Prrotein kinase C (PKC)-induced phosphorylation in macrophages is necessary for a full functional response to bacterial lipopolysaccharides (LPS).
The aim of this project is to understand the mechanism by which LPS regulates PKC-dependent signaling pathways in macrophages. Our focus is the molecular characterization of a novel 42K, macrophage-specific, PKC substrate whose synthesis, myristoylation and phosphorylation are regulated by LPS, and which is a likely effector of LPS-induced responses. The protein associates with the plasma membrane and is enriched in phagolysosomal membranes. In addition, we have demonstrated that 42K interacts with calcium/calmodulin and with actin, and that these interactions are modulated by phosphorylation of 42K. In order to study the mechanism of action of 42K, we have purified the protein and have used tryptic peptide sequences to clone the cDNA. We will sequence the cDNA encoding 42K, and identify the phosphorylation sites and the calmodulin and actin binding sites by mutagenesis and in vitro studies. Experiments with purified 42K and actin will further define the site of binding of 42K with actin, and will clarify the functional consequences of 42K phosphorylation on actin structure. In vitro experiments will also clarify the calmodulin binding site, and will define the role of calcium and phosphorylation in this binding interaction. The role of 42K in modulating the cytoskelatal rearrangement associated with phagocytosis and with bulk membrane flow in the macrophage will be, investigated in detail. We will mutate the calmodulin and actin-binding domains of 42K and determine whether the mutant protein acts as a dominant negative regulator of phagocytosis, secretion and chemotaxis. Site-specific mutagenesis will also be utilized to determine whether myristic acid directs 42K to the membrane and is required for its subsequent phosphorylation by PKC, as well as to define the membrane binding domain of 42K and the effect of altemate acylation signals on intracellular targeting of 42K. An in vitro membrane binding assay for 42K will be established and a candidate membrane receptor for myristoylated 42K will be identified in crosslinking experiments using synthetic peptides derived from the membrane binding domain of 42K. Deletional analysis of the 42K promoter region will define the regulatory elements necessary for macrophage-specific and LPS-inducible gene expression. We will also characterize the DNA binding proteins which interact with these regulatory elements and their cognate binding sequences. These experiments will define the biology of a novel, 42K, macrophage-specific PKC substrate which might serve to integrate information from the PKC and calcium/calmodulin signal transduction pathways in the regulation of the cytoskeleton.
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