Protein Kinase C in Macrophage Activation

描述（由申请方提供）：Fc γ受体（Fc γ R）依赖性吞噬作用是识别和破坏抗体（IgG）调理病原体的过程，是宿主防御的核心。FcgammaR信号转导失调导致多种慢性疾病状态，包括类风湿性关节炎、多发性硬化和动脉粥样硬化斑块的进展。治疗此类疾病的一种方法在于鉴定Fc γ R信号级联的关键组分，其可以是基因治疗或药物干预的新靶标。然而，这种策略是成功的，它是必要的，以了解潜在的吞噬过程的分子相互作用。PKC在Fc γ R介导的信号传导、介导吞噬作用、呼吸爆发和细胞因子产生中起核心作用。大部分的证据表明，PKC在这些功能依赖于使用PKC抑制剂，这是不是亚型特异性。因此，分子方法是必要的研究异构体特异性PKC信号。我们使用真实的时间共聚焦显微镜来跟踪用GFP缀合的PKC构建体转染的RAW 264.7巨噬细胞中的IgG依赖性吞噬作用。真实的时间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，并定量吞噬率和信号分子与靶标的积累。我们将操纵野生型或突变型PKC的水平，并量化这些操作对PKC定位和吞噬率的影响。这将确定PKC中包含信号决定簇的区域。一旦确定了区域，就可以确定将PKC与下游事件联系起来的结合伴侣和底物。在上游，我们将重点关注PKC定位/激活所必需的酶和第二信使。 我们将测试的假设，PKC定位到吞噬体介导的一个或多个区域内的监管结构域。这些可与存在于吞噬体膜中的活化C激酶受体（RACK）和/或脂质第二信使相互作用。PKC定位于吞噬体是伪足延伸所必需的蛋白质磷酸化所必需的。具体目的：1）PKC催化活性是否为吞噬所必需？2)绘制Fc γ R连接后膜定位所必需和足够的PKC区域，3）阐明DAG调节PKC定位的机制，以及4）鉴定PKC-n靶点和结合蛋白。确定PKC转导吞噬信号的机制对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.