Abstract
Hepatocyte growth factor (HGF)/scatter factor is a multifunctional cytokine that induces mitogenesis, motility, and morphogenesis in epithelial, endothelial, and neuronal cells. The receptor for HGF/scatter factor was identified as c-Met tyrosine kinase, and activation of the receptor induces multiple signaling cascades. To gain further insight into c-Met-mediated multiple events at a molecular level, we isolated several signaling molecules including a novel binding partner of c-Met, SH2 domain-containing inositol 5-phosphatase 1 (SHIP-1). Western blot analysis revealed that SHIP-1 is expressed in the epithelial cell line, Madin-Darby canine kidney (MDCK) cells. SHIP-1 binds at phosphotyrosine 1356 at the multifunctional docking site. Because a number of signaling molecules such as Grb2, phosphatidylinositol 3-kinase, and Gab1 bind to the multifunctional docking site, we further performed an in vitro competition study using glutathione S-transferase- or His-tagged signaling molecules with c-Met tyrosine kinase. Our binding study revealed that SHIP-1, Grb2, and Gab1 bound preferentially over phosphatidylinositol 3-kinase. Surprisingly, MDCK cells that overexpress SHIP-1 demonstrated branching tubulogenesis within 2 days after HGF treatment, whereas wild-type MDCK cells showed tubulogenesis only after 6 days following treatment without altering cell scattering or cell growth potency. Furthermore, overexpression of a mutant SHIP-1 lacking catalytic activity impaired HGF-mediated branching tubulogenesis.
Highlights
Met tyrosine kinase is the receptor for hepatocyte growth factor (HGF)1/scatter factor [1, 2], and signaling via the binding of this receptor to a ligand has been shown to affect a wide range of biological activities, including angiogenesis [3, 4], cel
A total of 800 cDNA clones of various length were obtained, together encoding seven different proteins. Five of these proteins were previously identified as c-Met-binding proteins including Gab1, PI 3-kinase, Grb2, PLC␥, and c-Src
We examined the binding of these mutants with the Src homology 2 (SH2) domain of SH2 domaincontaining inositol 5-phosphatase 1 (SHIP-1), PI 3-kinase, Grb2, Grb10, PLC␥, and c-Src, the Met binding domain (MBD) of Gab1, and the phosphotyrosine binding domain of Shc
Summary
Plasmid Constructions, Transfection, and Yeast Two-hybrid Screening—The construction of LexA fusion genes encoding the cytoplasmic domains of c-Met, c-Fms, TrkA, insulin receptor, and c-Kit downstream of LexA and the expression in Saccharomyces cerevisiae strain L40 were described previously [24, 31, 32]. Coimmunoprecipitation Study—After incubation for 8 h with medium containing 0.02% FCS, MDCK cells were stimulated with HGF (60 ng/ml) for 5, 10, or 30 min. Cells were extracted with lysis buffer containing 50 mM HEPES, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% trasylol, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 400 M sodium orthovanadate. Purified GST fusion proteins were bound for 1 h at 4 °C to glutathione-agarose beads (40 l slurry; Amersham Pharmacia Biotech) suspended in the binding buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% trasylol, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 20 mg/ml bovine serum albumin, and 200 M sodium orthovanadate). After incubation with 32P-labeled autophosphorylated c-Met overnight, beads were washed five times with binding buffer, and pellets were analyzed by SDS-PAGE
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