This application is for continuation of support for a project designed to characterize the interaction of Protein Kinase C (PKC) with its cofactors, and to determine the mechanisms by which it is activated. PKC is an important intracellular regulatory protein that is also the high affinity receptor for the tumor promotors, phorbol esters. The activity of PKC is calcium and phospholipid- dependent. At low calcium concentrations, the activity is also stimulated by phorbol esters and/or diacylglycerol. PKC binds to membranes in a calcium-dependent manner and it is also a member of a newly identified category of calcium response proteins that bind calcium only in the presence of membranes. PKC forms two membrane-bound states, one is reversible and calcium-dependent while the other has properties of an integral membrane protein. Formation of the latter is stimulated by phorbol esters. This study proposes to continue the elucidation of the mechanism of activation and interaction of PKC with these cofactors. The effect of phosphatidyl- ethanolamine, the major intracellular neutral phospholipid, on PKC and its various properties will be studied. Preliminary results show a great preference for this phospholipid over phosphatidyl-choline, the major neutral phospholipid on the cell surface. We plan to document and explain the basis for this difference using assays for both enzyme activity and protein-membrane binding. Other objectives include determination of the conditions and phospholipids that enhance formation of the integral membrane-bound form of PKC. This form of the protein will be detected by its cofactor- independent activity and by its elution with phospholipids on gel filtration chromatography in the presence of EGTA. A major goal will be to determine the mechanism by which Phorbol esters or diacylglycerol activate PKC in its reversible membrane-bound state. Protein-membrane interaction will be studied by fluorescence or light scattering techniques and activity will be measured by protein phosphorylation with radioactive ATP. The fluorescence and CD spectral changes in PKC that are induced by interaction with phospholipids, phorbol esters and calcium will be measured to determine the extent of protein structural change associated with these interactions. Another abundant group of proteins has been isolated that bind calcium and membranes in a manner similar to PKC. The first objective will be to determine their identity and if they are members of the annexin family of proteins. The effects of these various proteins on membrane properties will be studied to determine if they produce membrane permeability in either their reversible or irreversible membrane-bound forms. Extended goals are to continue to examine the mechanisms of various PKC activators and inhibitors, and to compare these various properties for the different isoforms of PKC. These results should help unravel the basis for the phorbol ester effect and the mechanism of action of some second messengers.
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