The Gab1 PH Domain Is Required for Localization of Gab1 at Sites of Cell-Cell Contact and Epithelial Morphogenesis Downstream from the Met Receptor Tyrosine Kinase

Abstract

Stimulation of the hepatocyte growth factor (HGF) receptor tyrosine kinase, Met, induces mitogenesis, motility, invasion, and branching tubulogenesis of epithelial and endothelial cell lines in culture. We have previously shown that Gab1 is the major phosphorylated protein following stimulation of the Met receptor in epithelial cells that undergo a morphogenic program in response to HGF. Gab1 is a member of the family of IRS-1-like multisubstrate docking proteins and, like IRS-1, contains an amino-terminal pleckstrin homology domain, in addition to multiple tyrosine residues that are potential binding sites for proteins that contain SH2 or PTB domains. Following stimulation of epithelial cells with HGF, Gab1 associates with phosphatidylinositol 3-kinase and the tyrosine phosphatase SHP2. Met receptor mutants that are impaired in their association with Gab1 fail to induce branching tubulogenesis. Overexpression of Gab1 rescues the Met-dependent tubulogenic response in these cell lines. The ability of Gab1 to promote tubulogenesis is dependent on its pleckstrin homology domain. Whereas the wild-type Gab1 protein is localized to areas of cell-cell contact, a Gab1 protein lacking the pleckstrin homology domain is localized predominantly in the cytoplasm. Localization of Gab1 to areas of cell-cell contact is inhibited by LY294002, demonstrating that phosphatidylinositol 3-kinase activity is required. These data show that Gab1 is an important mediator of branching tubulogenesis downstream from the Met receptor and identify phosphatidylinositol 3-kinase and the Gab1 pleckstrin homology domain as crucial for subcellular localization of Gab1 and biological responses.

ACKNOWLEDGMENTS

This research was supported by an operating grant from the National Cancer Institute of Canada with funds from the Canadian Cancer Society (to M.P.), an American Cancer Society Grant, and National Institutes of Health grants NS 34514 and CA69495 (to A.J.W.). Financial support was provided by the Medical Research Council as Fellowships (to C.R.M. and I.R.), a fellowship from the Ministerio de Educacion y Ciencia of Spain (to M.H.-M.), and the Royal Victoria Hospital Research Institute as a studentship (to T.M.F.). M.P. is a Scientist of the Medical Research Council of Canada.

We are grateful to Genetics Institute for recombinant CSF-1, T. Pawson for anti-p85, G. S. Feng for anti-SHP2, M. Pasdar for anti-E-cadherin, and members of the Park laboratory and A. Nepveu for helpful comments.