HEPATOCYTE GROWTH-FACTOR SCATTER FACTOR STIMULATES THE RAS-GUANINE NUCLEOTIDE EXCHANGER

Hepatocyte growth factor/scatter factor (HGF/SF) induces mitogenesis a nd cell dissociation upon binding to the protein-tyrosine kinase recep tor encoded by the MET proto-oncogene (p190MET). The signal transducti on pathways downstream from the receptor activation are largely unknow n. We show that HGF/SF activates Ras protein. HGF/SF stimulation of me tabolically labeled A549 cells raised the amount of Ras-bound radiolab eled guanine nucleotides by over 5-fold. Furthermore, following HGF/SF stimulation of these cells, 50% of Ras was in the GTP-bound active st ate. The uptake by Ras of radiolabeled GTP was also increased by 5-fol d following HGF/SF stimulation in digitonin-permeabilized A549 cells. Moreover, HGF/SF treatment of A549 cells leads to stimulation of the c ytosolic Ras-guanine nucleotide exchange activity, measured as acceler ated release of [H-3]GDP from purified recombinant Ras protein in vitr o, in a dose- and time-dependent manner. Likewise, treatment with the protein-tyrosine kinase inhibitor 3-(1',4'dihydroxytetralyl)methylene- 2-oxindole of GTL-16 cells (featuring a p190MET receptor constitutivel y active) significantly decreased the cytosolic Ras-guanine nucleotide exchange activity. These data demonstrate that HGF/SF activates Ras p rotein by shifting the equilibrium toward the GTP-bound state and incr eases the uptake of guanine nucleotides by Ras, through mechanism(s) i ncluding the activation of a Ras-guanine nucleotide exchanger.