Dominant inhibitory mutations in the Mg(2+)-binding site of RasH prevent its activation by GTP.
We have previously demonstrated that substitution of Asn for Ser at position 17 of RasH yields a dominant inhibitory protein whose expression in cells interferes with endogenous Ras function (L. A. Feig, and G. M. Cooper, Mol. Cell. Biol. 8:3235-3243, 1988). Subsequent structural studies have shown that the hydroxyl group of Ser-17 contributes to the binding of Mg2+ associated with bound nucleotide. In this report, we show that more subtle amino acid substitutions at this site that would be expected to interfere with complexing Mg2+, such as Cys or Ala, also generated dominant inhibitory mutants. In contrast, a Thr substitution that conserves a reactive hydroxyl group maintained normal Ras function. These results argue that the defect responsible for the inhibitory activity is improper coordination of Mg2+. Preferential affinity for GDP, observed in the original Asn-17 mutant, was found exclusively in inhibitory mutants. However, this binding specificity did not completely block the mutant proteins from binding GTP in vivo since introduction of the autophosphorylation site, Thr-59, in 17N Ras resulted in the phosphorylation of the double mutant in cells. Furthermore, inhibitory mutants failed to activate a model downstream target, yeast adenylate cyclase, even when bound to GTP. Thus, the consequence of improper complexing of Mg2+ was to lock the protein in a constitutively inactive state. A model is presented to explain how these properties could cause the mutant protein to inhibit the activation of endogenous Ras by competing for a guanine nucleotide-releasing factor.
Department of Biochemistry, Tufts University Health Sciences Campus, Boston, Massachusetts 02111.
C L
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Farnsworth
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Feig
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D006160:Guanosine Triphosphate / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism*; D008954:Models, Biological; D009154:Mutation; D015689:Oncogene Protein p21(ras) / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D051381:Rats
D006160:Guanosine Triphosphate; D015689:Oncogene Protein p21(ras); D000262:Adenylyl Cyclases; D008274:Magnesium