Ceramide inhibition of mammalian phospholipase D1 and D2 activities is antagonized by phosphatidylinositol 4,5-bisphosphate

Ceramides inhibit phospholipase D (PLD) activity in several mammalian cell types. These effects have been related to preventing activation by ARF1, RhoA, and protein kinase C-α and -β and therefore indicate that PLD1 is inhibited. In the present work, we investigated the effects of ceramides in inhibiting both PLD1 and PLD2 and the interaction with another activator, phosphatidylinositol 4,5-bisphosphate (PIP₂). PLD1 and PLD2 were overexpressed separately in Sf9 insect cells using baculovirus vectors. In our cell-free system, PLD1 activity was inhibited completely by C₂-ceramide at sub-optimum concentrations of PIP₂ (3 and 6 μM), whereas at supra-optimum PIP₂ concentrations (18 and 24 νM) C₂-ceramide did not inhibit PLD1 activity. Partially purified PLD2 exhibited an absolute requirement for PIP₂ when the activity was measured using Triton X-100 micelles. Ceramides inhibited PLD2 activity, and this inhibition was decreased as PIP₂ concentrations increased. However, C₂-ceramide also reversibly inhibited the activity of PLD1 and PLD2 mutants in which binding of PIP₂ was decreased, indicating that ceramides are interacting with the catalytic core of the mammalian PLDs. By contrast, C₂-ceramide failed to produce a significant inhibition of PLDs from bacteria and plants. Our results provide a novel demonstration that ceramides reversibly inhibit mammalian PLD2 as well as PLD1 activities and that both of these actions are more pronounced when PIP₂ concentrations are rate-limiting.