title: 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.
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.
authors.affiliation: Institut für Pharmakologie, Universitätsklinikum Essen, D-45122 Essen, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Schmidt
authors.forename: M
authors.initials: M
authors.lastname: Voss
authors.forename: M
authors.initials: M
authors.lastname: Thiel
authors.forename: B
authors.initials: B
authors.lastname: Bauer
authors.forename: A
authors.initials: A
authors.lastname: Grannass
authors.forename: E
authors.initials: E
authors.lastname: Tapp
authors.forename: R H
authors.initials: RH
authors.lastname: Cool
authors.forename: J
authors.initials: J
authors.lastname: de Gunzburg
authors.forename: C
authors.initials: C
authors.lastname: von Eichel-Streiber
authors.forename: K H
authors.initials: KH
authors.lastname: Jakobs
mesh_terms: D016475:3T3 Cells; D000818:Animals; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000494:pharmacology*; D002460:Cell Line; D016360:Clostridioides difficile; D003013:Clostridium; D004789:Enzyme Activation / Q000187:drug effects; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005964:Glucosyltransferases / Q000378:metabolism*; D006801:Humans; D051379:Mice; D010739:Phospholipase D / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic / Q000378:metabolism; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D020725:ral GTP-Binding Proteins
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic; C072292:lethal toxin LT, Clostridium sordellii; C031815:toxB protein, Clostridium difficile; D005964:Glucosyltransferases; D011493:Protein Kinase C; D010739:Phospholipase D; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020725:ral GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate