C2-Ceramide inhibits phospholipase dl and d2 activities

We tested the hypothesis that the ceramide inhibition of PLD activity (J. Biol. Chem. 272, 1997, 1069-1075) would depend on the concentration of phosphatidylinositol 4,5-bisphosphate (PIP2)- Membranes from HL 60 cells were incubated with cytosoj and GTPyS. The inhibition of PLD activity by C2-ceramide depended on a decrease translocation of ADP ribpsylation factor (ARF) and PKC-a and -β to the membrane fraction. Inhibition of PLD activity was relatively greater when the phosphatidylcholine substrate contained suboptimal (3 and 6 \LM) rather than optimum concentrations (12 \iM) of PIP2. Higher PIP2 concentrations (18 and 24 |iM) produced lower PLD activity and in this case C2-ceramide stimulated PLD activity. This work was extended using recombinant human PLD1 and mouse PLD2 obtained from membranes of Sf9 insect cells transfected with the corresponding cDNA using baculovirus vectors. The overexpressed PLD1 and PLD2 were identified by Western blot analysis. As expected, recombinant PLD1 and PLD2 both required PIP2 for activity. In addition, PLD1 required ARF and GTPyS for its activity, whereas PLD2 did not. Both PLD1 and PLD2 activities were inhibited by C2-ceramide and this inhibition was greater at lower concentrations of PI?2. The inhibitions of both recombinant PLDs by supra-optimum concentrations of PIP2 were again reversed by C2-ceramide. Our work therefore shows that PLD2, as well as PLD1. is a potential target for ceramide inhibition. Furthermore, the ceramide effects on both PLDs depend upon PIP2 concentrations.