Differential requirement of Grb2 and PI3-kinase in HGF/SF-induced cell motility and tubulogenesis.

Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional cytokine that induces mitogenesis, motility, invasion, and morphogenesis of several epithelial and endothelial cell lines in culture. The receptor for HGF/SF has been identified as the Met tyrosine kinase. To investigate the signaling pathways that are involved in these events, we have generated chimeric receptors containing the extracellular domain of the colony stimulating factor-1 (CSF-1) receptor fused to the transmembrane and intracellular domains of the Met receptor (MET). Madin-Darby canine kidney (MDCK) epithelial cells, expressing the CSF-MET chimera dissociate, scatter and form branching tubules in response to CSF-1. However, cells expressing a mutant CSF-MET receptor containing a phenylalanine substitution for tyrosine 1356 (Y1356F) are unable to scatter or form branching tubules following stimulation with CSF-1. Tyrosine 1356 is essential for the recruitment of multiple substrates including Grb2, the p85 subunit of PI3-kinase, and PLC gamma. To investigate the role of these signaling pathways, we have generated a mutant receptor that selectively fails to associate with Grb2, and have treated MDCK cells with potent inhibitors of PLC gamma, PI3-kinase, and p70S6K, a downstream target of PI3-kinase. Our results implicate pathways downstream from PI3-kinase in cell dissociation and scatter, whereas pathways downstream from Grb2 are required for branching tubulogenesis in MDCK cells.