Protein 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 PKC substrates whose synthesis, myristoylation and phosphorylation are regulated by LPS, and which are therefore primary candidates as effector molecules of LPS-induced responses. We have purified, cloned and sequenced a 68K PKC substrate whose myristoylation and membrane association is induced by LPS. We will determine whether 68K cycles to and from the membrane, directed by myristic acid, targeted by myristoylated-68K binding proteins, and regulated by the phosphorylation state of the protein. The precise point at which 68K myristoylation is regulated will be defined. Site-specific mutagenesis will be utilized to determine whether myristic acid directs 68K to the membrane and is required for its subsequent phosphorylation by PKC. We will define the phosphorylation sites involved in displacing myristoylated 68K from the membrane, and assess whether dephosphorylation of 58K promote reattachment to the membrane. At the membrane 68K resides in punctate structures corresponding to focal adhesions. We will identify the molecular components of these structures and define how they associate with the actin cytoskeleton. The role of 68K and PKC in regulating the interaction between focal adhesion and the actin cytoskeleton will be explored under a variety of conditions, including phagocytosis and chemotaxis. A permeabilized cell system will be established in which to further investigate the effect of 68K phosphorylation on actin-cytoskeleton organization. In vitro experiments with purified 68K and actin will define the site of binding of 68K with actin, and will clarify the functional consequences of 58K phosphorylation on actin structure. Two other PKC substrates whose myristoylation is induced by LPS also represent good candidates as effector molecules for LPS-dependent responses. We will purify these 40K and 42K proteins, clone and sequence the cDNA's encoding them, and study the effects of LPS-induced myristoylation and phosphorylation on their subcellular location. The mechanism by which LPS primes PKC-induced arachidonic acid metabolism will be further characterized. We will examine whether LPS increases the transcription, translation and activities of the cyclooxygenase and lipoxygenase enzymes and whether LPS promotes the association of these enzymes with the plasma membrane.
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