title: Interdependent action of RalGEF and Erk in Ras-induced primitive endoderm differentiation of F9 embryonal carcinoma cells.
abstract: Previous work by us and others has implicated a role for Ral guanine exchange factors (RalGEFs) in Ras-induced cell growth and oncogenic transformation. Here we show for the first time that RalGEFs are involved in Ras-induced differentiation as well. Expression of oncogenic Ras in F9 embryonal carcinoma (EC) cells is known to induce differentiation to a primitive endoderm (PrE)-like phenotype, but the downstream signal transduction mechanisms involved are unclear. We found that PrE differentiation is induced by the Ras effector domain mutants, RasV12G37 and RasV12E38, but not by RasV12C40. Accordingly, expression of constitutively active forms of RalGEF (Rlf-CAAX) or Rafl (Raf-CAAX) is sufficient to induce differentiation. Inhibition of RalGEF activity by expression of dominant negative Ral completely abolishes Rlf-CAAX- and RasV12G37-induced differentiation, while it reduces differentiation by RasV12 and Raf-CAAX. Finally, while Rlf-CAAX does not increase Erk activity, inhibition of MEK blocks both Ras- as well as Rlf-CAAX-induced differentiation, suggesting that RalGEFs induce PrE differentiation in a manner depending on basal MEK or Erk activity. Based on these results we conclude that Ras induces PrE differentiation of F9 EC cells via an interplay of Erk-and RalGEF-mediated pathways.
authors.affiliation: Laboratory for Physiological Chemistry and Centre for Biomedical Genetics, Utrecht University, The Netherlands.
authors.forename: M H
authors.initials: MH
authors.lastname: Verheijen
authors.forename: R M
authors.initials: RM
authors.lastname: Wolthuis
authors.forename: L H
authors.initials: LH
authors.lastname: Defize
authors.forename: J
authors.initials: J
authors.lastname: den Hertog
authors.forename: J L
authors.initials: JL
authors.lastname: Bos
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D018236:Carcinoma, Embryonal; D002454:Cell Differentiation; D004707:Endoderm / Q000166:cytology*; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D011494:Protein Kinases / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015682:Retroviridae Proteins, Oncogenic / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D014407:Tumor Cells, Cultured; D020668:rap GTP-Binding Proteins; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D015682:Retroviridae Proteins, Oncogenic; D011494:Protein Kinases; D019908:Proto-Oncogene Proteins c-raf; C107753:proto-oncogene protein v-RAF1, pig; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019204:GTP-Binding Proteins; D020668:rap GTP-Binding Proteins; D018631:ras Proteins