RESTORATION OF CLOSTRIDIUM-DIFFICILE TOXIN-B-INHIBITED PHOSPHOLIPASE-D BY PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE

Receptor signalling to phospholipase D (PLD) in human embryonic kidney (HEK) cells stably expressing the m3 muscarinic acetylcholine recepto r apparently involves Rho proteins. Since phosphatidylinositol 4,5-bis phosphate [PtdIns(4,5)P-2] has been recognized as an essential cofacto r for PLD activity and since activated Rho proteins have been reported to stimulate the synthesis of PtdIns(4,5)P-2, we studied whether in H EK cells PLD activity is regulated by PtdIns(4,5)P-2 and, in particula r, whether PtdIns(4,5)P-2 can restore PLD activity inhibited by Clostr idium difficile toxin B, which inactivates Rho proteins. Addition of M gATP to permeabilized HEK cells increased basal PLD activity and poten tiated PLD stimulation by the stable GTP analogue, guanosine 5'-[gamma -thio]triphosphate (GTP[S]), concomitant with a large increase in PtdI ns(4,5)P-2. On the other hand, neomycin, which binds to PtdIns(4,5)P-2 , inhibited basal and GTP[S]-stimulated PLD activities. Addition of Pt dIns(4,5)P-2 increased PLD activity in HEK cell membranes by 2-3-fold, whereas various other phospholipids were ineffective. Prior treatment of KEK cells with toxin B reduced the level of PtdIns(4,5)P-2, measur ed either in intact cells or in membrane preparations, by about 40%. I n membranes of toxin-B-treated cells, basal and GTP[S]-stimulated PLD activities were reduced, when measured with exogenous phosphatidylchol ine as enzyme substrate. Inclusion of PtdIns(4,5)P-2 with phosphatidyl choline in the substrate vesicles or addition of PtdIns(4,5)P-2 fully restored basal and GTP[S]-stimulated PLD activities in membranes of to xin-B-treated cells. In conclusion, the data indicate that PtdIns(4,5) P-2 is an essential cofactor for PLD activity in HEK cells and that in hibition of PLD activity by the Rho-inactivating toxin B is apparently caused by depletion of the PLD cofactor, PtdIns(4,5)P-2.