BIOLOGICAL-ACTIVITY OF HUMAN N-RAS AND K-RAS GENES CONTAINING THE ASN17 DOMINANT-NEGATIVE MUTATION

Substitution of asparagine for serine at position 17 of human H-ras re sults in an impaired GTP-binding activity, causing the mutant Ras prot ein to be locked in a constitutively inactive GDP-bound state. Express ion of this mutant in NIH 3T3 cells inhibits cell proliferation by blo cking endogenous ras function. Plasmids that encode the analogous domi nant negative mutation at position 17 in human N- and K-ras were const ructed. These mutant ras genes, driven by a heavy metal-inducible shee p metallothionein promoter, were introduced by transfection into a var iety of animal cell lines. All three mutant ras genes displayed an inh ibitory phenotype when expressed in NM 3T3 cells. This inhibition coul d be overcome by cotransfection with either activated H-ras or v-raf. These data indicate that the three human Ras proteins probably act thr ough the same signal transduction pathway in NIH 3T3 cells and suggest that these mutations may confer similar phenotypes to other GTP/GDP-b inding proteins.