Bcr and Raf form a complex in vivo via 14-3-3 proteins

EMBO J. 1995 Oct 2;14(19):4839-48.

Abstract

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.

MeSH terms

  • 14-3-3 Proteins
  • Animals
  • Cell Line
  • Cell Membrane / chemistry
  • Chlorocebus aethiops
  • Cytosol / chemistry
  • DNA-Binding Proteins
  • Fungal Proteins / genetics
  • Oncogene Proteins / genetics
  • Oncogene Proteins / metabolism*
  • Precipitin Tests
  • Protein-Serine-Threonine Kinases / analysis
  • Protein-Serine-Threonine Kinases / metabolism*
  • Protein-Tyrosine Kinases*
  • Proteins / genetics
  • Proteins / metabolism*
  • Proto-Oncogene Proteins / analysis
  • Proto-Oncogene Proteins / metabolism*
  • Proto-Oncogene Proteins c-bcr
  • Proto-Oncogene Proteins c-raf
  • Recombinant Fusion Proteins / metabolism
  • Saccharomyces cerevisiae Proteins*
  • Serine / metabolism
  • Threonine / metabolism
  • Transcription Factors*
  • Tyrosine 3-Monooxygenase*

Substances

  • 14-3-3 Proteins
  • DNA-Binding Proteins
  • Fungal Proteins
  • GAL4 protein, S cerevisiae
  • Oncogene Proteins
  • Proteins
  • Proto-Oncogene Proteins
  • Recombinant Fusion Proteins
  • Saccharomyces cerevisiae Proteins
  • Transcription Factors
  • Threonine
  • Serine
  • Tyrosine 3-Monooxygenase
  • Protein-Tyrosine Kinases
  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-bcr
  • Proto-Oncogene Proteins c-raf