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 ofde novosynthesis 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 (∼1.45-fold) stimulatory effect on the incorporation of [14C]choline phosphate into PtdCho was obtained with 10 nMPMA, while maximal effects required 100 nMPMA. 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.