Interleukin-2 causes an increase in saturated/monounsaturated phosphatidic acid derived from 1,2-diacylglycerol and 1-O-alkyl-2-acylglycerol.
Phosphatidic acid generation through activation of diacylglycerol kinase alpha has been implicated in interleukin-2-dependent T-lymphocyte proliferation. To investigate this lipid signaling in more detail, we characterized the molecular structures of the diradylglycerols and phosphatidic acids in the murine CTLL-2 T-cell line under both basal and stimulated conditions. In resting cells, 1,2-diacylglycerol and 1-O-alkyl-2-acylglycerol subtypes represented 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. Interleukin-2 stimulation did not alter the molecular species profiles, however, 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 radiolabeled CTLL-2 cells were challenged with interleukin-2, no change in the cellular content of phosphatidylcholine nor phosphatidylethanolamine was observed thereby ruling out phospholipase C activity as the source of diradylglycerol. In addition, interleukin-2 failed to stimulate de novo synthesis of diradylglycerol. Structural analysis revealed approximately equal amounts of 1,2-diacyl phosphatidic acid and 1-O-alkyl-2-acyl phosphatidic acid under resting conditions, both containing only saturated and monounsaturated fatty acids. After acute (2 and 15 min) interleukin-2 stimulation the total phosphatidic acid mass increased, almost entirely through the formation of 1-O-alkyl-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 alpha, the major isoform in CTLL-2 cells, and that the lipid kinase activity was almost totally inhibited by R59949. In conclusion, this investigation shows that, in CTLL-2 cells, 1,2-diacylglycerol kinase alpha specifically phosphorylates a pre-existing pool of 1-O-alkyl-2-acylglycerol to form the intracellular messenger 1-O-alkyl-2-acyl phosphatidic acid.
Jones
D R
DR
Department of Immunology and Oncology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Cantoblanco, 28049, Madrid, Spain.
Pettitt
T R
TR
Sanjuán
M A
MA
Mérida
I
I
Wakelam
M J
MJ
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D019852:Diacylglycerol Kinase / Q000378:metabolism; D004075:Diglycerides / Q000096:biosynthesis / Q000378:metabolism*; D005227:Fatty Acids / Q000032:analysis / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000032:analysis / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D007376:Interleukin-2 / Q000494:pharmacology*; D007527:Isoenzymes / Q000378:metabolism; D051379:Mice; D010712:Phosphatidic Acids / Q000096:biosynthesis* / Q000737:chemistry; D013601:T-Lymphocytes / Q000378:metabolism*
C011439:1,2-diacylglycerol; D004075:Diglycerides; D005227:Fatty Acids; D005231:Fatty Acids, Unsaturated; D007376:Interleukin-2; D007527:Isoenzymes; D010712:Phosphatidic Acids; C035561:diarachidonyl diglyceride; D019852:Diacylglycerol Kinase