Proliferative diseases, such as cancer, and inflammatory diseases, such as rheumatoid arthritis, result from aberrations in cellular signaling pathways. Many cellular proteins that participate in cellular signaling pathways, most notably several forms of phospholipase A2 (PLA2) and protein kinase C (PKC), interact with the cell membrane (interfacial binding) in response to cellular and lipid second messengers. Although studies to date have shown that the interfacial binding plays a key role in the regulation of these proteins, little is known about the mechanism of their interfacial binding. The primary objective of this research program is to understand the molecular basis of the interfacial binding of PLA2 and PKC isoforms with a special emphasis on determining the differences in their interfacial binding mechanisms. A long-term objective is to apply the principles learned from these studies to the development of therapeutic agents that can specifically block the uncontrolled activation of particular PLA2 and PKC isoforms.
Specific aims during this proposed period are: (1) to identify the protein residues of mammalian secretory PLA2s that are involved in the interfacial binding mechanisms, (2) to determine the interfacial binding mechanism of cytosolic PLA2 and identify its interfacial binding residues, (3) to elucidate the mechanisms whereby three different types of PKC interact with membranes, determine how the physical state of membrane affects the PKC activation, and identify PKC residues that are involved in the interfacial binding and activation, and finally (4) to determine the mechanism and functional roles of protein and lipid domain formation in the interfacial binding of PLA2 and PKC. The principal methodologies to be used include: (1) many recombinant DNA techniques for the site-directed mutagenesis and over-expression of PLA2s and PKCs, (2) kinetic and membrane-binding analyses of PLA2s and PKCs using the polymerized mixed liposome system developed in this laboratory which allows the systematic structure-function studies of interfacial binding, and (3) phospholipid monolayer technique elaborated for the analysis of interfacial binding mechanism.
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