MET, RON, and SEA are members of a gene family encoding tyrosine kinase receptors with distinctive properties. Besides mediating growth, they control cell dissociation, motility ("scattering"), and formation of branching tubules. While there are transforming counterparts of MET and SEA, no oncogenic forms of RON have yet been identified. A chimeric Tpr-Ron, mimicking the oncogenic form of Met (Tpr-Met) was generated to investigate its transforming potential. For comparison, a chimeric Tpr-Sea was also constructed. Fusion with Tpr induced constitutive activation of the Ron and Sea kinases. While Tpr-Sea was more efficient than Tpr-Met in transformation, Tpr-Ron did not transform NIH 3T3 cells. The differences in the transforming abilities of Tpr-Met and Tpr-Ron were linked to the functional features of the respective tyrosine kinases using the approach of swapping subdomains. Kinetic analysis showed that the catalytic efficiency of Tpr-Ron is five times lower than that of Tpr-Met. Moreover, constitutive activation of Ron resulted in activation of the MAP kinase signaling cascade approximately three times lower than that attained by Tpr-Met. However, constitutive activation of Ron did induce a mitogenic-invasive response, causing cell dissociation, motility, and invasion of extracellular matrices. Tpr-Ron also induced formation of long, unbranched tubules in tridimensional collagen gels. These data show that RON has the potential to elicit a motile-invasive rather than a transformed phenotype.In human malignancies a number of tyrosine kinase receptors are constitutively activated by gene rearrangements that fuse their kinase domains with N-terminal unrelated sequences (for a review, see reference 46). The hepatocyte growth factor (HGF) receptor is converted into an oncogene by rearrangement of the MET proto-oncogene with a gene designated TPR (6). Similar rearrangements involving TPR have also been reported for RAF and TRK (19,26). In Tpr-Met the extracellular, transmembrane, and part of the juxtamembrane domains of the HGF receptor are replaced by the N-terminal sequence of Tpr (36). The kinase activity of the resulting hybrid protein is deregulated, since two leucine-zipper motifs present in the Tpr moiety promote its constitutive dimerization (17,41). This conformation mimics receptor activation following ligand binding.A distinctive property of the HGF receptor (Met) is the ability to evoke a complex response including cell growth, "scattering," and tubulogenesis (for a review, see reference 5). Scattering involves cytoskeletal reorganization and loss of intercellular junctions, followed by active cell migration (14,16,55,61). Epithelial tubulogenesis results from polarized cell growth and invasion of collagen matrices (33). These pleiotropic effects are elicited by the activation of several signalling pathways. Met-mediated signal transduction depends on ligand-induced phosphorylation of two critical carboxy-terminal tyrosine residues, which act as docking sites for multiple SH2-containing cytoplasmic effecto...

Hepatocyte growth factor (HGF) is a paracrine cytokine that influences epithelial morphogenesis by modulating cell-cell adhesion and cell polarity. We have examined the role of HGF in the tight junction (TJ) formation. We followed the assembly and disassembly at the plasma membrane of the major component of the TJ, zonula occludens-1 (ZO-1) protein, after HGF treatment. We applied HGF to the basolateral compartment of MDCK cell monolayers grown on transwell filters to analyze the effect of HGF on polarized cells. Confocal laser scanning microscopy showed that HGF caused a marked reduction of ZO-1 at the lateral sites and a concomitant increase in the cytoplasm. We used the calcium switch assay to analyze the effect of HGF in early TJ development. In MDCK cells cultured in low calcium levels, ZO-1 is distributed intracellularly. The presence of HGF greatly retarded the movement of ZO-1 from the cytosol to the membrane after restoration of normal (1.8 mM) calcium levels for 1.5 and 3 hr. The presence of HGF during the calcium switch caused increased tyrosine phosphorylation of beta-catenin. The incubation of MDCK cells with vanadate, a potent tyrosine-specific phosphatase inhibitor, also affected the ZO-1 localization at the plasma membrane during the calcium switch. This was concomitant with increased tyrosine phosphorylation of beta-catenin. These results suggest that HGF affects the TJ assembly, and this phenomenon may be important in loosening of intercellular junctions and migration of epithelial cells during HGF-induced morphogenesis.

We have used the yeast two-hybrid system to identify proteins that interact with the intracellular portion of the hepatocyte growth factor (HGF) receptor (Met). We isolated a human cDNA encoding a novel protein of 68 kDa, which we termed FAP68. This protein is homologous to a previously described FK506-binding proteinassociated protein, FAP48, which derives from an alternative spliced form of the same cDNA, lacking an 85-nucleotide exon and leading to an early stop codon. Here we show that epithelial cells, in which the HGF receptor is naturally expressed, contain FAP68 and not FAP48 proteins. FAP68 binding to Met requires the last 30 amino acids of the C-terminal tail, which are unique to the HGF receptor. Indeed, FAP68 does not interact with related tyrosine kinases of the Met and insulin receptor families. FAP68 interacts specifically with the inactive form of HGF receptor, such as a kinase-defective receptor or a dephosphorylated wild type receptor. In vivo, endogenous FAP68 can be coimmunoprecipitated with the HGF receptor in the absence of stimuli and not upon HGF stimulation. Thus, FAP68 represents a novel type of effector that interacts with the inactive HGF receptor and is released upon receptor phosphorylation. Free FAP68 exerts a specific stimulatory activity toward the downstream target p70 S6 protein kinase (p70S6K). Significantly, nonphosphorylated HGF receptor prevents FAP68 from stimulating p70S6K. These data suggest a role for FAP68 in coupling HGF receptor signaling to the p70S6K pathway. Hepatocyte growth factor (HGF)1 is a cytokine controlling proliferation and cell-cell dissociation ("scattering") in a broad spectrum of cells. It also induces formation of tubular structures in epithelial and endothelial cells and axon sprouting in neurons (for a review, see Ref. 1 and references within). The HGF receptor is the transmembrane tyrosine kinase encoded by the c-MET proto-oncogene, which has been genetically linked with human cancer (2). The downstream signaling cascades linking the ligand-activated receptor to the control of cell proliferation, scattering, and differentiation have been carefully analyzed. The three biological responses are triggered by phosphorylation of a unique multifunctional docking site containing two tyrosines (Tyr 1349 and Tyr 1356 ), located in the receptor C-terminal tail (3, 4). The two phosphotyrosines interact with multiple cytoplasmic signal transducers either directly, or indirectly, via molecular adaptors such as Grb2 (5), Shc (6), and Gab-1 (7). After HGF stimulation, the receptor binds and activates phosphatidylinositol 3-kinase (PI3K; Refs. 8 and 9) and Src (3), recruits the Grb2/Sos complex stimulating Ras (10), and directly phosphorylates the transcriptional factor Stat-3 (11). The Gab-1 adaptor recruits additional signal transducers, such as PI3K, phospholipase C-␥, and the SHP2 phosphatase, and activates the Rac-c-Jun N-terminal kinase signaling pathway through the bound Crk adaptor (for a review, see Ref. 12 and references within). The use of dominant ne...

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