IDENTIFICATION, PARTIAL-PURIFICATION, AND CHARACTERIZATION OF A NOVELPHOSPHOLIPID-DEPENDENT AND FATTY ACID-ACTIVATED PROTEIN-KINASE FROM HUMAN PLATELETS

A novel lipid-dependent protein kinase in human platelets was partiall y purified and characterized. This enzyme was calcium-independent and was selective for phosphatidic acid as a cofactor/activator with initi al activation observed at approximately 2 mol % and peak activity achi eved at 4 mol % phosphatidic acid. In the presence of phosphatidylseri ne, enzyme activation was observed with concentrations of phosphatidic acid as low as 0.5 mol % with peak activity at 2 mol %. Other anionic phospholipids also activated the enzyme but to a lesser extent and wi th less potency. Enzyme activity was independent of diacylglycerol or phorbol esters and the enzyme did not bind [H-3]phorbol dibutyrate. In a soluble protein kinase assay, the enzyme was activated by cis-unsat urated fatty acids with maximum activation occurring at 5-10 muM sodiu m oleate. Western blot analysis showed that this enzyme did not cross- react immunologically with antibodies raised against the currently ide ntified isoenzymes of protein kinase C. A number of additional biochem ical criteria distinguished this enzyme from known isoenzymes of prote in kinase C. These biochemical and immunologic data define a novel lip id-dependent protein kinase in human platelets. The role of this enzym e in signal transduction as a phosphatidic acid-activated enzyme and a s a possible target for cis-unsaturated fatty acids is discussed.