LPS REGULATION OF MACROPHAGE FUNCTION

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Protein kinase C (PKC)- induced phosphorylation is necessary for macrophages to respondfully to bacterial lipopolysaccharides (LPS). The aim of this project is to investigate the mechanism by which LPS influences PKC-dependent signalling pathways such as those leading to the cytoskeletal rearrangements associated with phagocytosis, membrane traffic, cellular adherence and motility. Dr. Aderem's focus is the molecular characterization of the MARCKS protein, and LPS-inducible PKC substrate, which regulates actin structure at the membrane. He will functionally delete the murine MARCKS null mice. In another approach to determining the role(s) of MARCKS in vivo, a macrophage-specific promoter will be used to generate transgenic mice expressing mutant MARCKS proteins in macrophages. He will characterize the LPS- inducible MARCKS promoter by defining regulatory elements in the 5' upstream region. The intron and the 3' untranslated region will also be analyzed for the presence of regulatory elements. The mechanism by which MARCKS targets to specific membranes will be analyzed by biophysical techniques, and by characterizing MARCKS binding proteins. The role of MARCKS in phagocytosis, lysosome recruitment, and transcytosis will be defined using MARCKS mutants and microbes which modify the phagocytic pathway. He will characterize the role of MARCKS in cell motility by examining actin structure and dynamics, calmodulin, and polarized membrane insertion, immotile cells expressing MARCKS mutants. A novel 50 kDa protein which is induced during phagocytosis in macrophages, and which associates with phagosomes, will also be characterized.