Protein Kinase C in Macrophage Activation

描述(由申请人提供)：FcGamma受体(FcGammaR)依赖的吞噬作用，即抗体(LGG)调理病原体被识别和摧毁的过程，是宿主防御的中心。调节失调的FcGammaR信号导致多种慢性疾病状态，包括类风湿性关节炎、多发性硬化症和动脉粥样硬化斑块的进展。治疗此类疾病的一种方法是确定FcGammaR信号级联的关键成分，这些成分可能是基因治疗或药物干预的新靶点。然而，要使这种策略成功，有必要了解吞噬过程背后的分子相互作用。PKC在FcGammaR介导的信号转导、介导吞噬、呼吸爆发和细胞因子的产生中起核心作用。有关PKC参与这些功能的大部分证据依赖于PKC抑制剂的使用，而这些抑制剂并不是异构体特异性的。因此，有必要用分子手段来研究异构体特异性的PKC信号转导。我们正在使用实时共聚焦显微镜跟踪在RAW 264.7巨噬细胞中依赖于免疫球蛋白的吞噬作用，这些巨噬细胞被绿色荧光蛋白结合的蛋白激酶C构建。实时成像使我们能够跟踪单个颗粒的摄取和GFP信号随时间的定位，并定量测定吞噬细胞的速度和信号分子与靶的累积。我们将操纵野生型或突变型PKC的水平，并量化这些操纵对PKC定位和吞噬速率的影响。这将确定PKC内包含信号决定因素的区域。一旦定义了区域，就可以确定将PKC与下游事件联系起来的结合伙伴和底物。在上游，我们将重点介绍PKC定位/激活所需的酶和第二信使。我们将检验这一假设，即PKC定位到吞噬小体是由调控结构域中的一个或多个区域介导的。它们可能与吞噬体膜上存在的活化C激酶受体(RACK)和/或脂质第二信使相互作用。PKC定位于吞噬小体是伪足延伸所必需的蛋白质的磷酸化所必需的。具体目标：1)1)吞噬所需的PKC催化活性？，2)FcGammaR连接时膜定位所必需的和充分的PKC区域，3)阐明DAG调节PKC定位的机制，4)鉴定PKC-n靶标和结合蛋白。确定PKC转导吞噬细胞信号的机制对于我们理解宿主防御和导致自身免疫性疾病的缺陷至关重要。

DESCRIPTION (provided by applicant): Fcgamma receptor (FcgammaR)-dependent phagocytosis, the process by which antibody (lgG) opsonized pathogens are recognized and destroyed, is central to host defense. Dysregulated FcgammaR signaling contributes to a variety of chronic disease states, including rheumatoid arthritis, multiple sclerosis, and progression of atherosclerotic plaques. One approach for treating such diseases lies in the identification of critical components of the FcgammaR signaling cascade that may be novel targets for gene therapy or pharmaceutical intervention. However, for such strategies to be successful, it is necessary to understand the molecular interactions underlying the phagocytic process. PKC plays a central role in FcgammaR-mediated signaling, mediating phagocytosis, respiratory burst, and cytokine production. The bulk of the evidence implicating PKC in these functions relies on the use of PKC inhibitors, which are not isoform specific. Thus, molecular approaches are necessary to study isoform-specific PKC signaling. We are using real time confocal microscopy to follow lgG-dependent phagocytosis in RAW 264.7 macrophages transfected with GFP conjugated PKC constructs. Real time imaging allows us to follow the uptake of individual particles and localization of the GFP signal with time and to quantitate the rate of phagocytosis and the accumulation of signaling molecules with targets. We will manipulate levels of wild type or mutant PKCs and quantify the effect of these manipulations on PKC localization and the rate of phagocytosis. This will identify the regions within PKC that contain signaling determinants. Once the regions have been defined, binding partners and substrates can be identified that will link PKC with downstream events. Upstream, we will focus on the enzymes and second messengers necessary for PKC localization/ activation. We will test the hypothesis that PKC localization to phagosomes is mediated by one or more regions within the regulatory domain. These may interact with Receptor for Activated C Kinases (RACK) and/or lipid second messengers present in the phagosomal membrane. PKC localization to phagosomes is necessary for phosphorylation of proteins necessary for pseudopod extension. Specific aims: 1) ls PKC catalytic activity necessary for phagocytosis?, 2) Map the regions of PKC necessary and sufficient for membrane localization upon FcgammaR ligation, 3) Elucidate the mechanism by which DAG regulates PKC localization, and 4) Identify PKC-n targets and binding proteins. Defining the mechanisms by which PKC transduces the phagocytic signal is critical to our understanding of host defense and the defects that result in autoimmune diseases.