Met -/- kidneys express epithelial cells that chemotax and form tubules in response to EGF receptor ligands

Cells derived from the inner medullary collecting duct undergo in vitro branching tubulogenesis to both the c-met receptor ligand hepatocyte growth factor (HGF) as well as epidermal growth factor (EGF) receptor ligands. In contrast, many other cultured renal epithelial cells respond in this manner only to HGF, suggesting that these two receptors may use independent signaling pathways during morphogenesis. We have therefore compared the signaling pathways for mIMCD-3 cell morphogenesis in response to EGF and HGF. Inhibition of the p42/44 mitogen-activated protein kinase (MAPK) pathway with the mitogen-activated protein kinase kinase (MKK1) inhibitor PD98059 (50 M) markedly inhibits HGF-induced cell migration with only partial inhibition of EGF-induced cell motility. Similarly, HGF-dependent, but not EGF-dependent, branching morphogenesis was more greatly inhibited by the MKK1 inhibitor. Examination of EGF-stimulated cells demonstrated that extracellular-regulated kinase 5 (ERK5) was activated in response to EGF but not HGF, and that activation of ERK5 was only 60% inhibited by 50 M PD98059. In contrast, the MKK inhibitor U0126 markedly inhibited both ERK1/2 and ERK5 activation and completely prevented HGF-and EGF-dependent migration and branching process formation. Expression of dominant negative ERK5 (dn-BMK1) likewise inhibited EGF-dependent branching process formation, but did not affect HGF-dependent branching process formation. Our results indicate that activation of the ERK1/ERK2 signaling pathway is critical for HGF-induced cell motility/morphogenesis in mIMCD-3 cells, whereas ERK5 appears to be required for EGF-dependent morphogenesis.In renal epithelial cells, cell morphogenesis is an important process in embryonic development and repair of injured tubules. Two cell types commonly utilized to examine renal epithelial morphogenesis are murine mIMCD-3 collecting duct cells and canine MDCK tubular cells. In mIMCD-3 cells, both the c-met receptor ligand hepatocyte growth factor (HGF) 1 and the epidermal growth factor receptor (EGFR) ligands, transforming growth factor-␣ and epidermal growth factor (EGF), are capable of mediating branching morphogenesis (1, 2). In contrast, despite the presence of the EGFR in both cell types, only HGF is capable of mediating branching morphogenesis in MDCK cells, suggesting that these two receptors initiate morphogenesis utilizing similar, but not identical, signaling pathways. Upon activation, both the c-met receptor and the EGFR are tyrosine-phosphorylated and subsequently interact with GRB-2 and GAB1 (GRB-2-associated binding protein) (3, 4). These adapter molecules allow the activated receptor to initiate signaling through phosphoinositide 3-kinase (PI3K), phospholipase C-␥ (PLC␥), SHPTP and mitogen-activated protein kinase (MAPK) (3, 5-10). We have found that activation of the PI3K results in an increase in receptor-mediated activation of PLC␥ and that these signaling pathways then mediate PKC activation as an important component of the cell migratory response (11,12). H...

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Differential MAPK Pathways Utilized for HGF- and EGF-dependent Renal Epithelial Morphogenesis

Karihaloo

O'rourke²,

Nickel

et al. 2001

“…Immunoprecipitation was performed as described previously (10). Briefly, 24 h after transfection, the cells were washed once with ice-cold phosphate-buffered saline, scraped in 500 l of ice-cold 1% Nonidet P-40 lysis buffer (137 mM NaCl, 20 mM Trizma base, 1 mM MgCl, 1 mM CaCl, 10 mM NaF, 1 mM sodium orthovanadate, 10% glycerol, 1% Igepal CA-630, 1 mM phenylmethylsulfonyl fluoride, 0.5 g/ml leupeptin, 0.7 g/ml pepstatin A), and vortexed vigorously.…”

Section: Methodsmentioning

confidence: 99%

Activated ERK2 Interacts with and Phosphorylates the Docking Protein GAB1

Roshan¹,

Kjelsberg²,

Spokes³

et al. 1999

Grb2-associated binder 1 (GAB1) is a docking protein found to associate with the activated c-MET receptor via the MET-binding domain (MBD) and appears to be critical for the tubulogenic actions of this receptor. Pulldown experiments with bacterially expressed MBD and full-length GAB1 revealed the presence of c-MET as well as phosphorylated ERK2 (pERK2). By using purified pERK2 and non-pERK2, we found that GAB1 associates exclusively with the phosphorylated form of the enzyme and that this association does not require mediation by a third protein. When epitope-tagged GAB1 was cotransfected with constitutively active MEK1 into A293 cells, co-immunoprecipitation of GAB1 and pERK2 was observed, demonstrating that this interaction can occur in intact cells. In vitro, both the MBD and full-length GAB1 were found to be substrates for activated ERK2. In intact cells, epitope-tagged GAB1 was found to be basally phosphorylated on serine with an increase following co-transfection with constitutively active MEK1 and the appearance of novel phosphorylation sites detected by phosphopeptide mapping. Thus, it appears that GAB1 can associate directly with phosphorylated ERK2 via the MET-binding domain and that GAB1 then acts as a substrate for the enzyme.In cultured epithelial cells, the tyrosine kinase receptor c-MET and its ligand hepatocyte growth factor (HGF) 1 are capable of inducing mitogenesis, motogenesis (scattering/chemotaxis), and morphogenesis (formation of branching tubules) (1). In the kidney, these distinct actions may comprise the components of more complex events such as ureteric bud branching and repair of renal tubules after injury. Whereas mitogenesis and motogenesis have been described following activation of many tyrosine kinase receptors, epithelial tubulogenesis appears to be relatively specific for ligand activation of members of the MET receptor family and the epidermal growth factor receptor (EGFR) family (2). Recently a docking protein, GAB1 (Grb-2-associated binding protein 1), was found to associate with both c-MET and the EGFR in a ligand-dependent fashion and to be capable of inducing the tubulogenic phenotype (3-5).GAB1 was cloned by as a protein that associated with Grb-2 in an expression library assay. The amino acid sequence of GAB1 suggested that it was a docking protein similar to the insulin receptor substrate 1 family with an amino-terminal pleckstrin homology (PH) domain and a proline-rich carboxyl terminus, as well as tyrosine phosphorylation sites for potential SH2 binding by the phosphoinositide 3-kinase, Grb2, phospholipase C-␥, and SHPTP2. By using a yeast two-hybrid screen with the carboxyl terminus of c-MET as the bait, Weidner et al. (4), and Grb-2-mediated interaction between the proline-rich region of GAB1 and Y 1356 VNV of c-MET (6). The finding that the MBD of GAB1 associates directly with c-MET in a tyrosine-dependent fashion suggests that this domain might associate with other tyrosine-phosphorylated proteins and thus act in a novel fashion to mediate epithelial signaling ...

“…To determine whether autophosphorylation of these tyrosine residues is critical for cMet internalization, wt or mutant cMet was expressed in cells immortalized from the kidneys of cMet null (Ϫ/Ϫ) mice. These epithelial cMet Ϫ/Ϫ cells undergo chemotaxis and branching morphogenesis in response to EGF and transforming growth factor-␣ but not HGF, indicating that cMet signaling is specifically abrogated in these cells (49). We generated several cell lines stably expressing moderate levels of wt or mutant cMet, including tyrosine kinase-deficient K1110A cMet (KinD-cMet) and the multisubstrate-docking site mutant Y1349F,Y1356F (YF-cMet), which is deficient in Gab1 and Grb2 binding.…”

Section: The Tyrosine Kinase Activity and The Docking Site Phosphotyrmentioning

confidence: 99%

Specific Grb2-mediated Interactions Regulate Clathrin-dependent Endocytosis of the cMet-tyrosine Kinase

Lorinczi

Ireton

et al. 2007

Lysosomal degradation of the receptor-tyrosine kinase cMet requires receptor ubiquitination by the E3 ubiquitin ligase Cbl followed by clathrin-dependent internalization. A role for Cbl as an adaptor for cMet internalization has been previously reported. However, the requirement for Cbl ubiquitin ligase activity in this process and its mode of recruitment to cMet has yet to be determined. Cbl can directly bind cMet at phosphotyrosine 1003 or indirectly via Grb2 to phosphotyrosine 1356 in the multisubstrate binding domain of cMet. The direct binding of Cbl with cMet is critical for receptor degradation and not receptor internalization. Here we show a strict requirement for Grb2 and the ubiquitin ligase activity of Cbl for cMet endocytosis. Receptor internalization was impaired by small interfering RNA depletion of Grb2, overexpression of dominant negative Grb2 mutants, and point mutations in the cMet multisubstrate docking site that inhibits the direct association of Grb2 with cMet. The requirement for Grb2 was specific and did not involve the multiadaptor Gab1. cMet internalization was impaired in cells expressing an ubiquitin ligase-deficient Cbl mutant or conjugation-deficient ubiquitin but was unaffected in cells expressing a Cbl mutant that is unable to bind cMet directly. Expression of a Cbl-Grb2 chimera rescued impaired cMet endocytosis in cells depleted of endogenous Grb2. These results indicate that the ubiquitin ligase activity of Cbl is critical for clathrin-dependent cMet internalization and suggest a role for Grb2 as an intermediary linking Cbl ubiquitin ligase activity to this process.