IDENTIFICATION OF RESIDUES CRITICAL FOR RAS(17N) GROWTH-INHIBITORY PHENOTYPE AND FOR RAS INTERACTION WITH GUANINE-NUCLEOTIDE EXCHANGE FACTORS

The Ras(17N) dominant negative antagonizes endogenous Ras function by forming stable, inactive complexes with Ras nucleotide exchange factor s (GEFs; e.g., SOS1). We have used the growth-inhibitory phenotype of Ras(17N) to characterize two aspects of Ras interaction with GEFs. Fir st, we used a nonprenylated version of Ras(17N), designated Ras(17N/18 6S), which no longer associates with the plasma membrane and lacks the growth-inhibitory phenotype, to address the importance of Ras subcell ular location and posttranslational modification for its interaction w ith GEFs. We observed that addition of an N-terminal myristylation sig nal to Ras(17N/186S) restored the growth-inhibitory activity of nonpre nylated Ras(17N). Thus, membrane association, rather than prenylation, is critical for Ras interaction with Ras GEFs. Second, we used a biol ogical 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 th e growth-inhibitory activity of Ras(17N). Since GEF interaction is dis pensable for oncogenic but not normal Ras function, out demonstration that single-amino-acid substitutions at these three positions impaired the transforming activity of normal but not oncogenic Ras provides fu rther support for the role of these residues in Ras-GEF interactions. Finally, Ras(WT) proteins with mutations at these residues were no lon ger activated by mammalian SOS1. Altogether, these results suggest tha t the Ras intracellular location and Ras residues 75 to 78 are critica l for Ras-GEF interaction.