Analysis of the Ras p21/mitogen-activated protein kinase signaling in vitro and in Xenopus oocytes

Abstract

Ras p21 in the GTP-bound form was shown to act as an upstream activator for mitogen-activated protein (MAP) kinase kinase (MAPKK) and MAP kinase, and Raf-1 was reported to act as a MAPKK kinase. Further, physical association between Ras and Raf-1 was demonstrated. Here we have shown that incubation of Xenopus immature oocyte extracts with Ras enhances the ability of endogenous Raf-1 to activate MAPKK. Moreover, a dominant negative form of Raf-1 blocked the Ras-induced activation of MAPKK and MAP kinase in the extracts, but not the cyclin A-dependent activation of MAP kinase. When the extracts were depleted of 45-kDa MAPKK with polyclonal anti-MAPKK antibody, no activation of MAP kinase occurred even after incubation with Ras. These results suggest that Ras can activate the MAPKK kinase activity of Raf-1 in the extracts and that MAPKK is indispensable for the Ras-induced MAP kinase activation. It is well known that Ras can induce oocyte maturation when injected into immature Xenopus oocytes. Co-injection of Ras with an anti-MAPKK antibody that inhibits the MAPKK activity prevented the Ras-induced germinal vesicle breakdown, suggesting that MAPKK mediates, at least, one of cellular functions of Ras.