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.
Duke Scholars
Published In
ISSN
Publication Date
Volume
Issue
Start / End Page
Location
Related Subject Headings
- Substrate Specificity
- Sphingosine
- Protein Kinases
- Protein Kinase Inhibitors
- Piperazines
- Phospholipids
- Phorbol 12,13-Dibutyrate
- Oleic Acids
- Oleic Acid
- Kinetics
Citation
Published In
ISSN
Publication Date
Volume
Issue
Start / End Page
Location
Related Subject Headings
- Substrate Specificity
- Sphingosine
- Protein Kinases
- Protein Kinase Inhibitors
- Piperazines
- Phospholipids
- Phorbol 12,13-Dibutyrate
- Oleic Acids
- Oleic Acid
- Kinetics