Identification, partial purification, and characterization of a novel phospholipid-dependent and fatty acid-activated protein kinase from human platelets

• Published in: The Journal of biological chemistry 1994-04, Vol.269 (13), p.9729-9735
• Main Authors: KHAN, W. A, BLOBE, G. C, RICHARDS, A. L, HANNUN, Y. A
• Format: Article
• Language: English
• Subjects: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Blood Platelets - enzymology; Blotting, Western; Calcium - pharmacology; Chromatography; Chromatography, Affinity; Chromatography, Ion Exchange; Diglycerides - pharmacology; Durapatite; Enzyme Activation; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Humans; Isoquinolines - pharmacology; Kinetics; Oleic Acid; Oleic Acids - pharmacology; Phorbol 12,13-Dibutyrate - metabolism; Phospholipids - pharmacology; Piperazines - pharmacology; Protein Kinase Inhibitors; Protein Kinases - blood; Protein Kinases - isolation & purification; Sphingosine - pharmacology; Substrate Specificity; Transferases
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(17)36943-0

A novel lipid-dependent protein kinase in human platelets was partially purified and characterized. This enzyme was calcium-independent and was selective for phosphatidic acid as a cofactor/activator with initial activation observed at approximately 2 mol % and peak activity achieved at 4 mol % phosphatidic acid. In the presence of phosphatidylserine, 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 with less potency. Enzyme activity was independent of diacylglycerol or phorbol esters and the enzyme did not bind [3H]phorbol dibutyrate. In a soluble protein kinase assay, the enzyme was activated by cis-unsaturated fatty acids with maximum activation occurring at 5-10 microM sodium oleate. Western blot analysis showed that this enzyme did not cross-react immunologically with antibodies raised against the currently identified isoenzymes of protein kinase C. A number of additional biochemical criteria distinguished this enzyme from known isoenzymes of protein kinase C. These biochemical and immunologic data define a novel lipid-dependent protein kinase in human platelets. The role of this enzyme in signal transduction as a phosphatidic acid-activated enzyme and as a possible target for cis-unsaturated fatty acids is discussed.