Mutations in conserved regions 1, 2, and 3 of Raf-1 that activate transforming activity

Mol Carcinog. 2002 Apr;33(4):189-97. doi: 10.1002/mc.10031.


To investigate the role of Raf-1 in v-Ha-ras transformation, we have isolated and characterized a number of Raf-1 mutants that display increased transforming activity in Rat2 fibroblasts. A dipeptide deletion (Delta144-145) in the cysteine-rich domain (CRD) of conserved region (CR) 1 increased the interaction between Raf-1 and v-Ha-ras effector loop mutants in the yeast two-hybrid system, supporting the proposal that the CRD serves as a secondary ras-binding domain. Many activating mutations were located in CR2. Two representative CR2 mutants (Delta250-258 and S257L) displayed increased interaction with v-Ha-ras effector loop mutants and with mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) 1 in the two-hybrid system. One novel mutation in CR3 was recovered; G361S affected the third glycine of the GXGXXG protein kinase motif involved in ATP binding. Expression of G361S Raf-1 in Rat2 fibroblasts activated MEK and ERK. The CR1, CR2, and CR3 activating mutations, when combined in cis, cooperated in transforming Rat2 fibroblasts. Conversely, Raf-1 transforming activity was decreased when the S257L or G361S mutation was combined in cis with the R89E substitution, which disrupts ras-Raf interaction. This mutant analysis provides additional information about the distinct functions of individual Raf-1 regions and documents a novel genetic mechanism for activating an oncogenic kinase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • Amino Acid Sequence
  • Amino Acid Substitution
  • Animals
  • Cell Line
  • Cell Transformation, Neoplastic / genetics*
  • Conserved Sequence*
  • Fibroblasts
  • Genes, ras
  • MAP Kinase Signaling System / physiology
  • Microscopy, Phase-Contrast
  • Mitogen-Activated Protein Kinases / metabolism
  • Molecular Sequence Data
  • Mutation*
  • Protein Serine-Threonine Kinases / metabolism
  • Proto-Oncogene Proteins c-raf / genetics*
  • Rats
  • Recombinant Proteins / metabolism


  • Recombinant Proteins
  • Protein Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-raf
  • Mitogen-Activated Protein Kinases