Raf-1 serine/threonine protein kinase has the hallmarks of a critical switch that connects growth factor receptor activation at the cell membrane with transcriptional events in the nucleus. We have previously shown by the use of Raf-1-dominant negative mutants that Raf-1 is required for serum- and TPA-induced expression from the oncogene-responsive element in the polyomavirus enhancer. The minimal region of Raf-1 that displayed this dominant negative phenotype (Raf-C4) contains a cysteine finger motif. To insure that the Raf-C4 protein blocks AP-1/Ets-dependent transcription and to determine if the cysteine-rich region within conserved region 1 is necessary for this activity, we tested the ability of Raf-C4pm17 to block AP-1/Ets-driven expression in HepG2 cells. Raf- C4pm17 contains a single amino acid substitution (Cys to Ser at position 168) within the Raf-C4 cysteine-rich region. This substitution strongly decreases the dominant negative effect of Raf-C4. Thus, the conserved cysteine finger motif in Raf-1 is probably necessary for interactions with upstream regulatory factors. v-Ras also activates transcription from the oncogene-responsive element in the polyomavirus enhancer. To determine if Raf-1 mediates transactivation by Ras, we looked at the ability of Raf-C4 to block Ras-induced activation through the oncogene-responsive element. v-Ha-Ras efficiently transactivated pB4X CAT expression, and Raf-C4 completely blocked Ras- induced expression. These results demonstrate that Raf-1 is necessary for transactivation by Ras. Raf-C4 appears to function by titrating out a Raf-1 activating factor that is induced by Ras following serum or TPA treatment of NIH 3T3 cells. In addition, we show that Raf-1 and Ras cooperate in transactivation through the oncogene-responsive element and that the cysteine-rich region is necessary for this effect. The inability of the Raf-1 cysteine finger mutant (Raf-pm17) to cooperate with Ras suggests that it is incapable of binding the Ras-induced Raf-1 activator. Current work is aimed at the identification and characterization of this Ras-induced Raf-1 activator.
