Inhibition of small G proteins by Clostridium sordellii lethal toxin activates cdc2 and MAP kinase in Xenopus oocytes

The lethal toxin (LT) from Clostridium sordellii is a glucosyltransferase t hat modifies and inhibits small G proteins of the Ras family, Ras and Rap, as well as Rac proteins. LT induces cdc2 kinase activation and germinal ves icle breakdown (GVBD) when microinjected into full-grown Xenopus oocytes. T oxin B from Clostridium difficile, that glucosylates and inactivates Rac pr oteins, does not induce cdc2 activation, indicating that proteins of the Ra s family, Ras and/or Rap, negatively regulate cdc2 kinase activation in Xen opus oocyte. In oocyte extracts, LT catalyzes the incorporation of [C-14]gl ucose into a group of proteins of 23 kDa and into one protein of 27 kDa. Th e 23-kDa proteins are recognized by anti-Rap1 and anti-Rap2 antibodies, whe reas the 27-kDa protein is recognized by several anti-Ras antibodies and pr obably corresponds to K-Ras. Microinjection of LT into oocytes together wit h UDP-[C-14]glucose results in a glucosylation pattern similar to the in vi tro glucosylation, indicating that the 23- and 27-kDa proteins are in vivo substrates of LT. In vivo time-course analysis reveals that the 27-kDa prot ein glucosylation is completed within 2 h, well before cdc2 kinase activati on, whereas the 23-kDa proteins are partially glucosylated at GVBD. This ob servation suggests that the 27-kDa Ras protein could be the in vivo target of LT allowing cdc2 kinase activation. Interestingly, inactivation of Ras p roteins does not prevent the phosphorylation of c-Raf1 and the activation o f MAP kinase that occurs normally around GVBD. (C) 1998 Academic Press.