Protein Kinase C in Macrophage Activation

描述（由申请人提供）：Fcgamma受体（Fcgamma）依赖性吞噬作用是抗体（lgG）活化的病原体被识别和破坏的过程，是宿主防御的核心。失调的FcgammaR信号有助于多种慢性疾病状态，包括类风湿关节炎、多发性硬化和动脉粥样硬化斑块的进展。治疗这类疾病的一种方法在于鉴定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，为了使这种策略取得成功，有必要了解吞噬过程背后的分子相互作用。PKC在fcgammar介导的信号传导、吞噬、呼吸爆发和细胞因子产生中起核心作用。大量证据表明PKC在这些功能中依赖于PKC抑制剂的使用，而PKC抑制剂不是同工异构体特异性的。因此，分子方法是研究特异性PKC信号的必要手段。我们使用实时共聚焦显微镜观察转染GFP偶联PKC构建物的RAW 264.7巨噬细胞中lg依赖性吞噬。实时成像使我们能够随时间跟踪单个颗粒的摄取和GFP信号的定位，并量化吞噬速率和信号分子与目标的积累。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。这将识别PKC中包含信号决定因子的区域。一旦确定了这些区域，就可以确定将PKC与下游事件联系起来的结合伙伴和底物。上游，我们将重点关注PKC定位/激活所需的酶和第二信使。我们将检验PKC定位到吞噬体的假设是由调控域中的一个或多个区域介导的。这些可能与存在于吞噬体膜上的活化C激酶受体（RACK）和/或脂质第二信使相互作用。PKC定位到吞噬体是假足延伸所需蛋白磷酸化的必要条件。具体目标：1)PKC的催化活性是吞噬所必需的吗？2)绘制FcgammaR连接时PKC在膜上定位所必需和充分的区域；3)阐明DAG调控PKC定位的机制；4)鉴定PKC-n的靶点和结合蛋白。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.