Inhibition of Small G Proteins byClostridium sordelliiLethal Toxin Activates cdc2 and MAP kinase inXenopusOocytes

Abstract

The lethal toxin (LT) from Clostridium sordellii is a glucosyltransferase that 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 vesicle breakdown (GVBD) when microinjected into full-grown Xenopus oocytes. Toxin B from Clostridium difficile, that glucosylates and inactivates Rac proteins, does not induce cdc2 activation, indicating that proteins of the Ras family, Ras and/or Rap, negatively regulate cdc2 kinase activation in Xenopus oocyte. In oocyte extracts, LT catalyzes the incorporation of [14C]glucose into a group of proteins of 23 kDa and into one protein of 27 kDa. The 23-kDa proteins are recognized by anti-Rap1 and anti-Rap2 antibodies, whereas the 27-kDa protein is recognized by several anti-Ras antibodies and probably corresponds to K-Ras. Microinjection of LT into oocytes together with UDP-[14C]glucose results in a glucosylation pattern similar to the in vitro 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 protein glucosylation is completed within 2 h, well before cdc2 kinase activation, whereas the 23-kDa proteins are partially glucosylated at GVBD. This observation suggests that the 27-kDa Ras protein could be the in vivo target of LT allowing cdc2 kinase activation. Interestingly, inactivation of Ras proteins does not prevent the phosphorylation of c-Raf1 and the activation of MAP kinase that occurs normally around GVBD.

Regulation of Xenopus p21-activated Kinase (X-PAK2) by Cdc42 and Maturation-promoting Factor Controls Xenopus Oocyte Maturation

“…Recently, using lethal toxin, a glucosyl transferase that inactivates a large number of small G proteins, Rime et al (40) demonstrated that glucosylation of small GTPases spontaneously induces Xenopus oocyte maturation. In contrast, using toxin B, which inhibits more specifically Rho, Rac, and Cdc42 GTPase members, Rime et al (40) no longer observed spontaneous maturation, only major cytoplasmic rearrangements. The authors conclude that the maturation pathway in oocytes must involve the inhibition of endogenous Ras or Rap members.…”

“…In our model, such an event should start only once germinal vesicle breakdown has occurred; otherwise, germinal vesicle migration may occur in the absence of a spindle. It is interesting in this regard to note that Rime et al (40) observed strong cytoskeleton rearrangements upon treatment of the oocytes with Toxin B. We observed ourselves that microinjection of Cdc42N17, an inactive GDP-bound mutant, although it does not induce GVBD by itself in the absence of hormonal treatment, frequently induces migration of the intact germinal vesicle to the cortex (as verified by fixing and dissection of the oocyte), which induced ballooning of the oocyte at the animal cortex (data not shown).…”

Mos activates MAP kinase in mouse oocytes through two opposite pathways

“…Thus, the activation of MAPK by oncogenic Ras would occur through the classical Raf‐1/MEK1 pathway independently of c‐Mos synthesis (Daar et al ., 1991; Fabian et al ., 1993; Muslin et al ., 1993; Fukuda et al ., 1994). Whereas exogenous forms of Ras or Raf‐1 induce MAPK activation and meiosis resumption, the inhibition of endogenous small G proteins of the Ras family by the lethal toxin from Clostridium sordellii triggers meiotic maturation (Rime et al ., 1998). Despite all the studies performed in Xenopus , the relationships between the Mos and the Ras/Raf‐1 pathways, as well as the role of small G proteins of the Ras family in triggering MAPK activation and meiosis resumption, still require further investigations.…”

From progesterone to active Cdc2 in Xenopus oocytes: a puzzling signalling pathway

“…Some of the possible players implicated downstream PKA are the small GTPases of the Ras family Rime et al, 1998). Those proteins act as molecular switches in the transduction of many extracellular signals (Barbacid, 1987).…”