Regulation of Protein Kinase C Translocation

多形核中性粒细胞（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反应。趋化因子（CF）以这些能力引导PMN。它们与PMN受体结合，PMN受体发出细胞内信号，调节关键的反应诱导因子。我们将研究这些元件的一个家族，蛋白激酶C （PKC）。我们还将研究来自磷脂（PL）的信号，这些信号指导PKC从细胞质中的潜伏状态转移到活性的膜相关激酶。这种易位被认为是二次Ca2+瞬态信号。然而，在PMN中，CF刺激由Ca2+瞬态依赖性和Ca2+瞬态非依赖性成分组成的PKC运动。相比之下，5-氧-二十碳四烯酸仅引起Ca2+瞬态依赖性，而纳米摩尔水平的花生四烯酸（AA）主要诱导Ca2+瞬态非依赖性PKC运动。我们假设PMN中不同的PKC异构体在CF、5-氧-二十碳四烯酸和AA的作用下表现出不同的易位模式；CF刺激的PMN发出与Ca2+无关的瞬时易位信号；AA就是这样一个信号。我们将跟踪PMN中的PKC同工异构体；确定AA对PKC影响的机制；检查PMN代谢PL为PKC易位信号的Ca2+瞬时需求；并确定AA和其他脂质来源信号在介导PKC易位和细胞功能中的作用。实验系统包括人体PMN；HL-60细胞AA的可逆性耗竭；稳定表达野生型或突变型PKC与荧光蛋白融合的HEK 293细胞；后者细胞短暂表达一种CF受体；和一个无细胞模型。我们将通过Western blots和荧光激光共聚焦显微镜跟踪PKC；测定PL对AA、磷脂酰肌醇、二酰基甘油和磷脂酸的代谢质量；通过诱变开发编码单位点、缺失和截断PKC物种的构建体；并使用这些结构来定义PKC对AA或CF作出反应所需的结构域和结构域功能。我们将在超氧化物产生和其他反应的测定中测试PMN和HL-60细胞中这些生化研究的相关性。这项工作的结果将适用于所有哺乳动物细胞，刺激，如CF，作用于蛇形受体，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，其中Ca2+瞬态和Ca2+独立信号（例如AA）从细胞发出，以指导不同PKC异构体的运动。通过在PKC和AA之间建立这种联系，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.