Regulation of phospholipase D activity and phosphatidic acid production after purinergic (P2Y₆) receptor stimulation

Phosphatidic acid (PA) is a lipid second messenger located at the intersection of several lipid metabolism and cell signaling events including membrane trafficking, survival, and proliferation. Generation of signaling PA has long been primarily attributed to the activation of phospholipase D (PLD). PLD catalyzes the hydrolysis of phosphatidylcholine into PA. A variety of both receptor-tyrosine kinase and G-protein-coupled receptor stimulations have been shown to lead to PLD activation and PA generation. This study focuses on profiling the PA pool upon P2Y₆ receptor signaling manipulation to determine the major PA producing enzymes. Here we show that PLD, although highly active, is not responsible for the majority of stable PA being produced upon UDP stimulation of the P2Y₆ receptor and that PA levels are tightly regulated. By following PA flux in the cell we show that PLD is involved in an initial increase in PA upon receptor stimulation; however, when PLD is blocked, the cell compensates by increasing PA production from other sources. We further delineate the P2Y₆ signaling pathway showing that phospholipase Cβ3 (PLCβ3), PLCβ1, DGKζ and PLD are all downstream of receptor activation. We also show that DGKζ is a novel negative regulator of PLD activity in this system that occurs through an inhibitory mechanism with PKCα. These results further define the downstream events resulting in PA production in the P2Y₆ receptor signaling pathway.