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 (Δ144–145) 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 (Δ250–258 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. © 2002 Wiley‐Liss, Inc.