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.
Bustin
S A
SA
Department of Surgery, St. Bartholomew's and the Royal London School of Medicine and Dentistry, Queen Mary and Westfield College, England. s.a.bustin@mds.qmw.ac.uk
McKay
I A
IA
D048948:14-3-3 Proteins; D019943:Amino Acid Substitution; D001665:Binding Sites; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D015398:Signal Transduction; D055672:Static Electricity; D035381:TATA-Binding Protein Associated Factors; D035361:Transcription Factor TFIIIB; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014446:Tyrosine 3-Monooxygenase; D015003:Yeasts / Q000235:genetics*; D016335:Zinc Fingers
D048948:14-3-3 Proteins; C090956:BRF1 protein, S cerevisiae; C441028:BRF1 protein, human; D020033:Protein Isoforms; D011506:Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D035381:TATA-Binding Protein Associated Factors; D035361:Transcription Factor TFIIIB; D014157:Transcription Factors; D014446:Tyrosine 3-Monooxygenase; D019908:Proto-Oncogene Proteins c-raf