Concomitant activation of pathways downstream of Grb2 and PI 3-kinase is required for MET-mediated metastasis.
(1/1519)
The Met tyrosine kinase - the HGF receptor - induces cell transformation and metastasis when constitutively activated. Met signaling is mediated by phosphorylation of two carboxy-terminal tyrosines which act as docking sites for a number of SH2-containing molecules. These include Grb2 and p85 which couple the receptor, respectively, with Ras and PI 3-kinase. We previously showed that a Met mutant designed to obtain preferential coupling with Grb2 (Met2xGrb2) is permissive for motility, increases transformation, but - surprisingly - is impaired in causing invasion and metastasis. In this work we used Met mutants optimized for binding either p85 alone (Met2xPI3K) or p85 and Grb2 (MetPI3K/Grb2) to evaluate the relative importance of Ras and PI 3-kinase as downstream effectors of Met. Met2xPI3K was competent in eliciting motility, but not transformation, invasion, or metastasis. Conversely, MetP13K/Grb2 induced motility, transformation, invasion and metastasis as efficiently as wild type Met. Furthermore, the expression of constitutively active PI 3-kinase in cells transformed by the Met2xGrb2 mutant, fully rescued their ability to invade and metastasize. These data point to a central role for PI 3-kinase in Met-mediated invasiveness, and indicate that simultaneous activation of Ras and PI 3-kinase is required to unleash the Met metastatic potential. (+info)
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.
(2/1519)
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. (+info)
The five amino acid-deleted isoform of hepatocyte growth factor promotes carcinogenesis in transgenic mice.
(3/1519)
Hepatocyte growth factor (HGF) is a polypeptide with mitogenic, motogenic, and morphogenic effects on different cell types including hepatocytes. HGF is expressed as two biologically active isotypes resulting from alternative RNA splicing. The roles of each HGF isoform in development, liver regeneration and tumorigenesis have not yet been well characterized. We report the generation and analysis of transgenic mice overexpressing the five amino acid-deleted variant of HGF (dHGF) in the liver by virtue of an albumin expression vector. These ALB-dHGF transgenic mice develop normally, have an enhanced rate of liver regeneration after partial hepatectomy, and exhibit a threefold higher incidence of hepatocellular carcinoma (HCC) beyond 17 months of age. Moreover, overexpression of dHGF dramatically accelerates diethyl-nitrosamine induced HCC tumorigenesis. These tumors arise faster, are significantly larger, more numerous and more invasive than those appearing in non-transgenic littermates. Approximately 90% of female dHGF-transgenic mice had multiple macroscopic HCCs 40 weeks after injection of DEN; whereas the non-transgenic counterparts had only microscopic nodules. Liver tumors and cultured tumor cell lines from dHGF transgenics showed high levels of HGF and c-Met mRNA and protein. Together, these results reveal that in vivo dHGF plays an active role in liver regeneration and HCC tumorigenesis. (+info)
Three receptor genes for plasminogen related growth factors in the genome of the puffer fish Fugu rubripes.
(4/1519)
Plasminogen related growth factors (PRGFs) and their receptors play major roles in embryogenesis, tissue regeneration and neoplasia. In order to investigate the complexity and evolution of the PRGF receptor family we have cloned and sequenced three receptors for PRGFs in the teleost fish Fugu rubripes, a model vertebrate with a compact genome. One of the receptor genes isolated encodes the orthologue of mammalian MET, whilst the other two may represent Fugu rubripes orthologues of RON and SEA. This is the first time three PRGF receptors have been identified in a single species. (+info)
Heparan sulfate-modified CD44 promotes hepatocyte growth factor/scatter factor-induced signal transduction through the receptor tyrosine kinase c-Met.
(5/1519)
CD44 has been implicated in tumor progression and metastasis, but the mechanism(s) involved is as yet poorly understood. Recent studies have shown that CD44 isoforms containing the alternatively spliced exon v3 carry heparan sulfate side chains and are able to bind heparin-binding growth factors. In the present study, we have explored the possibility of a physical and functional interaction between CD44 and hepatocyte growth factor/scatter factor (HGF/SF), the ligand of the receptor tyrosine kinase c-Met. The HGF/SF-c-Met pathway mediates cell growth and motility and has been implicated in tumor invasion and metastasis. We demonstrate that a CD44v3 splice variant efficiently binds HGF/SF via its heparan sulfate side chain. To address the functional relevance of this interaction, Namalwa Burkitt's lymphoma cells were stably co-transfected with c-Met and either CD44v3 or the isoform CD44s, which lacks heparan sulfate. We show that, as compared with CD44s, CD44v3 promotes: (i) HGF/SF-induced phosphorylation of c-Met, (ii) phosphorylation of several downstream proteins, and (iii) activation of the MAP kinases ERK1 and -2. By heparitinase treatment and the use of a mutant HGF/SF with greatly decreased affinity for heparan sulfate, we show that the enhancement of c-Met signal transduction induced by CD44v3 was critically dependent on heparan sulfate moieties. Our results identify heparan sulfate-modified CD44 (CD44-HS) as a functional co-receptor for HGF/SF which promotes signaling through the receptor tyrosine kinase c-Met, presumably by concentrating and presenting HGF/SF. As both CD44-HS and c-Met are overexpressed on several types of tumors, we propose that the observed functional collaboration might be instrumental in promoting tumor growth and metastasis. (+info)
Reduced differentiation potential of primary MyoD-/- myogenic cells derived from adult skeletal muscle.
(6/1519)
To gain insight into the regeneration deficit of MyoD-/- muscle, we investigated the growth and differentiation of cultured MyoD-/- myogenic cells. Primary MyoD-/- myogenic cells exhibited a stellate morphology distinct from the compact morphology of wild-type myoblasts, and expressed c-met, a receptor tyrosine kinase expressed in satellite cells. However, MyoD-/- myogenic cells did not express desmin, an intermediate filament protein typically expressed in cultured myoblasts in vitro and myogenic precursor cells in vivo. Northern analysis indicated that proliferating MyoD-/- myogenic cells expressed fourfold higher levels of Myf-5 and sixfold higher levels of PEA3, an ETS-domain transcription factor expressed in newly activated satellite cells. Under conditions that normally induce differentiation, MyoD-/- cells continued to proliferate and with delayed kinetics yielded reduced numbers of predominantly mononuclear myocytes. Northern analysis revealed delayed induction of myogenin, MRF4, and other differentiation-specific markers although p21 was upregulated normally. Expression of M-cadherin mRNA was severely decreased whereas expression of IGF-1 was markedly increased in MyoD-/- myogenic cells. Mixing of lacZ-labeled MyoD-/- cells and wild-type myoblasts revealed a strict autonomy in differentiation potential. Transfection of a MyoD-expression cassette restored cytomorphology and rescued the differentiation deficit. We interpret these data to suggest that MyoD-/- myogenic cells represent an intermediate stage between a quiescent satellite cell and a myogenic precursor cell. (+info)
Clinical importance of c-Met protein expression in high grade astrocytic tumors.
(7/1519)
The clinical importance of the expression of c-Met protein, the receptor of hepatocyte growth factor/scatter factor, was evaluated in neuroepithelial tissue tumors. c-Met immunohistochemistry was performed using the streptavidin-biotin-peroxidase complex method with anti-c-Met polyclonal antibody. Specimens were classified as c-Met negative (< 30%) or c-Met positive (> or = 30%) according to the proportion of immunopositive cells under microscopic examination. All c-Met-positive cases occurred in high grade astrocytic tumors, not in other neuroepithelial tissue tumors. Most c-Met-positive astrocytic tumors were classified histologically as high grade tumors. Epidermal growth factor-receptor (EGFR) and MIB-1 immunohistochemistry were also performed for high grade astrocytic tumors. Survival analysis was performed for patients with these tumors with variables including c-Met positivity, EGFR positivity, and MIB-1 labeling index. Positivity of c-Met was independent from EGFR positivity and MIB-1 labeling index, and the c-Met-positive group showed a significant shorter survival (p < 0.05). c-Met immunopositivity may be a parameter of biological aggressiveness in high grade astrocytic tumors. Examination of c-Met expression in astrocytic tumors provides significant clinical information, especially as a prognostic factor. (+info)
The multisubstrate docking site of the MET receptor is dispensable for MET-mediated RAS signaling and cell scattering.
(8/1519)
The scatter factor/hepatocyte growth factor regulates scattering and morphogenesis of epithelial cells through activation of the MET tyrosine kinase receptor. In particular, the noncatalytic C-terminal tail of MET contains two autophosphorylation tyrosine residues, which form a multisubstrate-binding site for several cytoplasmic effectors and are thought to be essential for signal transduction. We show here that a MET receptor mutated on the four C-terminal tyrosine residues, Y1311F, Y1347F, Y1354F, and Y1363F, can induce efficiently a transcriptional response and cell scattering, whereas it cannot induce cell morphogenesis. Although the mutated receptor had lost its ability to recruit and/or activate known signaling molecules, such as GRB2, SHC, GAB1, and PI3K, by using a sensitive association-kinase assay we found that the mutated receptor can still associate and phosphorylate a approximately 250-kDa protein. By further examining signal transduction mediated by the mutated MET receptor, we established that it can transmit efficient RAS signaling and that cell scattering by the mutated MET receptor could be inhibited by a pharmacological inhibitor of the MEK-ERK (MAP kinase kinase-extracellular signal-regulated kinase) pathway. We propose that signal transduction by autophosphorylation of the C-terminal tyrosine residues is not the sole mechanism by which the activated MET receptor can transmit RAS signaling and cell scattering. (+info)