Both p21ras and pp60v-src are required, but neither alone is sufficient, to activate the Raf-1 kinase

Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):2922-6. doi: 10.1073/pnas.89.7.2922.


The raf genes encode a family of cytoplasmic proteins with intrinsic protein-serine/threonine kinase activity. The c-raf gene is the cellular homolog of v-raf, the transforming gene of murine sarcoma virus 3611. The constitutive kinase activity of the v-Raf protein has been implicated in transformation and mitogenesis. The activity of Raf-1, the protein product of the c-raf gene, is normally suppressed by a regulatory N-terminal domain. Activation of various tyrosine-kinase growth factor receptors results in activation of Raf-1 and its hyperphosphorylation. Further, Raf-1 has been shown to act either downstream or independently of the p21ras protein, as indicated by experiments involving microinjection of anti-Ras antibodies. To investigate the potential role of p21ras in the activation of Raf-1 by tyrosine kinases, we have used the baculovirus/Sf9 cell system to overproduce various wild-type and mutant forms of pp60src, p21ras, and Raf-1 proteins. We show that either pp60v-src or p21c-ras can independently activate the autokinase activity of Raf-1, but only to a limited extent. Surprisingly, both pp60v-src and p21c-ras are required to fully activate Raf-1. Analysis of the Raf-1 autokinase activity in vitro shows that Raf-1 autophosphorylation sites are distributed equally on serine and threonine residues. When Raf-1 is analyzed by immunoblotting, as previously reported for mammalian cell experiments, a marked increase in the apparent molecular weight of Raf-1 is seen only when it is coexpressed with both pp60v-src and p21ras.

Publication types

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

MeSH terms

  • Animals
  • Cells, Cultured
  • Enzyme Activation
  • In Vitro Techniques
  • Moths
  • Oncogene Protein pp60(v-src) / metabolism*
  • Phosphorylation
  • Phosphoserine / metabolism
  • Phosphothreonine / metabolism
  • Phosphotyrosine
  • Protein Kinases / metabolism*
  • Proto-Oncogene Proteins / metabolism*
  • Proto-Oncogene Proteins c-raf
  • Proto-Oncogene Proteins p21(ras) / metabolism*
  • Recombinant Proteins / metabolism
  • Tyrosine / analogs & derivatives
  • Tyrosine / metabolism


  • Proto-Oncogene Proteins
  • Recombinant Proteins
  • Phosphothreonine
  • Phosphoserine
  • Phosphotyrosine
  • Tyrosine
  • Protein Kinases
  • Oncogene Protein pp60(v-src)
  • Proto-Oncogene Proteins c-raf
  • Proto-Oncogene Proteins p21(ras)