Mutations in conserved regions 1, 2, and 3 of Raf-1 that activate transforming activity.
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.
Chan
Edmond Y W
EY
Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada.
Stang
Stacey L
SL
Bottorff
Drell A
DA
Stone
James C
JC
D000483:Alleles; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D017124:Conserved Sequence; D005347:Fibroblasts; D011905:Genes, ras; D020935:MAP Kinase Signaling System / Q000502:physiology; D008858:Microscopy, Phase-Contrast; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism
D011994:Recombinant Proteins; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D020928:Mitogen-Activated Protein Kinases