Distinct Protein Kinase C-Delta Signaling Modes in Cardiomyocytes

描述（由申请人提供）：蛋白激酶C-δ（PKCd）是一种信号调节酶，在控制心脏收缩、心室重塑、缺血-再灌注损伤和心脏保护中发挥多效性作用。PKCd传统上被认为是一种变构激活的酶，在脂质膜上发挥膜界定的作用。PKCd激活的这种传统模型不能充分解释PKCd在心脏中的作用，其中PKCd磷酸化非膜隔室中的蛋白质，并在缺血性损伤和心脏保护中发挥多种（并且在某些情况下相反）作用。我们以前的研究开始解决这个长期存在的困境，表明PKCd是在心肌细胞中以刺激特异性的方式激活。我们发现，PKCd在受到氧化应激的心肌细胞（但不是G蛋白偶联受体激动剂）的Y311磷酸化，Y311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。本申请中的新数据揭示了PKCd酶学中Y311磷酸化依赖性变化的机制。我们发现，Y311磷酸化产生的PKCd的磷酸-酪氨酸（pY）结合C2域的对接网站。C2结构域-pY 311相互作用又通过调节激酶结构域催化口袋中新位点（S357）的磷酸化间接控制PKCd活性。PKCd-S357磷酸化的氧化还原依赖性降低导致高水平的脂质非依赖性活性（允许整个细胞的底物磷酸化，而不仅仅是脂质膜上的底物磷酸化）和PKCd的底物磷酸受体位点（P-位点）特异性的变化。动态改变P-位点特异性的机制（通过激酶结构域磷酸化的变化）对于PKCd是新颖的，对于任何其他激酶都是前所未有的。本申请中的研究将考虑S357磷酸化的变化作为解释PKCd在氧化应激期间独特的细胞作用的机制。目的#1将使用体外生物化学方法来鉴定C2结构域和S357磷酸化在PKCd信号传导至调节心脏生长和凋亡反应的途径的控制中的作用。我们将使用生物化学方法来确定生长因子和ROS依赖性机制，调节PKCd-S357磷酸化，并利用遗传方法和过表达策略（包括类似物敏感形式的PKCd），以确定底物/效应器，独特激活的不同分子形式的PKCd和（结合研究目的2）检查他们在心脏损伤反应中的作用。目的#2将使用经工程改造以表达突变PKCdS 357 A或PKCdS 357 E等位基因代替WT-PKCd等位基因的小鼠模型，以确定PKCd-S357磷酸化/去磷酸化在体内缺血-再灌注损伤后的心脏功能和心脏发病机制中的作用。这些研究的首要目标是鉴定PKCd的新分子决定簇，其可以靶向预防或减轻缺血再灌注损伤和病理性心脏重塑。

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.