Diacylglycerol signals the translocation of CTP:choline-phosphate cytidylyltransferase in HeLa cells treated with 12-O-tetradecanoylphorbol-13-acetate.
The mechanism of 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated phosphatidylcholine biosynthesis in HeLa cells was investigated. TPA caused a 3-fold increase in particulate CTP:phosphocholine cytidylyltransferase activity in HeLa cells which correlated with decreased cytidylyltransferase activity in the cytosol. The increase in membrane-associated cytidylyltransferase was confirmed by immunoblotting. Immunoprecipitation studies suggested that TPA had no effect on the phosphorylation state of cytidylyltransferase. Enhanced binding of cytidylyltransferase to diacylglycerol-enriched membranes has previously been shown. Diacylglycerol levels in TPA-treated HeLa cells increased approximately 2-fold (2.29 to 4.02 nmol/mg of protein) after 1 h of TPA treatment. A time course experiment showed a temporal relationship in which production of diacylglycerol appeared to signal translocation of cytidylyltransferase to membranes followed by a stimulation of phosphatidylcholine biosynthesis. Diacylglycerol was further evaluated as a translocator of cytidylyltransferase by depleting HeLa cells of protein kinase C and incubating with dioctanoylglcerol. This treatment increased both membrane-associated cytidylyltransferase activity and the rate of phosphatidylcholine biosynthesis approximately 2-fold. A time course experiment with dioctanoylglycerol showed a strong positive correlation (r2 = 0.89) between the amount of particulate cytidylyltransferase activity and the rate of phosphatidylcholine biosynthesis. Therefore, TPA stimulates phosphatidylcholine biosynthesis by causing a translocation of cytidylyltransferase from the cytosol to membranes, which appears to be mediated by increased diacylglycerol.
Utal
A K
AK
Lipid and Lipoprotein Research Group, University of Alberta, Edmonton, Canada.
Jamil
H
H
Vance
D E
DE
D002463:Cell Membrane Permeability; D019916:Choline-Phosphate Cytidylyltransferase; D004072:Digitonin; D004075:Diglycerides / Q000302:isolation & purification / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D009713:Nucleotidyltransferases / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D004075:Diglycerides; D010713:Phosphatidylcholines; D011493:Protein Kinase C; D009713:Nucleotidyltransferases; D019916:Choline-Phosphate Cytidylyltransferase; D004072:Digitonin; D013755:Tetradecanoylphorbol Acetate