Direct proof that phorbol ester accelerates the use of choline phosphate for phosphatidylcholine synthesis in intact cells.
Phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinase C, stimulates both the cellular uptake of radiolabeled choline and the activity of CTP:phosphocholine cytidylyltransferase resulting in increased incorporation of choline into phosphatidylcholine (PtdCho). Because of these multiple actions, it is difficult to determine the contribution of de novo synthesis to PMA-stimulated incorporation of radiolabeled choline into PtdCho. To address this issue, in this work Ha-ras-transformed NIH 3T3 fibroblasts, which were found to readily accumulate radiolabeled choline phosphate from the medium, were used. PMA (100 nM) had no stimulatory effect on the uptake of [14C]choline phosphate by transformed cells, but it significantly (2.0- to 2.9-fold) enhanced the incorporation of this labeled precursor into cellular PtdCho during a 5-h incubation period. A well-detectable (approximately 1.45-fold) stimulatory effect on the incorporation of [14C]choline phosphate into PtdCho was obtained with 10 nM PMA, while maximal effects required 100 nM PMA. In transformed cells, PMA also stimulated incorporation of [14C]choline into PtdCho. However, when [14C]choline phosphate and [14C]choline were directly compared, PMA was found to exert slightly, but consistently, greater stimulatory effects on the incorporation of [14C]choline phosphate into PtdCho. The protein kinase C inhibitor GF 109203X inhibited PMA-induced synthesis of PtdCho from both [14C]choline and [14C]choline phosphate. These results directly demonstrate that PMA can stimulate PtdCho synthesis through the protein kinase C system at a step subsequent to the uptake of choline and the action of choline kinase.
Hormel Institute, University of Minnesota, Austin 55912, USA.
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Kiss
D016475:3T3 Cells; D000818:Animals; D002250:Carbon Radioisotopes; D002794:Choline / Q000378:metabolism*; D019916:Choline-Phosphate Cytidylyltransferase; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D007700:Kinetics; D008301:Maleimides / Q000494:pharmacology; D051379:Mice; D009713:Nucleotidyltransferases / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000096:biosynthesis*; D010767:Phosphorylcholine / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011865:Radioisotope Dilution Technique; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D002250:Carbon Radioisotopes; D004791:Enzyme Inhibitors; D007211:Indoles; D008301:Maleimides; D010713:Phosphatidylcholines; D010767:Phosphorylcholine; D011493:Protein Kinase C; D009713:Nucleotidyltransferases; D019916:Choline-Phosphate Cytidylyltransferase; C070515:bisindolylmaleimide I; D002794:Choline; D013755:Tetradecanoylphorbol Acetate