Dissociation of protein kinase C activation and sn-1,2-diacylglycerol formation. Comparison of phosphatidylinositol- and phosphatidylcholine-derived diglycerides in alpha-thrombin-stimulated fibroblasts.
Diacylglycerols (DAGs) derived from phosphatidylcholine (PC) hydrolysis have been shown to activate protein kinase C (PKC) in vitro, but it is not known whether this event occurs in response to DAGs generated via agonist-induced PC hydrolysis in intact cells. In this report we have addressed this question directly, using alpha-thrombin stimulation of IIC9 fibroblasts. PKC activation in intact cells was assessed in two ways, by measuring: 1) PKC membrane association as determined by kinase activity and Western blot analysis and 2) the phosphorylation of an endogenous PKC substrate, an 80-kDa protein. Treatment with 500 ng/ml alpha-thrombin has been shown to stimulate both phosphoinositide and PC hydrolysis, whereas treatment with 100 pg/ml alpha-thrombin stimulates only PC breakdown. Using these two conditions, we show that DAG produced from phosphoinositide, but not PC hydrolysis, is associated with the activation of PKC.
Leach
K L
KL
Department of Cell Biology, Upjohn Company, Kalamazoo, Michigan 49007.
Ruff
V A
VA
Wright
T M
TM
Pessin
M S
MS
Raben
D M
DM
D000818:Animals; D001345:Autoradiography; D015153:Blotting, Western; D006224:Cricetinae; D003412:Cricetulus; D004072:Digitonin / Q000494:pharmacology; D004075:Diglycerides / Q000096:biosynthesis*; D004789:Enzyme Activation; D005347:Fibroblasts / Q000187:drug effects; D010713:Phosphatidylcholines / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D013917:Thrombin / Q000494:pharmacology*
D004075:Diglycerides; D010713:Phosphatidylcholines; D010716:Phosphatidylinositols; D011493:Protein Kinase C; D013917:Thrombin; D004072:Digitonin