| 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. | |
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MedLine Citation:
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PMID: 9516439 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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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. |
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Authors:
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M Schmidt; M Voss; M Thiel; B Bauer; A Grannass; E Tapp; R H Cool; J de Gunzburg; C von Eichel-Streiber; K H Jakobs |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't |
Journal Detail:
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Title: The Journal of biological chemistry Volume: 273 ISSN: 0021-9258 ISO Abbreviation: J. Biol. Chem. Publication Date: 1998 Mar |
Date Detail:
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Created Date: 1998-04-23 Completed Date: 1998-04-23 Revised Date: 2007-11-15 |
Medline Journal Info:
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Nlm Unique ID: 2985121R Medline TA: J Biol Chem Country: UNITED STATES |
Other Details:
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Languages: eng Pagination: 7413-22 Citation Subset: IM |
Affiliation:
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Institut für Pharmakologie, Universitätsklinikum Essen, D-45122 Essen, Germany. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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3T3 Cells Animals Bacterial Proteins* Bacterial Toxins / pharmacology* Cell Line Clostridium Clostridium difficile Enzyme Activation / drug effects GTP Phosphohydrolases / metabolism* GTP-Binding Proteins / metabolism* Glucosyltransferases / metabolism* Humans Mice Phospholipase D / metabolism* Protein Kinase C / metabolism Receptor, Muscarinic M3 Receptors, Muscarinic / metabolism Signal Transduction Tetradecanoylphorbol Acetate / pharmacology* ral GTP-Binding Proteins |
| Chemical | |
Reg. No./Substance:
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0/Bacterial Proteins; 0/Bacterial Toxins; 0/Receptor, Muscarinic M3; 0/Receptors, Muscarinic; 0/lethal toxin LT, Clostridium sordellii; 0/toxB protein, Clostridium difficile; 16561-29-8/Tetradecanoylphorbol Acetate; EC 2.4.1.-/Glucosyltransferases; EC 2.7.11.13/Protein Kinase C; EC 3.1.4.4/Phospholipase D; EC 3.6.1.-/GTP Phosphohydrolases; EC 3.6.1.-/GTP-Binding Proteins; EC 3.6.5.2/ral GTP-Binding Proteins |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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