Regulation of Protein Kinase C Translocation

中性粒细胞(PMN)不仅介导宿主的监视和防御，而且还介导炎症和其他损伤性反应。趋化因子(CF)在这些能力上指导PMN。它们与PMN受体结合，PMN受体发出细胞内信号，调节关键的反应诱发元件。我们将研究这些元件中的一个家族，即蛋白激酶C(PKC)。我们还将研究来自磷脂(PL)的信号，这些信号指示PKC从胞浆中的潜伏状态移动到活性的膜相关激酶。这种易位被认为是次要的钙瞬变信号。然而，在PMN中，CF刺激由钙瞬变依赖和非钙瞬变两部分组成的PKC运动。相反，5-氧代二十碳四烯酸酯只引起钙瞬变依赖性的PKC运动，而纳分子水平的花生四烯酸(AA)主要引起钙瞬变非依赖性的PKC运动。我们假设PMN中不同的PKC亚型对CF、5-氧代二十碳四烯酸和AA表现出不同的易位模式；被CF刺激的PMN发出钙瞬变非依赖性的易位信号；AA是这样的信号之一。我们将追踪PMN中的PKC亚型；确定AA对PKC影响的机制；研究PMN将磷脂酰胆碱代谢为PKC转位信号的钙瞬变需求；并确定AA和其他脂质来源的信号在介导PKC转位和细胞功能中所起的作用。实验系统包括人PMN；可逆性缺失AA的HL-60细胞；稳定表达野生型或突变PKC融合到荧光蛋白的HEK 293细胞；后者瞬时表达CF受体的细胞；以及无细胞模型。我们将通过Western blotts和荧光激光共聚焦显微镜跟踪PKC；测量PL到AA、磷脂酰肌醇、二酰甘油和磷脂酸的质量代谢；通过突变开发编码单一位点、缺失和截短的PKC物种的构建体；并使用这些构建体来定义PKC中响应AA或CF所需的结构域和域功能。我们将测试这些生化研究在PMN和HL-60细胞的超氧化物产生和其他反应的检测中的相关性。这项工作的结果将适用于所有哺乳动物细胞，像CF一样作用于蛇纹石受体的刺激，以及许多与PKC结构同源的调节蛋白。这些研究应该揭示一个观察PKC调控的框架，在这个框架中，细胞发出钙瞬变和钙非依赖性信号(例如AA)来指导不同的PKC亚型的运动。通过将PKC和AA联系起来，我们的研究将提出新的药理和营养策略，以减少PKC易位，从而减少导致自我损伤和其他病理反应的不良细胞反应。

Polymorphonuclear neutrophils (PMN) mediate not only host surveillance and defense but also inflammatory and other injurious reactions. Chemotactic factors (CF) guide PMN in these capacities. They bind to PMN receptors that issue intracellular signals that regulate key response-eliciting elements. We will investigate one family of such elements, the protein kinases C (PKC). We will also examine the signals derived from phospholipids (PL) that direct PKC to move from a latent state in cytosol to an active membrane-associated kinase. This translocation is regarded as secondary to a Ca2+ transient signal. In PMN, however, CF stimulate PKC movements comprised of both Ca2+ transient-dependent and Ca2+ transient-independent components. In contrast, 5-oxo-eicosatetraenoate causes only Ca2+ transient-dependent and nanomolar levels of arachidonic acid (AA) induce mainly Ca2+ transient-independent PKC movements. We hypothesize that the various PKC isoforms in PMN manifest different patterns of translocation in response to CF, 5-oxo- eicosatetraenoate, and AA; that PMN stimulated with CF issue Ca2+-transient-independent translocation signals; and that AA is one such signal. We will track PKC isoforms in PMN; determine the mechanism of AA's effect on PKC; examine the Ca2+-transient requirements for PMN to metabolize PL into signals for PKC translocation; and define the role AA and other lipid-derived signals play in mediating PKC translocation as well as cell function. Systems for experimentation include human PMN; HL-60 cells reversibly depleted of AA; HEK 293 cells stably expressing wild type or mutant PKC fused to a fluorescent protein; the latter cells transiently expressing a CF receptor; and a cell- free model. We will track PKC by Western blots and fluorescent laser confocal microscopy; measure the metabolism of PL to AA, phosphatidyl-inositols, diacylglycerol, and phosphatidic acid by mass; develop constructs encoding single site, deletion, and truncated PKC species by mutagenesis; and use these constructs to define the structural domains and domain functions in PKC required for PKC to respond to AA or CF. We will test the relevancy of these biochemical studies on PMN and HL-60 cells in assays of superoxide production and other responses. Results of this work will apply to all mammalian cells, to stimuli that, like CF, act on serpentine receptors, and to the many regulatory proteins with structural homology to PKC. The studies should reveal a framework in which to view PKC regulation, one where Ca2+ transients and Ca2+-independent signals (e.g. AA) issue from cells to direct the movement of different PKC isoforms. By making this link between PKC and AA, our studies will suggest new pharmacological and nutritional strategies for the abatement of PKC translocation and thereby the untoward cellular responses which cause self-injury and other pathological reactions.