Influence of phosphatidylinositol 4,5-bisphosphate on human phospholipase D1 wild-type and deletion mutants: is there evidence for an interaction of phosphatidylinositol 4,5-bisphosphate with the putative Pleckstrin homology domain?

Abstract Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) is an essential cofactor of phospholipase D (PLD) enzymes. In order to further characterize its role in PLD activation, we have constructed N-terminal deletion mutants of the human PLD1 (hPLD1) and a mutant lacking the putative pleckstrin homology domain (ΔPH), which has been proposed to be involved in PIP 2 binding. For the N-terminal deletion mutants (up to 303 amino acids) and the ΔPH mutant we found no significant differences compared to the hPLD1 wild-type, except changes in the specific activities: the K m values were about 20 μM for the substrate phosphatidylcholine, and PIP 2 activated the PLD enzymes maximally between 5 and 10 μM. In contrast, preincubation of the PLD proteins with 5–10 μM PIP 2 or PIP 2 -containing lipid vesicles inhibited the PLD activity. This inhibition was neither abolished by n -octyl-β- D -glucopyranoside or neomycin nor by the ADP-ribosylation factor, another activator of PLD enzymes. All tested PLD proteins were active without PIP 2 in the presence of 1 M ammonium sulfate. The 303 N-terminal amino acids of hPLD1 are not involved in substrate binding or the interaction with PIP 2 . Our data indicate further that the putative PH domain of hPLD1 is not responsible for the essential effects of PIP 2 on PLD activity.