Mutant ras-encoded proteins with altered nucleotide binding exert dominant biological effects.
We report that residues Lys-16 and Asp-119 play critical roles in the guanine nucleotide binding and, consequently, the biological function of the Ha-ras-encoded protein (Ha). Substitution of an asparagine residue for Lys-16 reduces the affinity of Ha for GDP and GTP by a factor of 100 but does not alter the specificity of nucleotide binding. The replacement of Asp-119 with an alanine residue reduces the affinity of Ha for GDP and GTP by a factor of 20 and reduces the relative affinity of Ha for GDP over IDP from 200-500 to 10. Based on these observations, a structural model for the GDP/GTP-binding site of Ha is proposed. By microinjecting purified proteins into NIH 3T3 cells, we observed that the ability of [Ala119]Ha to induce changes characteristic of cellular transformation was much greater than that of normal Ha and similar to that of the oncogenic [Val12, Thr59]Ha. In this assay, [Asn16]Ha and [Val12, Asn16, Thr59]Ha were similar in potency to normal Ha. In yeast cells, Ha proteins with reduced nucleotide affinity exert a dominant temperature-dependent lethality that is avoided by the coexpression of the activated yeast ras gene [Ala18, Val19]RAS2. We interpret the biological consequences of reducing the nucleotide affinity of ras proteins in terms of two opposing factors: a growth-promoting effect, resulting from an increase in the GDP-GTP exchange rate, and a growth-limiting effect, resulting from an increase in the nucleotide-free ras protein species.
D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D004926:Escherichia coli; D005347:Fibroblasts / Q000378:metabolism; D005799:Genes, Dominant; D006150:Guanine Nucleotides / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D009154:Mutation; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras); D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013379:Substrate Specificity
D006150:Guanine Nucleotides; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D016283:Proto-Oncogene Proteins p21(ras)