Genetic definition of ras effector elements.

Abstract

The products of ras genes may function as GTP-binding signal transducers, but the nature of their targets is largely unknown. To define genetically the cellular effector(s) of ras in rat fibroblast transformation, somatic variants that suppress the nontransforming phenotype of v-H-ras effector domain mutations were sought. Variant cell lines perturbed in the ras effector pathway were recovered, and the properties of one suggest that the primary target of ras action may be altered. In this cell variant, no single residue in the ras protein effector domain must be wild type to bring about transformation. In parental rat cells, conservative substitutions are tolerated in six of nine residues. Functional interaction with the target may not require a high degree of structural specificity in the ras protein effector domain.

Interaction of Activated Ras with Raf-1 Alone May Be Sufficient for Transformation of rat2 Cells

“…On the other hand, we have found that in rat2 cells, transformation-defective v-H-ras effector mutants, the activated version of H-Ras found in the Harvey sarcoma virus, can be suppressed by somatic mutations in MEK1 (8,68). The mutant Ras species all interact very weakly with Raf-1, while the selected mutant MEK species act as coupled amplifiers that increase Ras signals above the threshold needed for transformation.…”

“…Included in this study were the v-H-ras effector domain substitutions described previously (68,70). Also studied were new v-H-ras effector domain mutations, including a complete set of codon 32 mutations constructed by the cassette mutagenesis technique (70).…”

“…In selected cases, DNA was isolated from the yeast and plasmid Ras sequences were then recovered by PCR and subjected to DNA sequence analysis. Each of the three Ras mutations selected for further study was reconstructed in v-H-ras by the cassette mutagenesis technique and transferred to a neo retrovirus vector for transformation studies (68). The three possible double-mutation alleles and the triple mutation were similarly constructed and studied.…”

“…The expression of all new species of v-H-ras was documented by immune precipitation of [ 35 S]methionine-labeled protein with monoclonal antibody Y13-238 followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as described previously (68). v-H-ras products expressed in E. coli were purified as described previously (69), except that anion-exchange chromatography involved fast protein liquid chromatography on Mono-Q matrix.…”

“…Helper-free retrovirus stocks were used to engineer drug-resistant rat2 clones that expressed mutant Ras species, and transforming activity was scored by examining the morphology of cells in drug-resistant (Neo ϩ ) colonies as previously described (8,68). The somatic mutant rv68BUR (68) was used as a more sensitive assay of Ras transforming activity.…”