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伤害性反应。 趋化因子（CF）引导PMN发挥这些功能。 它们与PMN受体结合，PMN受体发出细胞内信号，调节关键的反应引发元件。 我们将研究这样的元素，蛋白激酶C（PKC）的一个家庭。 我们还将研究来自磷脂（PL）的信号，这些信号指导PKC从胞质溶胶中的潜伏状态转变为活性膜相关激酶。 这种易位被认为是Ca 2+瞬时信号的次要作用。 而在PMN中，CF刺激PKC运动，包括Ca 2+瞬时依赖性和Ca 2+瞬时非依赖性成分。 相比之下，5-氧代-二十碳四烯酸仅引起Ca 2+瞬时依赖性，而纳摩尔水平的花生四烯酸（AA）主要诱导Ca 2+瞬时非依赖性PKC运动。 我们假设，在PMN的各种PKC亚型表现出不同的模式易位在CF，5-氧代-二十碳四烯酸，和AA的，与CF刺激的PMN问题钙瞬时独立的易位信号，AA是这样的信号之一。 我们将跟踪PKC亚型在PMN;确定AA对PKC的作用机制;检查PMN代谢PL转化为PKC易位信号的Ca 2+瞬时需求;并确定AA和其他脂质衍生信号在介导PKC易位以及细胞功能中的作用。 用于实验的系统包括人PMN;可逆地耗尽AA的HL-60细胞;稳定表达与荧光蛋白融合的野生型或突变型PKC的HEK 293细胞;后者瞬时表达CF受体的细胞;和无细胞模型。 我们将通过Western印迹和荧光激光共聚焦显微镜跟踪PKC;测量PL代谢为AA、磷脂酰肌醇、二酰甘油和磷脂酸的质量;通过诱变开发编码单位点、缺失和截短PKC物种的构建体;并使用这些构建体来定义PKC响应AA或CF所需的PKC结构域和结构域功能。 我们将测试这些生化研究的相关性PMN和HL-60细胞的超氧化物产生和其他反应的测定。 这项工作的结果将适用于所有的哺乳动物细胞，刺激，如CF，作用于蛇形受体，并与PKC的结构同源的许多调节蛋白。 这些研究应该揭示一个框架，在其中查看PKC调节，其中Ca 2+瞬变和Ca 2+非依赖性信号（例如AA）从细胞发出，以指导不同的PKC亚型的运动。 通过建立PKC和AA之间的联系，我们的研究将提出新的药理学和营养学策略，以减少PKC易位，从而减少导致自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.