Regulation of c-myc expression by Ras/Raf signalling
Regulation of c-myc expression by Ras/Raf signalling
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DOI：
10.1038/sj.onc.1201520
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发表时间：
1998-01-15
期刊：
影响因子：
8
通讯作者：
Rapp, UR
中科院分区：
文献类型：
--
作者：
Kerkhoff, E;Houben, R;Rapp, UR
C-myc基因是在生长因子刺激停滞细胞后被诱导的，有丝分裂原与跨膜受体的相互作用触发了一系列平行的信号转导通路。为了分析Ras/Raf级联在c-myc表达调控中的作用，我们建立了含有激活或抑制这一途径的条件系统的成纤维细胞系，c-Raf-1激动域与雌激素受体的激素结合域(c-Raf-1-BXB-ER(TM))融合提供了4-羟基他莫昔芬调节形式的致癌c-Raf-1激酶，我们已经产生了稳定表达嵌合Raf蛋白的NIH3T3细胞(N-BXB-ER(TM))。4-羟基他莫昔芬介导的融合蛋白在饥饿的血清N-BXB-ER(TM)中的激活在2-6h内诱导c-myc基因的表达，c-Raf-1激动域的缺失产生突变的c-Raf-1蛋白(c-Raf-1-C4b)，它可以直接与RAS蛋白的效应域相互作用，从而阻断RAS介导的信号转导。我们建立了四环素应答启动子(N-C4b-tet)控制下的NIH3T3细胞株，与不表达Ras抑制剂的细胞相比，表达c-Raf-1-C4b蛋白的血清饥饿细胞在血清刺激后对c-myc表达的诱导显著降低，在Ras失活c-Raf-1-C4b突变体的存在下，激活分离的Raf/MEK/Erk级联反应后c-myc mRNA的诱导，以及在存在Ras失活c-Raf-1-C4b突变体的情况下，c-myc表达的诱导被部分抑制，强烈表明Ras/Raf途径参与了c-myc表达的调节。
The c-myc gene is induced upon growth factor stimulation of arrested cells, The interaction of a mitogen with a transmembrane receptor triggers a variety of parallel signal transduction cascades. In order to analyse the role of the Ras/Raf cascade in the regulation of c-myc expression we have established fibroblast cell lines harboring conditional systems activating or inhibiting this pathway, Fusion of the c-Raf-1 kinase domain with the hormone binding domain of the estrogen receptor (c-Raf-1-BxB-ER(TM)) provides a 4-hydroxytamoxifen regulated form of the oncogenic c-Raf-1 kinase, We have generated NIH3T3 cells stably expressing the chimeric Raf protein (N-BxB-ER(TM)). 4-hydroxytamoxifen mediated activation of the fusion protein in serum starved N-BxB-ER(TM) induces the expression of the c-myc gene within 2-6 h, Deletion of the c-Raf-1 kinase domain generates a mutant c-Raf-1 protein (c-Raf-1-C4B), which can directly interact with the effector domain of the Ras protein and thereby block Ras mediated signalling, We have established a NIH3T3 based cell line expressing the c-Raf-1-C4B protein under the control of a tetracycline responsive promoter (N-C4B-tet), Serum starved cells expressing the c-Raf-1-C4B protein exhibit a significantly reduced induction of c-myc expression following serum stimulation compared to the same cells not expressing the Ras inhibitor, The induction of c-myc mRNA following the activation of the isolated Raf/Mek/Erk cascade in addition to the partial inhibition of serum mediated induction of c-myc expression in the presence of the Ras inactivating c-Raf-1-C4B mutant strongly indicates an involvement of the Ras/Raf pathway in the regulation of c-myc expression.