14-3-3 proteins are required for maintenance of Raf-1 phosphorylation and kinase activity

Mol Cell Biol. 1998 Sep;18(9):5229-38. doi: 10.1128/MCB.18.9.5229.

Abstract

By binding to serine-phosphorylated proteins, 14-3-3 proteins function as effectors of serine phosphorylation. The exact mechanism of their action is, however, still largely unknown. Here we demonstrate a requirement for 14-3-3 for Raf-1 kinase activity and phosphorylation. Expression of dominant negative forms of 14-3-3 resulted in the loss of a critical Raf-1 phosphorylation, while overexpression of 14-3-3 resulted in enhanced phosphorylation of this site. 14-3-3 levels, therefore, regulate the stoichiometry of Raf-1 phosphorylation and its potential activity in the cell. Phosphorylation of Raf-1, however, was insufficient by itself for kinase activity. Removal of 14-3-3 from phosphorylated Raf abrogated kinase activity, whereas addition of 14-3-3 restored it. This supports a paradigm in which the effects of phosphorylation on serine as well as tyrosine residues are mediated by inducible protein-protein interactions.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 14-3-3 Proteins
  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Cell Line
  • Chloramphenicol O-Acetyltransferase / biosynthesis
  • Cloning, Molecular
  • Glutathione Transferase
  • Humans
  • Mice
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Phosphorylation
  • Phosphoserine
  • Phosphotyrosine
  • Polymerase Chain Reaction
  • Protein Biosynthesis
  • Protein Structure, Secondary*
  • Proteins / chemistry*
  • Proteins / metabolism*
  • Proto-Oncogene Proteins c-raf / chemistry*
  • Proto-Oncogene Proteins c-raf / metabolism*
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / metabolism
  • Spectrum Analysis
  • Transfection
  • Tyrosine 3-Monooxygenase*

Substances

  • 14-3-3 Proteins
  • Proteins
  • Recombinant Fusion Proteins
  • Phosphoserine
  • Phosphotyrosine
  • Tyrosine 3-Monooxygenase
  • Chloramphenicol O-Acetyltransferase
  • Glutathione Transferase
  • Proto-Oncogene Proteins c-raf