We show that Gab2 recruitment to the Met receptor is dependent on the Grb2 adapter protein. into CRA-026440 Gab2 increases Gab2 association with Met, but does not confer on Gab2 the ability to promote epithelial morphogenesis. We propose that the Grb2-independent recruitment of Gab proteins to Met is necessary but not sufficient to promote epithelial morphogenesis. INTRODUCTION Hepatocyte growth CRA-026440 factor (HGF) is a mesenchymally derived factor with pleiotropic activities. In vivo, HGF stimulates cell proliferation and survival, as well as cell dispersal, motility, and a morphogenic program in a wide range of cellular targets, including epithelial, endothelial, and hematopoietic cells, and neurons (reviewed in Gherardi and Stoker, 1991 ; Michalopoulos, 1995 ; Zarnegar and Michalopoulos, 1995 ). These effects are fundamental for the diverse biological functions of HGF observed in vivo, including embryogenesis, organogenesis, angiogenesis, tissue regeneration, axonal outgrowth, wound healing, and invasion by tumor CRA-026440 cells (reviewed in Rosen strain, by induction with isopropyl-1-thio–d-galactopyranoside. GST fusion proteins (0.5C1 g) immobilized on glutathione-Sepharose beads were incubated with either 300 g of lysate from 293T cells transiently expressing the Met receptor, or 350 g of lysate from MDCK cells expressing Gab1 or Gab2, stimulated or not with HGF for 15 min. After rocking in 0.5% Triton X-100 lysis buffer for at least 2 h at 4C, bound proteins were washed three times with lysis buffer. Approximately 1 mg of protein was used for immunoprecipitations from 293T or MDCK cell lysates. Immunoprecipitation and Western blotting were performed as described in Maroun (Nepean, Ontario Canada). Rabbit anti-SHP-2 and rabbit anti-MAPK were kindly provided by Dr. Nicole Beauchemin (McGill Cancer Centre, Montreal, PQ, Canada), and Dr. John Blenis (Harvard Medical School, Boston, MA), respectively. HGF and recombinant CSF were generously provided by Dr. George Van de Woude (Van Andel Research Institute, Grand Rapids, MI) and Genetics Institute (Boston, MA), respectively. HA-tagged Gab2, Gab2MBD-pGEX 4T-1, and anti-Gab2 sera were gifts from Dr. Ben Neel (Beth Isreal-Deaconess Medical Center, Boston, MA). SH2 domain containing GST fusion proteins was generously provided by Dr. Bruce Mayer (University of Connecticut Health Center, Farmington, CT) (GST-Crk II SH2); Dr. Tony Pawson (Samuel Lunenfeld Research Institute, Toronto, Ontario, Cananda) (GST-PLC and GST-p85 SH2); and Dr. Gen-Sheng Feng (Burnham Institute, La Jolla, CA) (GST-SHP-2). RESULTS Gab1 and Gab2 Show a Similar Localization to Cell-Cell Junctions in MDCK Cells To assess whether Gab family proteins mediate similar biological responses, we have examined the functional Kcnj12 role of Gab2 in MDCK epithelial cells. Using Gab2 and Gab1 specific sera, we show that Gab2 is endogenously expressed in MDCK cells, and that Gab2 is phosphorylated within 5 min after HGF stimulation and is sustained for at least 2 h, similar to the time course of endogenous Gab1 phosphorylation (Figure ?(Figure1A).1A). Because both Gab1 and Gab2 are expressed in MDCK cells and are phosphorylated upon HGF stimulation, this indicates a potential redundancy of function. To investigate this possibility, we generated stable cell lines expressing HA-tagged Gab2. We showed previously that Gab1 is localized to sites of cell-cell attachment in the presence of serum but localizes diffusely to the cytosol after serum starvation (Maroun (2000) , who showed that insertion of 13 amino acids, GMQVPPPAHMGFR (aa 487C499), into Gab2 was sufficient to mediate Met association in a yeast two-hybrid assay (Schaeper G(2000) , these amino acids are critical for the Grb2-independent recruitment of Gab1 to the Met receptor and their insertion confers Grb2-independent recruitment of Gab2 to Met (Gab2/1/2*; Figure ?Figure6,6, ACD). The mutation of three proline residues within this region of Gab1 (Gab13P, 491C493) abolishes Grb2-independent recruitment of Gab1 (Figure ?(Figure5,5, B and C) and decreases HGF-induced tyrosine phosphorylation of Gab1 (Figure ?(Figure6E).6E). Noteably, the Gab13P mutant fails to rescue the.
