Regulation of Raf-1 kinase activity by the 14-3-3 family of proteins

EMBO J. 1995 Feb 15;14(4):685-96.

Abstract

We have identified the beta (beta) isoform of the 14-3-3 family of proteins as an activator of the Raf-1 protein kinase. 14-3-3 was isolated in a yeast two-hybrid screen for Raf-1 kinase domain binding proteins. Purified bovine brain 14-3-3 interacted specifically with both c-Raf-1 and the isolated Raf-1 kinase domain. Association was sensitive to the activation status of Raf-1; 14-3-3 bound to unactivated Raf-1, but not Raf-1 activated by protein kinase C alpha or Ras and Lck. The significance of these interactions under physiological conditions was demonstrated by co-immunoprecipitation of Raf-1 and 14-3-3 from extracts of quiescent, but not mitogen-stimulated, NIH 3T3 cells. 14-3-3 was not a preferred Raf-1 substrate in vitro and did not significantly affect Raf-1 kinase activity in a purified system. However, in cell-free extracts 14-3-3 acted as a Ras-independent activator of both c-Raf-1 and the Raf-1 kinase domain. The same results were obtained in vivo using transfection assays; 14-3-3 enhanced both c-Raf-1- and Raf-1 kinase domain-stimulated expression of AP-1- and NF-kappa B-dependent reporter genes and accelerated Raf-1 kinase domain-triggered differentiation of PC12 cells. We conclude that 14-3-3 is a latent co-activator bound to unactivated Raf-1 in quiescent cells and mediates mitogen-triggered but Ras-independent regulatory effects aimed directly at the kinase domain.

Publication types

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

MeSH terms

  • 14-3-3 Proteins
  • 3T3 Cells
  • Animals
  • Base Sequence
  • Cell-Free System
  • Cloning, Molecular
  • Enzyme Activation
  • Growth Substances / blood
  • Mice
  • Molecular Sequence Data
  • Protein Binding
  • Protein-Serine-Threonine Kinases / metabolism*
  • Proteins / metabolism*
  • Proto-Oncogene Proteins / metabolism*
  • Proto-Oncogene Proteins c-raf
  • Tyrosine 3-Monooxygenase*

Substances

  • 14-3-3 Proteins
  • Growth Substances
  • Proteins
  • Proto-Oncogene Proteins
  • Tyrosine 3-Monooxygenase
  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-raf