Distinct Protein Kinase C-Delta Signaling Modes in Cardiomyocytes

描述(申请人提供)：蛋白激酶C-增量(PKCd)是一种信号调节酶，在控制心脏收缩、心室重构、缺血再灌注损伤和心脏保护方面发挥多效性作用。PKCd传统上被认为是一种变构激活的酶，它在脂膜上发挥膜分隔作用。这种传统的PKCd激活模型不能很好地解释PKCd在心脏中的作用，PKCd在非膜室中磷酸化蛋白质，在缺血损伤和心脏保护中发挥不同的(在某些情况下是相反的)作用。我们之前的研究开始解决这一长期的困境，通过显示PKCd在心肌细胞中以刺激特异性的方式被激活。我们发现，在氧化应激(但不是G蛋白偶联受体激动剂)作用下的心肌细胞中，PKCd在Y311被磷酸化，并且Y311的磷酸化改变了PKCd对肌节调节蛋白心肌肌钙蛋白I和心肌肌钙蛋白T的活性。我们发现Y311的磷酸化为PKCd的磷酸酪氨酸(Py)结合C2结构域产生了一个对接位点。C2结构域与pY311的相互作用通过调节激酶域催化口袋中新位点(S357)的磷酸化来间接调控PKCd的活性。氧化还原依赖的PKCd-S357磷酸化水平的降低导致高水平的脂非依赖性活性(允许底物在整个细胞内磷酸化，而不仅仅是在脂膜上)，并改变了PKCd底物磷受体位点(P-Site)的特异性。一种动态改变P-位点特异性的机制(通过改变激酶结构域的磷酸化)对PKCd来说是新的，对其他任何一种激酶来说都是前所未有的。在这一应用中的研究将考虑将S357磷酸化的变化作为解释PKCd在氧化应激期间独特的细胞行为的机制。目的#1将使用体外生化方法来确定C2结构域和S357磷酸化在控制PKCd信号通路中的作用，该信号通路调节心脏生长和细胞凋亡反应。我们将使用生化方法来确定调节PKCd-S357磷酸化的生长因子和ROS依赖的机制，并利用遗传方法和过度表达策略(包括类似敏感形式的PKCd)来识别由不同分子形式的PKCd唯一激活的底物/效应器，并(与研究目标2一起)研究它们在心脏损伤反应中的作用。目的#2将利用表达突变的PKCDS357A或PKCDS357E等位基因的小鼠模型来替代WT-PKCd等位基因，以确定PKCd-S357磷酸化/去磷酸化在体内缺血再灌注损伤后心功能和心脏发病机制中的作用。这些研究的首要目标是确定新的PKCd分子决定因素，可以靶向预防或减轻缺血再灌注损伤和病理性心脏重构。

DESCRIPTION (provided by applicant): 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.