Protein Kinase C in Macrophage Activation

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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.