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, Rh oA, and protein kinase C-alpha and -beta and therefore indicate that PLD1 i s 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 (PIPA PLD I and PLD2 were overexpre ssed separately in Sf9 insect cells using baculovirus vectors. In our cell- free system, PLD1 activity was inhibited completely by C-2-ceramide at sub- optimum concentrations of PIP2 (3 and 6 muM), whereas at supra-optimum PIP2 concentrations (18 and 24 muM) 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 PLD 2 activity, and this inhibition was decreased as PIP2 concentrations increa sed. However, C2-ceramide also reversibly inhibited the activity of PLD1 an d PLD2 mutants in which binding Of PIP2 was decreased, indicating that cera mides are interacting with the catalytic core of the mammalian PLDs. By con trast, C2-ceramide failed to produce a significant inhibition of PLDs from bacteria and plants. Our results provide a novel demonstration that ceramid es reversibly inhibit mammalian PLD2 as well as PLD1 activities and that bo th of these actions are more pronounced when PIP2 concentrations are rate-l imiting.