Maintenance of Cdc42 GDP-bound state by Rho-GDI inhibits MAP kinase activation by the exchange factor Ras-GRF - Evidence for Ras-GRF function being inhibited by Cdc42-GDP but unaffected by Cdc42-GTP

The function of the Pas guanine nucleotide exchange factor Ras-GRF/cdc25(Mn ) is subject to tight regulatory processes. We have recently shown that the activation of the Ras/MAPK pathway by Ras-GRF is controlled by the Rho fam ily GTPase Cdc42 through still unknown mechanisms. Here, we report that ret aining Cdc42 in its GDP-bound state by overexpressing Rho-GDI inhibits Ras- GRF-mediated MAPK activation. Conversely, Ras-GRF basal and LPA- or ionomyc in-stimulated activities were unaffected by a constitutively active GTP-bou nd Cdc42, Moreover, the Cdc42 downstream effecters MLK3, ACK1, PAK1, and WA SP had no detectable influence on Ras-GRF-mediated MAPK activation. In cont rast, promoting GDP release from Cdc42 with the Rho family GEF Dbl or with ionomycin suppressed the restraint exerted by Cdc42 on Ras-GRF activity. We conclude that Cdc42-GDP inhibits Ras-GRF-induced MAPK activation, but neit her Cdc42-GTP nor the Cdc42 downstream effecters affect Ras-GRF performance , Interestingly, the loss of the GDP-bound state by Cdc42 abolishes its inh ibitory effects on Ras-GRF function. These results suggest that the Cdc42 m echanism of action may not be solely restricted to activation of downstream signaling cascades when GTP-loaded, Furthermore, the GDP-bound form may be acting as an inhibitory molecule down-modulating parallel signaling routes such as the Ras/MAPK pathway.