The Product of the Primary Response Gene BRF1 Inhibits the Interaction between 14-3-3 Proteins and cRaf-1 in the Yeast Trihybrid System

The 14-3-3 proteins are small abundant cytosolic eukaryotic proteins that associate with and modulate the activity of numerous other proteins. The 14-3-3 beta isoform has been shown to bind to the product of the protooncogene cRaf-1 and to facilitate its activation by Ras. Using the yeast two-hybrid system, we have demonstrated that 14-3-3 beta and another isoform, 14-3-3 tau, bind to the product of the primary response gene BRF1 and that the interaction between each isoform and BRF1 is significantly stronger than that with cRaf-1. We further demonstrated that the charge of residue 187 in 14-3-3 beta regulates its affinity for both BRF1 and cRaf-1. The interaction of either isoform with BRF1 requires both proteins to be fully intact. When all three proteins are coexpressed in a yeast trihybrid system, BRF1 interferes significantly with the binding of 14-3-3 to full-length cRaf-1 as well as to its regulatory and kinase domains. Using quantitative reverse transcription-polymerase chain reaction, 14-3-3 beta and BRF1 were found to be coexpressed in four different human tissues, suggesting a biologic role for their interaction in the regulation of cRaf-1-mediated signal transduction processes.