Interdependent action of RalGEF and Erk in Ras-induced primitive endoderm differentiation of F9 embryonal carcinoma cells

Previous work by us and others has implicated a role for Ral guanine exchan ge factors (RalGEFs) in Ras-induced cell growth and oncogenic transformatio n. Here we show for the first time that RalGEFs are involved in Ras-induced differentiation as well. Expression of oncogenic Ras in F9 embryonal carci noma (EC) cells is known to induce differentiation to a primitive endoderm (PrE)-like phenotype, but the downstream signal transduction mechanisms inv olved are unclear. We found that PrE differentiation is induced by the Ras effector domain mutants, RasV12G37 and RasV12E38, but not by RasV12C40. Acc ordingly, expression of constitutively active forms of RalGEF (Rlf-CAAX) or Raf1 (Raf-CAAX) is sufficient to induce differentiation. Inhibition of Ral GEF activity by expression of dominant negative Ral completely abolishes Rl f-CAAX- and RasV12G37-induced differentiation, while it reduces differentia tion by RasV12 and Raf-CAAX. Finally, while Rlf-CAAX does not increase Erk activity, inhibition of MEK blocks both Ras- as web as Rlf-CAAX-induced dif ferentiation, suggesting that RalGEFs induce PrE differentiation in a manne r depending on basal MEK or Erk activity. Based on these results we conclud e that Ras induces PrE differentiation of F9 EC cells via an interplay of E rk- and RalGEF-mediated pathways.