SPECIFIC-INHIBITION OF PHORBOL ESTER-STIMULATED PHOSPHOLIPASE-D BY CLOSTRIDIUM-SORDELLII LETHAL TOXIN AND CLOSTRIDIUM-DIFFICILE TOXIN B-1470 IN HEK-293 CELLS - RESTORATION BY RAL GTPASES

Activation of m3 muscarinic acetylcholine receptor (mAChR), stably exp ressed in human embryonic kidney (HEK)-293 cells, leads to phospholipa se D (PtD) stimulation, a process apparently involving Rho GTPases, as shown by studies with Clostridium botulinum C3 exoenzyme and Clostrid ium difficile toxin B (TcdB). Direct activation of protein kinase C (P HC) by phorbol esters, such as phorbol 12-myristate 13-acetate (PMA), also induces PLD stimulation, which is additive to the mAChR action an d which is only poorly sensitive to inactivation of Rho proteins by Tc dB. To study whether Ras-like GTPases are involved in PLD regulation, we studied the effects of the TcdB variant TcdB-1470 and Clostridium s ordellii lethal toxin (Test), known to inactivate Rac and some members of the Bus protein family, on PLD activities. TcdB-1470 and Test did not affect basal PLD activity and PLD stimulation by mAChR or direct G ; protein activation. In contrast, PMA-induced PtD stimulation was inh ibited by TcdB-1470 and Test in a time- and concentration-dependent ma nner, without alteration in immunologically detectable PKC isozyme lev els. In membranes of HEK-293 cells pretreated with TcdB-1470 or Test, basal and stable GTP analog-stimulated PLD activities measured with ex ogenous phosphatidylcholine, in the presence or absence of phosphatidy linositol 4,5-bisphosphate, were not altered, In contrast, pretreatmen t with TcdB-1470 and Test, but not TcdB, strongly reduced PMA-stimuIat ed PLD activity. The addition of recombinant Rad, serving as glucosyla tion substrate for TcdB, Test, and TcdB-1470, did not restore PtD stim ulation by PMA. Furthermore, PMA-stimulated PLD activity, suppressed b y prior treatment with TcdB-1470 or Test, was not rescued by the addit ion of recombinant Ras (RasG12V) or Rap proteins, acting as glucosylat ion substrates for Test only (Das) or TcdB-1470 and Test (Rap). In con trast, the addition of recombinant Ral proteins (RalA and RalB), gluco sylation substrates for TscL and TcdB-1470, but not for TcdB, to membr anes of TcdB-1470- or TcsL-treated cells fully restored PtD stimulatio n by PMA. without altering the strict MgATP dependence of PMA-induced PLD stimulation. RalA-mediated restoration of PMA-stimulated PLD activ ity in membranes of Test-treated cells was not enhanced by coaddition of RasG12V. In conclusion, the data presented indicate that TcdB-1470 and Test selectively interfere with phorbol ester stimulation of PLD a nd suggest an essential role of Ral proteins in PKC signaling to PLD i n HEK-293 cells.