Identification of signalling proteins interacting with B-Raf in the yeast two-hybrid system

Oncogene. 1996 May 16;12(10):2213-21.

Abstract

Recent studies suggested the existence of Ras/B-Raf/ MEK-1 complexes and a critical role for B-Raf in regulating the MAP kinase/ERKs signalling pathway. We report, here, that both Ras and MEK-1 proteins interact physically with B-Raf proteins in the yeast two-hybrid system. In addition, by screening a mouse brain cDNA library, we isolated additional B-Raf interacting proteins. These include three members of the 14-3-3 proteins family (eta, theta and zeta) and the MEK-2 protein. We also show that c-Raf-1, previously reported to interact with beta and zeta 14-3-3 proteins, also interacts with eta and theta 14-3-3 proteins in the two-hybrid system. By using different portions of the B-Raf protein, we mapped the regions of the protein involved in these interactions. Specifically, we have characterized B-Raf specific sequences required for an efficient interaction with MEK proteins. We show that, consequently, B-Raf interacts with MEK-1 and MEK-2 with a better affinity than does c-Raf-1, thus strengthening the notion that B-Raf is a stronger MEK activator than c-Raf-l. Our results also suggest that a MEK specific sequence, not present in MAP kinase kinases which are not activated by members of the Raf family, is required for the interaction with Raf proteins.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 14-3-3 Proteins
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Calcium-Calmodulin-Dependent Protein Kinases / metabolism
  • Cricetinae
  • Humans
  • MAP Kinase Kinase 1
  • MAP Kinase Kinase 2
  • Mice
  • Mitogen-Activated Protein Kinase Kinases*
  • Molecular Sequence Data
  • Protein Sorting Signals / metabolism*
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism*
  • Protein-Tyrosine Kinases / genetics
  • Protein-Tyrosine Kinases / metabolism
  • Proteins / genetics
  • Proteins / metabolism
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins / metabolism*
  • Proto-Oncogene Proteins c-raf
  • Saccharomyces cerevisiae / enzymology
  • Saccharomyces cerevisiae / metabolism*
  • Sequence Homology, Amino Acid
  • Signal Transduction / physiology*
  • Tyrosine 3-Monooxygenase*
  • ras Proteins / genetics
  • ras Proteins / metabolism

Substances

  • 14-3-3 Proteins
  • Protein Sorting Signals
  • Proteins
  • Proto-Oncogene Proteins
  • Tyrosine 3-Monooxygenase
  • MAP2K2 protein, human
  • Protein-Tyrosine Kinases
  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-raf
  • Calcium-Calmodulin-Dependent Protein Kinases
  • MAP Kinase Kinase 1
  • MAP Kinase Kinase 2
  • MAP2K1 protein, human
  • Map2k1 protein, mouse
  • Mitogen-Activated Protein Kinase Kinases
  • ras Proteins