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

Edmond Y W Chan, Stacey L Stang, Drell A Bottorff, James C Stone

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10 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)189-97
Number of pages9
JournalMolecular Carcinogenesis
Volume33
Issue number4
DOIs
Publication statusPublished - Apr 2002

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Keywords

  • alleles
  • amino acid sequence
  • amino acid substitution
  • animals
  • cell line
  • cell transformation, neoplastic
  • conserved sequence
  • fibroblasts
  • genes, ras
  • MAP kinase signaling system
  • microscopy, phase-contrast
  • mitogen-activated protein kinases
  • molecular sequence data
  • mutation
  • protein-serine-threonine kinases
  • proto-oncogene proteins c-raf
  • rats
  • recombinant proteins

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