Involvement of the Switch 2 Domain of Ras in Its Interaction with Guanine Nucleotide Exchange Factors

While Ras proteins are activated by stimulated GDP release, which enables acquisition of the active GTP-bound state, little is known about how guanine nucleotide exchange factors (GEFs) interact with Ras to promote this exchange reaction. Here we report that mutations within the switch 2 domain of Ras (residues 62-69) inhibit activation of Ras by the mammalian GEFs, Sos1, and GRF/CDC25Mm. While mutations in the 62-69 region blocked upstream activation of Ras, they did not disrupt Ras effector functions, including transcriptional activation and transformation of NIH 3T3 cells. Biochemical analysis indicated that the loss of GEF responsiveness of a Ras(69N) mutant was due to a loss of GEF binding, with no change in intrinsic nucleotide exchange activity. Furthermore, structural analysis of Ras(69N) using NMR spectroscopy indicated that mutation of residue 69 had a very localized effect on Ras structure that was limited to alpha-helix 2 of the switch 2 domain. Together, these results suggest that the switch 2 domain of Ras forms a direct interaction with GEFs.