Interleukin-2 causes an increase in saturated monounsaturated phosphatidicacid derived from 1,2-diacylglycerol and 1-O-alkyl-2-acylglycerol

Phosphatidic acid generation through activation of diacylglycerol kinase cu has been implicated in interleukin-2-dependent T-lymphocyte proliferation. To investigate this lipid signaling in more detail, we characterized the m olecular structures of the diradylglycerols and phosphatidic acids in the m urine CTLL-2 T-cell line under both basal and stimulated conditions. In res ting cells, 1,2-diacylglycerol and 1-O-alkyl-2-acylglycerol subtypes repres ented 44 and 55% of total diradylglycerol, respectively, and both showed a highly saturated profile containing primarily 16:0 and 18:1 fatty acids. 1- O-Alk-1'-enyl-2-acylglycerol represented 1-2% of total diradylglycerol, Int erleukin-2 stimulation did not alter the molecular species profiles, howeve r, it did selectively reduce total 1-O-alkyl-2-acylglycerol by over 50% at 15 min while only causing a 10% drop in 1,2-diacylglycerol, When radiolabel ed CTLL-2 cells were challenged with interleukin-a, no change in the cellul ar content of phosphatidylcholine nor phosphatidylethanolamine was observed thereby ruling out phospholipase C activity as the source of diradylglycer ol, In addition, interleukin-a failed to stimulate de novo synthesis of dir adylglycerol, Structural analysis revealed approximately equal amounts of 1 ,2-diacyl phosphatidic acid and 1-O-alkyl-2-acyl phosphatidic acid under re sting conditions, both containing only saturated and monounsaturated fatty acids. After acute (2 and 15 min) interleukin-a stimulation the total phosp hatidic acid mass increased, almost entirely through the formation of 1-O-a lkyl-2-acyl species. In vitro assays revealed that both 1,2-diacylglycerol and 1-O-alkyl-2-acylglycerol were substrates for 1,2-diacylglycerol kinase cu, the major isoform in CTLL-2 cells, and that the lipid kinase activity w as almost totally inhibited by R59949, In conclusion, this investigation sh ows that, in CTLL-2 cells, 1,2-diacylglycerol kinase ct specifically phosph orylates a pre-existing pool of 1-O-alkyl-2-acylglycerol to form the intrac ellular messenger 1-O-alkyl-2-acyl phosphatidic acid.