Ras, Rap, and Rac small GTP-binding proteins are targets for Clostridium sordellii lethal toxin glucosylation.
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.
Popoff
M R
MR
Institut Pasteur, Unité des Toxines Microbiennes, 75724 Paris, Cedex 15, France.
Chaves-Olarte
E
E
Lemichez
E
E
von Eichel-Streiber
C
C
Thelestam
M
M
Chardin
P
P
Cussac
D
D
Antonny
B
B
Chavrier
P
P
Flatau
G
G
Giry
M
M
de Gunzburg
J
J
Boquet
P
P
D016475:3T3 Cells; D008841:Actin Cytoskeleton / Q000648:ultrastructure; D000199:Actins / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins / Q000378:metabolism* / Q000633:toxicity; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D003013:Clostridium / Q000472:pathogenicity*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D005964:Glucosyltransferases / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006367:HeLa Cells / Q000187:drug effects / Q000648:ultrastructure; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism; D014532:Uridine Diphosphate Glucose / Q000378:metabolism; D020744:rac GTP-Binding Proteins; D020668:rap GTP-Binding Proteins
D000199:Actins; D001427:Bacterial Toxins; C072292:lethal toxin LT, Clostridium sordellii; D013912:Threonine; D004815:Epidermal Growth Factor; D006160:Guanosine Triphosphate; D005964:Glucosyltransferases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D020744:rac GTP-Binding Proteins; D020668:rap GTP-Binding Proteins; D005947:Glucose; D014532:Uridine Diphosphate Glucose