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. 1991 Oct;11(10):4822–4829. doi: 10.1128/mcb.11.10.4822

Dominant inhibitory mutations in the Mg(2+)-binding site of RasH prevent its activation by GTP.

C L Farnsworth 1, L A Feig 1
PMCID: PMC361448  PMID: 1922022

Abstract

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.

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Selected References

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