Direct interaction of Ras and the amino-terminal region of Raf-1 in vitro

Nature. 1993 Jul 22;364(6435):352-5. doi: 10.1038/364352a0.

Abstract

The Ras proteins are key regulators of the growth of eukaryotic cells, but their direct target enzymes, or 'effectors', are unknown. The protein encoded by the c-raf-1 proto-oncogene is thought to function downstream of p21ras because disruption of Raf blocks signalling by Ras in a number of systems. Here we report that the amino-terminal cysteine-rich regulatory region of p74c-raf-1 expressed as a glutathione-S-transferase (GST) fusion protein binds directly to Ras with relatively high affinity (50 nM). The binding is strictly dependent on the Ras protein being in the active GTP-bound conformation rather than the inactive GDP-bound state. Raf-GST interacts with wild-type and oncogenic Ras (Val 12) but fails to interact with a biologically inert effector mutant of Ras (Ala 38) and a dominant negative mutant (Asn 17). A peptide based on the effector region of Ras inhibits the interaction. Raf-GST acts as a potent competitive inhibitor of the GTPase-activating proteins p120GAP and neurofibromin. In addition, Raf itself displays weak GTPase-stimulating activity towards Ras. It is therefore likely that Raf is a direct effector of Ras.

MeSH terms

  • Glutathione Transferase / metabolism
  • Guanosine Triphosphate / metabolism
  • Mutation
  • Peptide Fragments / metabolism
  • Protein Binding
  • Proto-Oncogene Proteins / metabolism*
  • Proto-Oncogene Proteins c-raf
  • Proto-Oncogene Proteins p21(ras) / metabolism*
  • Recombinant Fusion Proteins / metabolism
  • Signal Transduction / physiology
  • Zinc / physiology

Substances

  • Peptide Fragments
  • Proto-Oncogene Proteins
  • Recombinant Fusion Proteins
  • Guanosine Triphosphate
  • Glutathione Transferase
  • Proto-Oncogene Proteins c-raf
  • Proto-Oncogene Proteins p21(ras)
  • Zinc