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 fami ly of proteins that can bind to Bcr. 14-3-3 beta binds to the serine/t hreonine 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 dime rs, based on the following findings. First, 14-3-3 isoforms bind to bo th Raf and Bcr. Second, Bcr does not bind to Raf directly in the two-h ybrid system, but co-expression of 14-3-3 beta allows complex formatio n. Third, Bcr, 14-3-3 proteins and Raf co-elute in gel filtration and in sequential ion exchange chromatography and the three proteins can b e co-immunoprecipitated from the separate fractions, indicating that t hey are present in a ternary complex. Moreover, similar to 10 times mo re Raf is bound to Bcr, and vice versa, in the membrane fraction (wher e Raf is activated) than in the cytosolic fraction. We suggest a new f unction for 14-3-3 proteins as a novel type of adaptor which acts by d imerization and binding to different proteins.