Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP.
Substitution of asparagine for serine at position 17 decreased the affinity of rasH p21 for GTP 20- to 40-fold without significantly affecting its affinity for GDP. Transfection of NIH 3T3 cells with a mammalian expression vector containing the Asn-17 rasH gene and a Neor gene under the control of the same promoter yielded only a small fraction of the expected number of G418-resistant colonies, indicating that expression of Asn-17 p21 inhibited cell proliferation. The inhibitory effect of Asn-17 p21 required its localization to the plasma membrane and was reversed by coexpression of an activated ras gene, indicating that the mutant p21 blocked the endogenous ras function required for NIH 3T3 cell proliferation. NIH 3T3 cells transformed by v-mos and v-raf, but not v-src, were resistant to inhibition by Asn-17 p21, indicating that the requirement for normal ras function can be bypassed by these cytoplasmic oncogenes. The Asn-17 mutant represents a novel reagent for the study of ras function by virtue of its ability to inhibit cellular ras activity in vivo. Since this phenotype is likely associated with the preferential affinity of the mutant protein for GDP, analogous mutations might also yield inhibitors of other proteins whose activities are regulated by guanine nucleotide binding.
Feig
L A
LA
Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111.
Cooper
G M
GM
D000818:Animals; D001216:Asparagine; D002455:Cell Division; D002478:Cells, Cultured; D003001:Cloning, Molecular; D006150:Guanine Nucleotides / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics*; D051379:Mice; D008815:Mice, Inbred Strains; D009154:Mutation; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D016283:Proto-Oncogene Proteins p21(ras); D012694:Serine; D014162:Transfection
D006150:Guanine Nucleotides; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D006153:Guanosine Diphosphate; D012694:Serine; D001216:Asparagine; D006160:Guanosine Triphosphate; D016283:Proto-Oncogene Proteins p21(ras)