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Hepatocyte growth factor/scatter factor stimulates the Ras-guanine nucleotide exchanger
A Graziani1, D Gramaglia, P dalla Zonca
1Department of Biomedical Sciences and Oncology, University of Torino Medical School, Italy.
Abstract:
Hepatocyte growth factor/scatter factor (HGF/SF) induces mitogenesis and cell dissociation upon binding to the protein-tyrosine kinase receptor encoded by the MET proto-oncogene (p190MET). The signal transduction pathways downstream from the receptor activation are largely unknown. We show that HGF/SF activates Ras protein. HGF/SF stimulation of metabolically labeled A549 cells raised the amount of Ras-bound radiolabeled guanine nucleotides by over 5-fold. Furthermore, following HGF/SF stimulation of these cells, 50% of Ras was in the GTP-bound active state. The uptake by Ras of radiolabeled GTP was also increased by 5-fold following HGF/SF stimulation in digitonin-permeabilized A549 cells. Moreover, HGF/SF treatment of A549 cells leads to stimulation of the cytosolic Ras-guanine nucleotide exchange activity, measured as accelerated release of [3H]GDP from purified recombinant Ras protein in vitro, 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 constitutively active) significantly decreased the cytosolic Ras-guanine nucleotide exchange activity. These data demonstrate that HGF/SF activates Ras protein by shifting the equilibrium toward the GTP-bound state and increases the uptake of guanine nucleotides by Ras, through mechanism(s) including the activation of a Ras-guanine nucleotide exchanger.
Insights
Hepatocyte growth factor/scatter factor (HGF/SF) activates Ras protein, a key signaling molecule. This study reveals HGF/SF shifts Ras to its active GTP-bound state, impacting cell growth and movement.
Area of Science:
- Cellular signaling pathways
- Molecular biology
- Oncogenesis
Background:
- Hepatocyte growth factor/scatter factor (HGF/SF) binds the MET receptor tyrosine kinase, initiating cellular responses.
- Downstream signaling pathways activated by MET receptor are not fully understood.
Purpose of the Study:
- To investigate the role of HGF/SF in activating Ras protein.
- To elucidate the mechanism by which HGF/SF influences Ras guanine nucleotide binding.
Main Methods:
- Metabolic labeling of A549 cells to quantify Ras-bound guanine nucleotides.
- Assessing Ras GTP-bound state following HGF/SF stimulation.
- Measuring guanine nucleotide exchange activity in digitonin-permeabilized cells and cell lysates.
- Utilizing a MET receptor tyrosine kinase inhibitor to confirm pathway involvement.
Main Results:
- HGF/SF stimulation increased Ras-bound guanine nucleotides by over 5-fold.
- Approximately 50% of Ras was in the GTP-bound active state after HGF/SF stimulation.
- HGF/SF treatment stimulated cytosolic Ras-guanine nucleotide exchange activity in a dose- and time-dependent manner.
- A MET inhibitor reduced Ras-guanine nucleotide exchange activity.
Conclusions:
- HGF/SF activates Ras protein by promoting its GTP-bound state.
- HGF/SF enhances guanine nucleotide uptake by Ras, potentially via Ras-guanine nucleotide exchange factors.
- These findings clarify a crucial step in MET receptor-mediated signal transduction.