The 85 kDa cytosolic phospholipase As (cPLA2) mediates agonist- induced arachidonic acid release, the first step in the production of eicosanoids and platelet-activating factor. cPLA2 is regulated by phosphorylation and by calcium, which binds to a C2 domain and induces its translocation to the nuclear envelope. However, certain agonists (PMA and okadaic acid) induce cPLA2-mediated arachidonic acid release in macrophages without increasing intracellular calcium implicating novel mechanisms for cPLA2 activation. A role for specific cytosolic regulatory proteins, novel phosphorylation events, and local changes in levels of phosphatidylinositol-bisphosphate (PIP2) in the membrane are hypothesized. Various cell models including macrophages, Sf9 insect bisphospate (PIP2) in the membrane are hypothesized. Various cell models including macrophages, Sf9 insect cells and mouse lung fibroblasts will be used to elucidate these novel mechanisms. The ability of agonists that promote arachidonic acid release to induce synthesis of PIP2 isomers will be measured. The effect of increasing synthesis of PIP2 by over-expressing phosphatidylinositol transfer protein and type I phosphatidylinositol-4- phosphate 5-kinase on cPLA2-mediated arachidonic acid release will be determined. A permeabilized cell system will be developed to reconstitute agonist-induced, cPLA2-mediated arachidonic acid release to explore the role of PIP2 and to identify cytosolic regulatory proteins. A role for protein kinase C in regulating cPLA2 by both direct and indirect mechanisms is also proposed. The ability of PKC to directly phosphorylate and regulate cPLA2 will through activation of phospholipase D by increasing production of diacylglycerol or PIP2 will be tested. A 54 kDa kinase specifically associates with cPLA2 in okadaic acid treated macrophages suggesting the importance of novel phosphorylation events. The 54 kDa kinase specifically associates with cPLA2 in okadaic acid treated macrophages suggesting the importance of novel phosphorylation events. The 54 kDa kinase will be identified and its role in regulating PLA2 determined. Finally, the observed inhibition of agonist-induced arachidonic acid release in macrophages by cyclohexamide and actinomycin D suggests the involvement of a rapidly turning-over protein, and the basis for this will be determined. These studies will lead to a better understanding of the regulation of arachidonic acid release and production of inflammatory lipid mediators.
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