REGULATION OF C-MYC EXPRESSION BY RAS RAF SIGNALING/

The c-myc gene is induced upon growth factor stimulation of arrested c ells, The interaction of a mitogen with a transmembrane receptor trigg ers a variety of parallel signal transduction cascades. In order to an alyse the role of the Ras/Raf cascade in the regulation of c-myc expre ssion we have established fibroblast cell lines harboring conditional systems activating or inhibiting this pathway, Fusion of the c-Raf-1 k inase domain with the hormone binding domain of the estrogen receptor (c-Raf-1-BxB-ER(TM)) provides a 4-hydroxytamoxifen regulated form of t he oncogenic c-Raf-1 kinase, We have generated NIH3T3 cells stably exp ressing the chimeric Raf protein (N-BxB-ER(TM)). 4-hydroxytamoxifen me diated 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 pro tein and thereby block Ras mediated signalling, We have established a NIH3T3 based cell line expressing the c-Raf-1-C4B protein under the co ntrol of a tetracycline responsive promoter (N-C4B-tet), Serum starved cells expressing the c-Raf-1-C4B protein exhibit a significantly redu ced 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 cascad e 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.