CD44 is required for two consecutive steps in HGF/c-Met signaling.
ABSTRACT The tyrosine kinase receptor c-Met and its ligand HGF/SF, ezrin, and splice variants of CD44 have independently been identified as tumor metastasis-associated proteins. We now show that these proteins cooperate. A CD44 isoform containing variant exon v6 sequences is strictly required for c-Met activation by HGF/SF in rat and human carcinoma cells, in established cell lines as well as in primary keratinocytes. CD44v6-deficient tumor cells were unable to activate c-Met unless they were transfected with a CD44v6-bearing isoform. Antibodies to two v6-encoded epitopes inhibited autophosphorylation of c-Met by interfering with the formation of a complex formed by c-Met, CD44v6, and HGF/SF. In addition, signal transduction from activated c-Met to MEK and Erk required the presence of the cytoplasmic tail of CD44 including a binding motif for ERM proteins. This suggests a role for ERM proteins and possibly their link to the cortical actin cytoskeleton in signal transfer.
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ABSTRACT: The extracellular protease urokinase is known to be crucially involved in morphogenesis, tissue repair and tumor invasion by mediating matrix degradation and cell migration. Hepatocyte growth factor/scatter factor (HGF/SF) is a secretory product of stromal fibroblasts, sharing structural motifs with enzymes of the blood clotting cascade, including a zymogen cleavage site. HGF/SF promotes motility, invasion and growth of epithelial and endothelial cells. Here we show that HGF/SF is secreted as a single-chain biologically inactive precursor (pro-HGF/SF), mostly found in a matrix-associated form. Maturation of the precursor into the active alpha beta heterodimer takes place in the extracellular environment and results from a serum-dependent proteolytic cleavage. In vitro, pro-HGF/SF was cleaved at a single site by nanomolar concentrations of pure urokinase, generating the active mature HGF/SF heterodimer. This cleavage was prevented by specific urokinase inhibitors, such as plasminogen activator inhibitor type-1 and protease nexin-1, and by antibodies directed against the urokinase catalytic domain. Addition of these inhibitors to HGF/SF responsive cells prevented activation of the HGF/SF precursor. These data show that urokinase acts as a pro-HGF/SF convertase, and suggest that some of the growth and invasive cellular responses mediated by this enzyme may involve activation of HGF/SF.The EMBO Journal 01/1993; 11(13):4825-33. · 9.82 Impact Factor
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ABSTRACT: Hepatocyte growth factor (HGF) has a strong affinity for heparin. About one fourth of HGF secreted from MRC-5 human embryonic lung fibroblast cells was found to be associated with heparin and heparan sulfate proteoglycan on the cell surface and extracellular matrix. To identify heparin-binding sites within the HGF molecule, we constructed variously deleted mutant HGFs and examined their binding ability to an immobilized heparin column. Native HGF and mutant HGFs, including d-K1 (deletion of the first kringle domain), d-K3 (deletion of the third kringle domain), d-K4 (deletion of the fourth kringle domain), d-beta (deletion of beta-chain), and HK1K2 (consisting of the N-terminal hairpin loop and the first two kringle domains), tightly bound to a heparin column, but d-H (deletion of the N-terminal hairpin loop) and d-K2 (deletion of the second kringle domain) markedly decreased binding ability to the column. These observations suggest that the N-terminal hairpin loop and the second kringle domain are essential for the heparin-binding of HGF. The finding that HK1K2 competed the binding of 125I-HGF to immobilized heparin provided additional evidence that the N-terminal half of HGF alpha-chain is the principal heparin-binding site. The hairpin loop in HGF possesses a cluster of basic amino acid residues and a highly positive net charge, when compared with hairpin loop structures in the other proteins, plasminogen and HGF-like protein. The second kringle domain in HGF has the basic amino acid cluster in the central region. Thus, it is likely that the basic clusters in these domains cooperatively contribute to the binding of HGF to the anionic heparin or heparan sulfate molecule.Journal of Biological Chemistry 02/1994; 269(2):1131-6. · 4.65 Impact Factor
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ABSTRACT: The dissociation, migration, and remodeling of epithelial monolayers induced by hepatocyte growth factor (HGF) entail modifications in cell adhesion and in the actin cytoskeleton through unknown mechanisms. Here we report that ezrin, a membrane-cytoskeleton linker, is crucial to HGF-mediated morphogenesis in a polarized kidney-derived epithelial cell line, LLC-PK1. Ezrin is a substrate for the tyrosine kinase HGF receptor both in vitro and in vivo. HGF stimulation causes enrichment of ezrin recovered in the detergent-insoluble cytoskeleton fraction. Overproduction of wild-type ezrin, by stable transfection in LLC-PK1 cells, enhances cell migration and tubulogenesis induced by HGF stimulation. Overproduction of a truncated variant of ezrin causes mislocalization of endogenous ezrin from microvilli into lateral surfaces. This is concomitant with altered cell shape, characterized by loss of microvilli and cell flattening. Moreover, the truncated variant of ezrin impairs the morphogenic and motogenic response to HGF, thus suggesting a dominant-negative mechanism of action. Site-directed mutagenesis of ezrin codons Y145 and Y353 to phenylalanine does not affect the localization of ezrin at microvilli, but perturbs the motogenic and morphogenic responses to HGF. These results provide evidence that ezrin displays activities that can control cell shape and signaling.The Journal of Cell Biology 08/1997; 138(2):423-34. · 10.82 Impact Factor