Identification, partial purification, and characterization of a novel phospholipid-dependent and fatty acid-activated protein kinase from human platelets.
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.
Khan
W A
WA
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
Blobe
G C
GC
Richards
A L
AL
Hannun
Y A
YA
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D001792:Blood Platelets / Q000201:enzymology*; D015153:Blotting, Western; D002118:Calcium / Q000494:pharmacology; D002845:Chromatography; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004075:Diglycerides / Q000494:pharmacology; D017886:Durapatite; D004789:Enzyme Activation; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D019301:Oleic Acid; D009829:Oleic Acids / Q000494:pharmacology*; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism; D010743:Phospholipids / Q000494:pharmacology*; D010879:Piperazines / Q000494:pharmacology; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000097:blood* / Q000302:isolation & purification*; D013110:Sphingosine / Q000494:pharmacology; D013379:Substrate Specificity
D004075:Diglycerides; D007546:Isoquinolines; D009829:Oleic Acids; D010743:Phospholipids; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D019301:Oleic Acid; D015240:Phorbol 12,13-Dibutyrate; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D017886:Durapatite; D011494:Protein Kinases; D013110:Sphingosine; D002118:Calcium