Structure-function studies of cytosolic phospholipase A2 and protein kinase C

The main objective of this thesis is to elucidate the mechanisms of membrane-protein interactions for two classes of enzymes; cytosolic phospholipase A2 and protein kinase C-alpha, which play important roles in cellular signaling.;Cytosolic phospholipase A2 (cPLA2) is a member of a large family of lipolytic enzymes that catalyze the hydrolysis of the fatty acid ester at the sn-2 position of phospholipids. cPLA 2 selectively liberates arachidonic acid from membrane phospholipids which can be converted to potent inflammatory mediators. To determine the mechanism of interfacial binding of cPLA2, we measured the interaction of this enzyme with phospholipid monolayers and polymerizable mixed liposomes. The results indicate that cPLA2 has a unique interfacial binding mode that involves Ca2+-dependent membrane penetration, which is important for its interfacial activities. Cytosolic PLA2 contains a membrane-targeting module (C2 domain) that is responsible for Ca 2+-dependent membrane binding of the protein. To identify protein residues in the C2 domain essential for its Ca2+ and membrane binding, Ca2+ ligands and putative membrane-binding residues of cPLA2 were mutated and the effects of mutations on enzyme activity, membrane binding affinity and monolayer penetration were determined. Results indicate that Ca2+ triggers conformational changes in the calcium binding loops, exposing several hydrophobic residues, which then drive the membrane binding of cPLA2.;Protein kinase C (PKC) is a family of serine/threonine kinases which plays a central role in many signal transduction pathways in the cell. PKC is activated upon translocation to the phosphatidylserine-containing membranes, which is governed by the regulatory domain. The regulatory domain of conventional PKCs contains two membrane targeting modules, the C2 domain responsible for Ca2+-dependent membrane binding of the protein, and the C1 domain composed of two cystein-rich zinc fingers that bind diacylglycerol and phorbol esters. To elucidate the interplay between the C1 and C2 domains during PKC activation, we mutated several charged residues in the C1 domain of PKC-alpha. We then measured the membrane binding affinities, activities and monolayer penetration of these mutants. The results suggest the existence of specific interactions between C1 and C2 domain that are important for the selective recognition of phosphatidylserine during the PKC activation.