyocyte survival. This application focuses on a revised model of PKCd activation that allows for agonist-specific PKCd signaling 'modes' that link to functionally distinct cellular responses. The proposed studies examine the role of novel molecular determinants on PKCd that specify its cellular actions and represent targets for the design of small-molecule inhibitors tailored to abrogate PKCd-dependent pathological cardiac remodeling.
