INHIBITION OF SDC25-C-DOMAIN-INDUCED GUANINE-NUCLEOTIDE EXCHANGE BY GUANINE RING BINDING DOMAIN MUTANTS OF V-H-RAS

Guanine-nucleotide exchange is the reaction that controls the activati on of H-ras p21. This reaction is stimulated by the guanine-nucleotide exchange factor. In this study we show that H-ras p21 harboring guani ne ring binding domain (the conserved NKXD motif) mutations, such as N 116I or K117E, are potent inhibitors of H-ras p21 guanine-nucleotide e xchange reaction promoted by SDC25C (Saccharomyces cerevisiae SDC25 C- domain gene product), a guanine-nucleotide exchange factor. The inhibi tion is due to the formation of a stable but catalytically inactive co mplex consisting of the H-ras mutant and SDC25C. By examining the inte raction of v-H-ras(N116I) or v-H-ras(K117E) with SDC25C at different c oncentrations of guanine-nucleotide, we demonstrate that the mechanism of SDC25C-promoted guanine-nucleotide exchange proceeds through the f ollowing pathway. First, SDC25C binds to H-ras and forms an intermedia te H-ras.SDC25C complex; subsequently, an incoming guanine-nucleotide dissociates the complex, releasing SDC25C from H-ras and causes guanin e-nucleotide exchange. This mechanism is similar to the one proposed f or Escherichia coli elongation factor Ts-catalyzed guanine-nucleotide exchange.