An inhibitory Raf-1 mutant suppresses expression of a subset of v-raf-activated genes.
The proto-oncogene Raf-1 is a cytoplasmic serine/threonine kinase implicated in the signaling process in cell proliferation. To determine if Raf-1 is sufficient and necessary to transmit mitogenic signals to growth-responsive genes, we examined the effect of constitutively activated (v-raf) or inhibitory (Raf-C4) Raf-1 proteins on reporter gene activation in transient expression assays of NIH 3T3 cells. In serum-starved cells, v-raf strongly activated transcription from the promoters of the immediate-early genes c-fos and egr-2, as well as the proximal or B promoter of the late growth response gene rep-3 (rep-3b). Two other late response gene promoters, cad and dhfr, were only modestly activated by v-raf, however. An individual serum response element from the c-fos or egr-2 promoter conferred both serum-inducibility and v-raf-responsiveness to a heterologous promoter. Consistent with the degree to which antisense c-raf-1 RNA and dominant-negative Raf-1 proteins interfere with NIH 3T3 cell proliferation, Raf-C4 reduced serum-induced transcription from the egr-2 and rep-3b promoters in a dose-dependent manner by 50%. In contrast, Raf-C4 did not significantly reduce transcription from the c-fos or cad promoters or the serum response element-driven heterologous promoters. We conclude that Raf-1 is both sufficient and necessary to activate a subset of early and late growth response genes.
Miltenberger
R J
RJ
McArdle Laboratory for Cancer Research, University of Wisconsin-Madison 53706.
Cortner
J
J
Farnham
P J
PJ
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001769:Blood; D005786:Gene Expression Regulation; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009838:Oligodeoxyribonucleotides; D027384:Oncogene Proteins v-raf; D011401:Promoter Regions, Genetic; D011505:Protein-Tyrosine Kinases / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D019908:Proto-Oncogene Proteins c-raf; D015682:Retroviridae Proteins, Oncogenic / Q000235:genetics*; D015398:Signal Transduction; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D009838:Oligodeoxyribonucleotides; D011518:Proto-Oncogene Proteins; D015682:Retroviridae Proteins, Oncogenic; D011505:Protein-Tyrosine Kinases; D027384:Oncogene Proteins v-raf; D019908:Proto-Oncogene Proteins c-raf