A dissection of signal transduction pathways involved in the Ras-mediated inhibition of skeletal myogenesis and the identification of a novel Ras -interacting protein
The ability of bHLH muscle regulatory factors (MRFs) to convert non-muscle cells to a myogenic lineage is regulated by numerous growth factor and oncoprotein signaling pathways. Oncogenic Ras inhibits skeletal muscle differentiation by disrupting MRF function via a mechanism that is independent of the dimerization, DNA binding, and transcriptional activation properties of the proteins. To investigate the signaling pathway that mediates the inhibition of myogenesis by oncogenic Ras, we tested two transformation-defective, H-Ras G12V effector domain variants for their ability to alter terminal differentiation. H-Ras G12V,T35S activates the Raf/MEK/MAPK cascade whereas H-Ras G12V,Y40C does not activate this cascade but influences other signaling intermediates, including Rac and Rho. Expression of each Ras variant in C3H10T1/2 cells abrogates MyoD-induced activation of the myogenic program, suggesting that MAPK-dependent and -independent signaling pathways individually block myogenesis in this model system. However, additional studies with constitutively activated Rac 1 and RhoA proteins reveal no negative effect on myogenesis. Similarly, treatment of Ras-inhibited myoblasts with the MEK1 inhibitor PD98059 revealed that elevated MAPK activity is not a significant contributor to the Ras effect. A non-Raf binding Ras variant (H-Ras G12V,E37G), which retains interaction with the RalGDS protein, inhibits the conversion of MyoD-expressing C3H10T1/2 fibroblasts to skeletal muscle. H-Ras G12V,E37G and RalGDS inhibit the activity of a muscle-specific reporter gene containing E-box and serum response factor (SRF) binding sites. H-Ras G12V,E37G does not activate endogenous MAPK, but does increase SRF-dependent transcription. RalGDS inhibits H-Ras G12V,E37G-induced expression of an SRF-regulated reporter gene, demonstrating that signaling through RalGDS does not duplicate the action of H-Ras G12V,E37G in this system. Furthermore, H-Ras G12V,E37G inhibits the expression of troponinI-Luc, an SRF-independent muscle-specific reporter gene, while RalGDS does not. These studies provide strong evidence that additional signaling molecules activated by Ras are required to achieve the complete inhibition of the myogenic differentiation program. A yeast two-hybrid screen of a skeletal muscle cDNA library using H-Ras G12V,C186S as the bait protein resulted in the identification of a novel Ras- interacting protein, Rain. This protein shares limited sequence homology with the Ras effector AF-6. The Rain message is expressed ubiquitously. Future studies will address the potential role of Rain in the Ras-mediated inhibition of skeletal myogenesis. ^
Major Professor: Elizabeth J. Taparowsky, Purdue University.
Biology, Molecular|Biology, Cell