A ROLE FOR RALGDS AND A NOVEL RAS EFFECTOR IN THE RAS-MEDIATED INHIBITION OF SKELETAL MYOGENESIS

Oncogenic Ras inhibits the differentiation of skeletal muscle cells th rough the activation of multiple downstream signaling pathways, includ ing a Raf-dependent, mitogen-activated or extracellular signal-regulat ed kinase kinase/mitogen-activated protein kinase (MEK/MAPK)-independe nt pathway. Here we report that a non-Raf binding Ras effector-loop va riant (H-Ras G12V,E37G), which retains interaction with the Ral guanin e nucleotide dissociation stimulator (RalGDS), inhibits the conversion of MyoD-expressing C3H10T1/2 mouse fibroblasts to skeletal muscle. We show that H-Ras G12V,E37G, RalGDS, and the membrane-localized RalGDS CAAX protein inhibit the activity of alpha-actin-Luc, a muscle-specifi c reporter gene containing a necessary E-box and serum response factor (SRF) binding site, while a RalGDS protein defective for Ras interact ion has no effect on alpha-actin-Luc transcription. H-Ras G12V,E37G do es not activate endogenous MAPK, but does increase SRI-dependent trans cription. Interestingly, RalGDS, RalGDS CAAX, and RalA G23V inhibit H- Ras G12V,E37G-induced expression of an SRF-regulated reporter gene, de monstrating that signaling through RalGDS does not duplicate the actio n of H-Ras G12V,E37G in this system. As additional evidence for this, we show that H-Ras G12V,E37G inhibits the expression of troponin I-Luc , an SRF-independent muscle-specific reporter gene, whereas RalGDS and RalGDS CAAX do not. Although our studies show that signaling through RalGDS can interfere with the expression of reporter genes dependent o n SRF activity (including alpha-actin-Luc), our studies also provide s trong evidence that an additional signaling molecule(s) activated by H -Ras G12V,E37G is required to achieve the complete inhibition of the m yogenic differentiation program.