Ramocki M B, White M A, Konieczny S F, Taparowsky E J
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907-1392, USA.
J Biol Chem. 1998 Jul 10;273(28):17696-701. doi: 10.1074/jbc.273.28.17696.
Oncogenic Ras inhibits the differentiation of skeletal muscle cells through the activation of multiple downstream signaling pathways, including a Raf-dependent, mitogen-activated or extracellular signal-regulated kinase kinase/mitogen-activated protein kinase (MEK/MAPK)-independent pathway. Here we report that a non-Raf binding Ras effector-loop variant (H-Ras G12V,E37G), which retains interaction with the Ral guanine 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-specific reporter gene containing a necessary E-box and serum response factor (SRF) binding site, while a RalGDS protein defective for Ras interaction has no effect on alpha-actin-Luc transcription. H-Ras G12V,E37G does not activate endogenous MAPK, but does increase SRF-dependent transcription. Interestingly, RalGDS, RalGDS CAAX, and RalA G23V inhibit 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. 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 on SRF activity (including alpha-actin-Luc), our studies also provide strong evidence that an additional signaling molecule(s) activated by H-Ras G12V,E37G is required to achieve the complete inhibition of the myogenic differentiation program.
致癌性Ras通过激活多个下游信号通路抑制骨骼肌细胞的分化，这些通路包括一条依赖Raf、丝裂原激活或细胞外信号调节激酶激酶/丝裂原激活蛋白激酶（MEK/MAPK）非依赖的通路。在此，我们报告一种非Raf结合的Ras效应环变体（H-Ras G12V，E37G），它保留了与Ral鸟嘌呤核苷酸解离刺激因子（RalGDS）的相互作用，抑制表达MyoD的C3H10T1/2小鼠成纤维细胞向骨骼肌的转化。我们表明，H-Ras G12V，E37G、RalGDS和膜定位的RalGDS CAAX蛋白抑制α-肌动蛋白-Luc的活性，α-肌动蛋白-Luc是一个含有必要E盒和血清反应因子（SRF）结合位点的肌肉特异性报告基因，而对Ras相互作用有缺陷的RalGDS蛋白对α-肌动蛋白-Luc转录没有影响。H-Ras G12V，E37G不激活内源性MAPK，但确实增加SRF依赖的转录。有趣的是，RalGDS、RalGDS CAAX和RalA G23V抑制H-Ras G12V，E37G诱导的SRF调节报告基因的表达，表明通过RalGDS的信号传导在该系统中不重复H-Ras G12V，E37G的作用。作为对此的额外证据，我们表明H-Ras G12V，E37G抑制肌钙蛋白I-Luc的表达，肌钙蛋白I-Luc是一个不依赖SRF的肌肉特异性报告基因，而RalGDS和RalGDS CAAX则不然。尽管我们的研究表明通过RalGDS的信号传导可以干扰依赖SRF活性的报告基因（包括α-肌动蛋白-Luc）的表达，但我们的研究也提供了强有力的证据，即H-Ras G12V，E37G激活的另一种信号分子是实现对成肌分化程序的完全抑制所必需的。
