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?

摘要

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.