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?
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 (delta PH), which has been proposed to be involved in PIP(2) binding. For the N-terminal deletion mutants (up to 303 amino acids) and the delta PH 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 microM for the substrate phosphatidylcholine, and PIP(2) activated the PLD enzymes maximally between 5 and 10 microM. In contrast, preincubation of the PLD proteins with 5-10 microM PIP(2) or PIP(2)-containing lipid vesicles inhibited the PLD activity. This inhibition was neither abolished by n-octyl-beta-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.
Höer
A
A
Institut für Pharmakologie, Fachbereich Humanmedizin der Freien Universität Berlin, Thielallee 67-73, 14195 Berlin, Germany. adhoer@zedat.fu-berlin.de
Cetindag
C
C
Oberdisse
E
E
D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D001798:Blood Proteins / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004789:Enzyme Activation / Q000187:drug effects; D017353:Gene Deletion; D005982:Glutathione Transferase / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D009154:Mutation; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000494:pharmacology*; D010739:Phospholipase D / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D018411:Spodoptera
D001798:Blood Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C039077:platelet protein P47; D005982:Glutathione Transferase; D010739:Phospholipase D; C106801:phospholipase D1