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

Research output: Contribution to journalArticle

9 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.

LanguageEnglish
Pages189-97
Number of pages9
JournalMolecular Carcinogenesis
Volume33
Issue number4
DOIs
Publication statusPublished - Apr 2002

Fingerprint

Mutation
Fibroblasts
Extracellular Signal-Regulated MAP Kinases
Cysteine
MAP Kinase Kinase Kinase 1
Amino Acid Motifs
Two-Hybrid System Techniques
Dipeptides
Mitogen-Activated Protein Kinase Kinases
Mitogen-Activated Protein Kinases
Glycine
Protein Kinases
Phosphotransferases
Adenosine Triphosphate

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

Cite this

Chan, Edmond Y W ; Stang, Stacey L ; Bottorff, Drell A ; Stone, James C. / Mutations in conserved regions 1, 2, and 3 of Raf-1 that activate transforming activity. In: Molecular Carcinogenesis. 2002 ; Vol. 33, No. 4. pp. 189-97.
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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.",
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Mutations in conserved regions 1, 2, and 3 of Raf-1 that activate transforming activity. / Chan, Edmond Y W; Stang, Stacey L; Bottorff, Drell A; Stone, James C.

In: Molecular Carcinogenesis, Vol. 33, No. 4, 04.2002, p. 189-97.

Research output: Contribution to journalArticle

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AU - Bottorff, Drell A

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AB - 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.

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KW - amino acid sequence

KW - amino acid substitution

KW - animals

KW - cell line

KW - cell transformation, neoplastic

KW - conserved sequence

KW - fibroblasts

KW - genes, ras

KW - MAP kinase signaling system

KW - microscopy, phase-contrast

KW - mitogen-activated protein kinases

KW - molecular sequence data

KW - mutation

KW - protein-serine-threonine kinases

KW - proto-oncogene proteins c-raf

KW - rats

KW - recombinant proteins

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DO - 10.1002/mc.10031

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SP - 189

EP - 197

JO - Molecular Carcinogenesis

T2 - Molecular Carcinogenesis

JF - Molecular Carcinogenesis

SN - 0899-1987

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ER -