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、心室重构、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活化酶，在脂质膜上发挥膜分隔作用。这种PKCd激活的传统模型并不能充分解释PKCd在心脏中的作用，PKCd磷酸化非膜室中的蛋白质，并在缺血性损伤和心脏保护中发挥不同（在某些情况下相反）的作用。我们之前的研究通过表明PKCd在心肌细胞中以刺激特异性的方式激活，开始解决这个长期存在的困境。我们发现PKCd在氧化应激下的心肌细胞中在Y311位点被磷酸化（而不是G蛋白偶联受体激动剂），并且Y311磷酸化改变PKCd对肌肉调节蛋白心肌肌钙蛋白I和心肌肌钙蛋白t的活性。这项应用的新数据揭示了PKCd酶学中Y311磷酸化依赖性变化的机制。我们发现Y311磷酸化产生了PKCd的磷酸化- tyr （pY）结合C2结构域的对接位点。C2结构域- py311相互作用反过来通过调节激酶结构域催化口袋中一个新位点（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，而不仅仅是在脂质膜上）和PKCd底物磷酸化受体位点（p位点）特异性的变化。动态改变p位点特异性的机制（通过改变激酶结构域磷酸化）对PKCd来说是新的，对任何其他激酶来说都是前所未有的。本研究将考虑S357磷酸化的变化作为一种机制来解释PKCd在氧化应激过程中的独特细胞行为。Aim #1将使用体外生化方法来确定C2结构域和S357磷酸化在PKCd信号通路控制中的作用，这些信号通路调节心脏生长和凋亡反应。我们将使用生化方法来识别调节PKCd- s357磷酸化的生长因子和ros依赖机制，并利用遗传方法和过表达策略（包括PKCd的类似敏感形式）来识别被PKCd的不同分子形式唯一激活的底物/效应物，并（与研究目标2一起）检查它们在心脏损伤反应中的作用。Aim #2将使用表达突变型PKCdS357A或PKCdS357E等位基因的小鼠模型来代替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。这些研究的总体目标是确定新的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.