Identification of residues critical for Ras(17N) growth-inhibitory phenotype and for Ras interaction with guanine nucleotide exchange factors

Lawrence A. Quilliam, Kiyoko Kato, Kelly M. Rabun, Mark M. Hisaka, Shayne Y. Huff, Sharon Campbell-Burk, Channing J. Der

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76 Scopus citations

Abstract

The Ras(17N) dominant negative antagonizes endogenous Ras function by forming stable, inactive complexes with Ras guanine nucleotide exchange factors (GEFs; e.g., SOS1). We have used the growth-inhibitory phenotype of Ras(17N) to characterize two aspects of Ras interaction with GEFs. First, we used a nonprenylated version of Ras(17N), designated Ras(17N/186S), which no longer associates with the plasma membrane and lacks the growth-inhibitory phenotype, to address the importance of Ras subcellular location and posttranslational modification for its interaction with GEFs. We observed that addition of an N-terminal myristylation signal to Ras(17N/186S) restored the growth-inhibitory activity of nonprenylated Ras(17N). Thus, membrane association, rather than prenylation, is critical for Ras interaction with Ras GEFs. Second, we used a biological selection approach to identify Ras residues which are critical for Ras(17N) growth inhibition and hence for interaction with Ras GEFs. We identified mutations at residues 75, 76, and 78 that abolished the growth-inhibitory activity of Ras(17N). Since GEF interaction is dispensable for oncogenic but not normal Ras function, our demonstration that single-amino-acid substitutions at these three positions impaired the transforming activity of normal but not oncogenic Ras provides further support for the role of these residues in Ras-GEF interactions. Finally, Ras(WT) proteins with mutations at these residues were no longer activated by mammalian SOS1. Altogether, these results suggest that the Ras intracellular location and Ras residues 75 to 78 are critical for Ras-GEF interaction.

Original languageEnglish (US)
Pages (from-to)1113-1121
Number of pages9
JournalMolecular and cellular biology
Volume14
Issue number2
DOIs
StatePublished - Feb 1994

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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