Ras, Rap, and Rac Small GTP-binding Proteins Are Targets for Clostridium sordellii Lethal Toxin Glucosylation (*)
- Michel R. Popoff(1),
- Esteban Chaves-Olarte(2),
- Emmanuel Lemichez(1)(4),
- Christoph von Eichel-Streiber(6),
- Monica Thelestam(2),
- Pierre Chardin(3),
- Didier Cussac(3),
- Bruno Antonny(3),
- Philippe Chavrier(5),
- Gilles Flatau(4),
- Murielle Giry(1),
- Jean de Gunzburg(7) and
- Patrice Boquet(1)(§)(4)
- From the (1)Institut Pasteur, Unité des Toxines Microbiennes, 75724 Paris, Cedex 15, France, the
- (2)Microbiology and Tumorbiology Center, Karolinska Institute, P. O. Box 280, S171-77 Stockholm, Sweden,
- (3)CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, Sophia-Antipolis, 06560 Valbonne, France,
- (4)INSERM, U452 Faculté de Médecine de Nice, 06107 Nice, Cedex 2, France,
- (5)Centre d'Immunologie Marseilles Luminy, Case 906, 13286 Marseille Cedex, France, the
- (6)Institut für Medizinische Mikrobiologie und Hygiene Verfugungsgebaude für Forschung und Entwicklung, Obere Zahlbacher Strasse 63, Johannes Gutenberg-Universität, 55101 Mainz, Federal Republic of Germany, and
- (7)INSERM U.248, 10 avenue de Verdun, 75010 Paris, France
- § To whom correspondence should be addressed. Tel.: 33-93-37-77-09; Fax: 33-93-53-35-09.
Abstract
Lethal toxin (LT) from Clostridium sordellii is one of the high molecular mass clostridial cytotoxins. On cultured cells, it causes a rounding of cell bodies and a disruption of actin stress fibers. We demonstrate that LT is a glucosyltransferase that uses UDP-Glc as a cofactor to covalently modify 21-kDa proteins both in vitro and in vivo. LT glucosylates Ras, Rap, and Rac. In Ras, threonine at position 35 was identified as the target amino acid glucosylated by LT. Other related members of the Ras GTPase superfamily, including RhoA, Cdc42, and Rab6, were not modified by LT. Incubation of serum-starved Swiss 3T3 cells with LT prevents the epidermal growth factor-induced phosphorylation of mitogen-activated protein kinases ERK1 and ERK2, indicating that the toxin blocks Ras function in vivo. We also demonstrate that LT acts inside the cell and that the glucosylation reaction is required to observe its dramatic effect on cell morphology. LT is thus a powerful tool to inhibit Ras function in vivo.
Footnotes
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↵* This work was supported in part by Swedish Medical Research Council Grant 05969 (to M. T.) and Deutsche Forschungsgemeinschaft Grant Ei 206/3-1 (to C. v. E.-S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- LT
-
C. sordellii lethal toxin
- GST
-
glutathione S-transferase
- MAP
-
mitogen-activated protein
- PBS
-
phosphate-buffered saline
- HPLC
-
high pressure liquid chromatography
- EGF
-
epidermal growth factor
- GTP
S -
guanosine 5′-3-O-(thio)triphosphate.
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↵2 Selzer, J., Just, I., Mann, M., and Aktories, K., Seventh European Workshop Conference on Bacterial Protein Toxins, Hindsgavl, Middelfart, Denmark, 7 2-7, 1995 (Poster Abstr. 50).
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↵3 C. von Eichel-Streiber, P. Boquet, M. Sauerborn, and M. Thelestam, manuscript in preparation.
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- Received August 2, 1995.
- Revision received January 16, 1996.
