l-O-Hexadecyl-2-O-methyl-sn-glycero-3-phosphocholine inhibits diacylglycerol kinase in WEHI-3B cells

Abstract

The effects of 1-O-hexadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-16-OCH3-GPC) and its metabolite 1-O-hexadecyl-2-O-methyl-sn-glycerol (AMG) on the activity of diacylglycerol kinase (DGK) in WEHI-3B cells were investigated. Treatment of WEHI-3B cells with 200 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) for 5 min leads to the activation of cytosolic DGK without significant effect on microsomal DGK. When these cells were first exposed to 50 μM ET-16-OCH3-GPC for 30 min prior to activation with TPA, the activity of DGK was inhibited by about 70%, as measured by the ability of enzyme to form [32P]phosphatidic acid ([32P]PA). Addition of either ET-16-OCH3-GPC or AMG to the preparation of enzyme in vitro also inhibited 1,2-dioleoyl-sn-glycerol (DG) phosphorylation in the presence of [γ-32P]ATP. The IC50 value for inhibition of cytosolic DGK by ET-16-OCH3-GPC and AMG were about 8.5 and 15 μM, respectively. ET-16-OCH3-GPC also inhibited the ability of guanosine 5′-O-(3-thiophosphate) (GTP-7S) to activate DGK in vitro. The potency of ET-16-OCH3-GPC at 10 μM in inhibiting DGK was greater than that of sphingosine at 50 μM, but less than that of R59022 (a specific DGK inhibitor) at 10 μM. The abilities of ET-16-OCH3-GPC and AMG to inhibit cytosolic DGK in intact WEHI-3B cells and enzyme preparations in vitro suggest that the cytotoxic activity of ether lipids may in part result from interference with this vital enzyme involved in the synthesis of phospholipids from DG and in cell-signaling systems.Key words: diacylglycerol, kinases, second messengers, ether lipids, cancer, WEHI-3B cells.