Abstract
Activation of m3 muscarinic acetylcholine receptor (mAChR), stably expressed in human embryonic kidney (HEK)-293 cells, leads to phospholipase D (PLD) stimulation, a process apparently involving Rho GTPases, as shown by studies with Clostridium botulinum C3 exoenzyme and Clostridium difficile toxin B (TcdB). Direct activation of protein kinase C (PKC) by phorbol esters, such as phorbol 12-myristate 13-acetate (PMA), also induces PLD stimulation, which is additive to the mAChR action and which is only poorly sensitive to inactivation of Rho proteins by TcdB. To study whether Ras-like GTPases are involved in PLD regulation, we studied the effects of the TcdB variant TcdB-1470 and Clostridium sordellii lethal toxin (TcsL), known to inactivate Rac and some members of the Ras protein family, on PLD activities. TcdB-1470 and TcsL did not affect basal PLD activity and PLD stimulation by mAChR or direct G protein activation. In contrast, PMA-induced PLD stimulation was inhibited by TcdB-1470 and TcsL in a time- and concentration-dependent manner, without alteration in immunologically detectable PKC isozyme levels. In membranes of HEK-293 cells pretreated with TcdB-1470 or TcsL, basal and stable GTP analog-stimulated PLD activities measured with exogenous phosphatidylcholine, in the presence or absence of phosphatidylinositol 4,5-bisphosphate, were not altered. In contrast, pretreatment with TcdB-1470 and TcsL, but not TcdB, strongly reduced PMA-stimulated PLD activity. The addition of recombinant Rac1, serving as glucosylation substrate for TcdB, TcsL, and TcdB-1470, did not restore PLD stimulation by PMA. Furthermore, PMA-stimulated PLD activity, suppressed by prior treatment with TcdB-1470 or TcsL, was not rescued by the addition of recombinant Ras (RasG12V) or Rap proteins, acting as glucosylation substrates for TcsL only (Ras) or TcdB-1470 and TcsL (Rap). In contrast, the addition of recombinant Ral proteins (RalA and RalB), glucosylation substrates for TscL and TcdB-1470, but not for TcdB, to membranes of TcdB-1470- or TcsL-treated cells fully restored PLD stimulation by PMA without altering the strict MgATP dependence of PMA-induced PLD stimulation. RalA-mediated restoration of PMA-stimulated PLD activity in membranes of TcsL-treated cells was not enhanced by coaddition of RasG12V. In conclusion, the data presented indicate that TcdB-1470 and TcsL selectively interfere with phorbol ester stimulation of PLD and suggest an essential role of Ral proteins in PKC signaling to PLD in HEK-293 cells.
Highlights
Activation of m3 muscarinic acetylcholine receptor, stably expressed in human embryonic kidney (HEK)-293 cells, leads to phospholipase D (PLD) stimulation, a process apparently involving Rho GTPases, as shown by studies with Clostridium botulinum C3 exoenzyme and Clostridium difficile toxin B (TcdB)
We demonstrate here that TcsL and TcdB-1470, which glucosylates Rac, Rap, and Ral GTPases, potently and selectively inhibit phorbol 12-myristate 13-acetate (PMA)-induced PLD stimulation in HEK-293 cells and that this inhibition is selectively reversed by Ral proteins, suggesting an essential role for Ral GTPases in protein kinase C (PKC) signaling to PLD in HEK-293 cells
We have demonstrated that PLD stimulation in HEK-293 cells by the G protein-coupled m3 muscarinic acetylcholine receptor (mAChR) and phorbol ester-activated PKC are apparently independent and that m3 mAChR- but not PKC-mediated PLD stimulation involves ADP-ribosylation factor (ARF) and Rho proteins (17, 27, 42, 43)
Summary
(Received for publication, April 30, 1997, and in revised form, January 7, 1998). Martina Schmidt‡, Matthias Vo߇, Markus Thiel‡, Bettina Bauer§, Andreas Granna߇, Eva Tapp‡, Robbert H. GTPase cascades are apparently involved in cell proliferation and cytoskeleton organization, but they may participate in PLD activation (34) Another pathway for PLD stimulation involves isozymes of the protein kinase C (PKC) family, members of the classical forms of PKC, as shown by studies in human neutrophils, in membranes of porcine brain, and with purified PLD (7, 35, 36). We have recently shown that mAChR-mediated PLD stimulation in these cells involves small molecular weight GTPases of both the ARF and Rho protein families (17, 27). Direct activation of PKC by phorbol esters, such as phorbol 12-myristate 13-acetate (PMA), induces marked PLD stimulation in HEK-293 cells, which is additive to the mAChR action and apparently does not involve ARF (17, 42, 43). We demonstrate here that TcsL and TcdB-1470, which glucosylates Rac, Rap, and Ral GTPases, potently and selectively inhibit PMA-induced PLD stimulation in HEK-293 cells and that this inhibition is selectively reversed by Ral proteins, suggesting an essential role for Ral GTPases in PKC signaling to PLD in HEK-293 cells
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