Activated Ras Displaces 14-3-3 Protein From the Amino Terminus of c-Raf-1

Oncogene. 1996 Feb 1;12(3):609-19.

Abstract

The serine/threonine protein kinase c-Raf-1 interacts with a number of cellular proteins including 14-3-3 isoforms which may be regulators or substrates of c-Raf-1 in signal transduction pathways. In vivo and in vitro binding analyses of c-Raf-1 and mutant proteins with 14-3-3 zeta indicate bivalent binding of 14-3-3 zeta to the amino terminus as well as to the carboxy terminus of c-Raf-1. Although 14-3-3 zeta and Ras use different binding regions on the amino terminal regulatory domain of c-Raf-1 (c-Raf-NT), 14-3-3 zeta is displaced from the amino terminus upon binding of activated Ras. In contrast, if c-Raf-1 full length is analysed instead of the separately expressed c-Raf-NT, binding of 14-3-3 zeta is only slightly effected by co-expression of activated Ras. This is explained by a second binding site of 14-3-3 zeta at the carboxy terminus of c-Raf-1. The mutant c-Raf-NT (S259A) cannot bind 14-3-3 zeta, suggesting a regulatory role of this in vivo phosphorylation site. However, c-Raf-NT phosphorylated or unphosphorylated at S259, is able to bind 14-3-3 zeta. Even though 14-3-3 zeta can be phosphorylated in vivo, only the unphosphorylated form binds to the amino terminus of c-Raf-1. The data presented indicate, that 14-3-3 zeta binds to c-Raf-1 in a bivalent fashion in unstimulated cells. 14-3-3 zeta is displaced from the amino terminus but not from the carboxy terminus of c-Raf-1 by binding of activated Ras to c-Raf-1.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 14-3-3 Proteins
  • Adenoviruses, Human
  • Animals
  • Binding Sites
  • Binding, Competitive
  • Cell Line, Transformed
  • Cloning, Molecular
  • Enzyme Inhibitors / metabolism
  • Glutathione Transferase
  • Humans
  • Kidney
  • Mutagenesis
  • Peptide Fragments / chemistry
  • Peptide Fragments / isolation & purification
  • Peptide Fragments / metabolism
  • Phosphopeptides / chemistry
  • Phosphopeptides / isolation & purification
  • Phosphorylation
  • Protein-Serine-Threonine Kinases / isolation & purification
  • Protein-Serine-Threonine Kinases / metabolism*
  • Proteins / isolation & purification
  • Proteins / metabolism*
  • Proto-Oncogene Proteins / isolation & purification
  • Proto-Oncogene Proteins / metabolism*
  • Proto-Oncogene Proteins c-raf
  • Rats
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism
  • Transfection
  • Tyrosine 3-Monooxygenase*
  • ras Proteins / metabolism*

Substances

  • 14-3-3 Proteins
  • Enzyme Inhibitors
  • Peptide Fragments
  • Phosphopeptides
  • Proteins
  • Proto-Oncogene Proteins
  • Recombinant Fusion Proteins
  • Tyrosine 3-Monooxygenase
  • Glutathione Transferase
  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-raf
  • ras Proteins