Plasma membrane recruitment of RalGDS is critical for Ras-dependent Ral activation

In COS cells, Ral GDP dissociation stimulator (RalGDS)-induced Ral activation was stimulated by Ras(G12V) or a Rap1/Ras chimera in which the IV-terminal region of Rap1 was ligated to the C-terminal region of Ras but not by Rap1(G12V) or a Ras/Rap1 chimera in which the IV-terminal region of Ras was ligated to the C-terminal region of Rap1, although RalGDS interacted with these small GTP-binding proteins, When Ras(G12V), Ral and the Rap1/Ras chimera were individually expressed in NIH3T3 cells, they localized to the plasma membrane. Rap1(Q63E) and the Ras/Rap1 chimera were detected in the perinuclear region, When RalGDS was expressed alone, it was abundant in the cytoplasm, When coexpressed with Ras(G12V) or the Rap1/Ras chimera, RalGDS was detected at the plasma membrane, whereas when coexpressed with Rap1(Q63E) or the Ras/Rap1 chimera, RalGDS was observed in the perinuclear region. RalGDS which was targeted to the plasma membrane by the addition of Ras farnesylation site (RaLGDS-CAAX) activated Ral in the absence of Ras(G12V). Although RalGDS did not stimulate the dissociation of GDP from Ral in the absence of the GTP-bound form of Ras in a reconstitution assay using the liposomes, RalGDS-CAAX could stimulate it without Pas, Ras(G12V) activated Raf-1 when they were coexpressed in Sf9 cells, whereas Ras(G12V) did not affect the RalGDS activity, These results indicate that Pas recruits RalGDS to the plasma membrane and that the translocated RalGDS induces the activation of Ral, but that Rap1 does not activate Pal due to distinct subcellular localization.