Bcr and Raf form a complex in vivo via 14-3-3 proteins.
In a yeast two-hybrid screen we identified a member of the 14-3-3 family of proteins that can bind to Bcr. 14-3-3 beta binds to the serine/threonine rich region B in the kinase domain encoded by the first exon. In this paper we show by co-immunoprecipitation that Bcr binds to Raf in vivo and we argue that this interaction is mediated by 14-3-3 dimers, based on the following findings. First, 14-3-3 isoforms bind to both Raf and Bcr. Second, Bcr does not bind to Raf directly in the two-hybrid system, but co-expression of 14-3-3 beta allows complex formation. Third, Bcr, 14-3-3 proteins and Raf co-elute in gel filtration and in sequential ion exchange chromatography and the three proteins can be co-immunoprecipitated from the the separate fractions, indicating that they are present in a ternary complex. Moreover, approximately 10 times more Raf is bound to Bcr, and vice versa, in the membrane fraction (where Raf is activated) than in the cytosolic fraction. We suggest a new function for 14-3-3 proteins as a novel type of new function for 14-3-3 proteins as a novel type of adaptor which acts by dimerization and binding to different proteins.
Braselmann
S
S
Onyx Pharmaceuticals Inc., Richmond, CA 94806, USA.
McCormick
F
F
D048948:14-3-3 Proteins; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D002522:Chlorocebus aethiops; D003600:Cytosol / Q000737:chemistry; D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000235:genetics; D015513:Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000032:analysis / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000032:analysis / Q000378:metabolism*; D051562:Proto-Oncogene Proteins c-bcr; D019908:Proto-Oncogene Proteins c-raf; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D014157:Transcription Factors; D014446:Tyrosine 3-Monooxygenase
D048948:14-3-3 Proteins; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D015513:Oncogene Proteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D013912:Threonine; D012694:Serine; D014446:Tyrosine 3-Monooxygenase; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D051562:Proto-Oncogene Proteins c-bcr; D019908:Proto-Oncogene Proteins c-raf