Phagocytic leukocytes (monocytes, macrophages, and neutrophils) perform essential functions in the immune response to microbial infection, but also contribute to host tissue damage in both infectious and non-infectious inflammatory disorders. Therefore, definition of the biochemical mechanism that regulate the activation of phagocytes is required for development of improved therapies to augment antimicrobial defenses and limit deleterious consequences to host tissues. Phospholipase D (PLD) is an important signal-transducing enzyme that regulates both antimicrobial and host-damaging functions of phagocytes. However, the biochemical mechanisms that regulate the assembly and activation of a functional PLD complex at the membrane substrate, remain incompletely defined. A common feature of the leukocyte effector functions regulated by PLD is their absolute dependence on the actin cytoskeleton. We recently demonstrated that stimulation of PLD1 in U937 promonocytes is accompanied by its stable association with a detergent-insoluble fraction containing F-actin and other cytoskeletal proteins. Currently unknown are: (1) the physical basis for association of PLD1 and the w cytoskeleton, and (2) its consequences for regulation of PLD activity. Our general hypothes' is that interactions between PLO and the actin cytoskeleton regulate the assembly and activation of a multi-component PLD complex at the membrane interface. The proposed studies test the novel specific hypotheses that (a) PLD1 binds to membrane-associated actin filaments and monomeric actin, and (b) these physical interactions between PLD1 and actin regulate PLD activity. We will utilize both highly purified in vitro systems for biochemical analysis as well as physiologic in vivo studies of phagocyte functional responses to evaluate these hypotheses in the context of pursuing three Specific Aims: (1) Characterize the physical association of PLD1 with actin filaments (2) Define the binding of PLD1 to G-actin, and (3) Determine whether association with actin modulates PLD activity. These studies will further both our specific knowledge of signal transduction mechanisms that regulate inflammation as well as our more general understanding of the spatial and temporal determinants of cellular activation and motility.
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