Regulation of c-myc expression by Ras/Raf signalling.

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) 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). 4-hydroxytamoxifen mediated activation of the fusion protein in serum starved N-BxB-ER 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.