Dominant inhibitory mutations in the Mg(2+)-binding site of RasH prevent its activation by GTP.
Mol Cell Biol
; 11(10): 4822-9, 1991 Oct.
Article
em En
| MEDLINE
| ID: mdl-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. 83235-3243, 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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteína Oncogênica p21(ras)
/
Guanosina Trifosfato
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Magnésio
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Mutação
Tipo de estudo:
Prognostic_studies
Limite:
Animals
Idioma:
En
Revista:
Mol Cell Biol
Ano de publicação:
1991
Tipo de documento:
Article