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The c-Cbl protooncogene is a negative regulator for several receptor tyrosine kinases (RTKs) through its ability to promote their polyubiquitination. Hence, uncoupling c-Cbl from RTKs may lead to their deregulation. In testing this, we show that c-Cbl promotes ubiquitination of the Met RTK. This requires the c-Cbl tyrosine kinase binding (TKB) domain and a juxtamembrane tyrosine residue on Met. This tyrosine provides a direct binding site for the c-Cbl TKB domain, and is absent in the rearranged oncogenic Tpr-Met variant. A Met receptor, where the juxtamembrane tyrosine is replaced by phenylalanine, is not ubiquitinated and has transforming activity in fibroblast and epithelial cells. We propose the uncoupling of c-Cbl from RTKs as a mechanism contributing to their oncogenic activation.

Hepatocyte growth factor/scatter factor is a multifunctional factor that induces mitogenesis, motility, invasion, and branching tubulogenesis of several epithelial and endothelial cell lines in culture. The receptor for hepatocyte growth factor has been identified as the Mettyrosine kinase. Upon stimulation with hepatocyte growth factor, the Met ␤ subunit becomes highly phosphorylated on tyrosine residues, one of which, tyrosine 1356 within the carboxyl terminus, is crucial for dissociation, motility, and branching tubule formation in Madin-Darby canine kidney epithelial cells. Tyrosine 1356 forms a multisubstrate binding site for the Grb2 and Shc adaptor proteins, the p85 subunit of phosphatidylinositol 3-kinase, phospholipase C␥, and a phosphatase, SHP2. To investigate additional signaling molecules that are activated by the Met receptor, we have identified hepatocyte growth factor-induced phosphoproteins in tubular epithelial cells. We have established that proteins of 100 -130 kDa are highly phosphorylated following stimulation of epithelial cells and that one of these is the Grb2-associated binding protein Gab1, a possible insulin receptor substrate-1-like signal transducer. We show that Gab1 is the major substrate for the Met kinase in vitro and in vivo. Association of Gab1 with Met requires a functional Grb2 binding site involving tyrosine 1356 and to a lesser extent tyrosine 1349. Met receptor mutants that fail to induce branching tubulogenesis are impaired in their ability to interact with Gab1, suggesting that Gab1 may play a role in these processes.

The Tpr-Met oncoprotein, which is a member of a family of tyrosine kinase oncoproteins generated following genomic rearrangement, consists of the catalytic kinase domain of the hepatocyte growth factor/scatter factor receptor tyrosine kinase (Met) fused downstream from sequences encoded by the tpr gene. We have previously demonstrated that a single tyrosine residue in the carboxyl terminus, Tyr 489 , is highly phosphorylated and is essential for efficient transformation of Fr3T3 fibroblasts by Tpr-Met and for the association of Tpr-Met with the Grb2 adaptor protein and phosphatidylinositol 3-kinase. We show here that Tyr 489 is also required for association of Tpr-Met with phospholipase C␥ and the tyrosine phosphatase, SHPTP2/Syp. To distinguish which of these substrates are required for cell transfor-

The Tpr-Met oncoprotein consists of the catalytic kinase domain of the hepatocyte growth factor/scatter factor receptor tyrosine kinase (Met) fused downstream from sequences encoded by the tpr gene. Tpr-Met is a member of a family of tyrosine kinase oncoproteins generated following genomic rearrangement and has constitutive kinase activity. We have previously demonstrated that a single carboxyl-terminal tyrosine residue, Tyr 489 , is essential for efficient transformation of Fr3T3 fibroblasts by Tpr-Met and forms a multisubstrate binding site for Grb2, phosphatidylinositol 3 kinase, phospholipase C␥, SHP2, and an unknown protein of 110 kDa. A mutant Tpr-Met protein that selectively fails to bind Grb2 has reduced transforming activity, implicating pathways downstream of Grb2 in Tpr-Met mediated cell transformation. We show here that the 110-kDa TprMet substrate corresponds to the recently identified Grb2-associated protein, Gab1. Moreover, we show that tyrosine phosphorylation of the Cbl protooncogene product as well as Gab1 required Tyr 489 and correlated with the ability of Tpr-Met to associate with Grb2 and to transform cells, providing evidence that pathways downstream of Gab1 and/or Cbl may play a role in TprMet-mediated cell transformation.

Hepatocyte growth factor is a multifunctional cytokine that induces mitogenesis, motility, invasion, and branching tubulogenesis of several epithelial and endothelial cell lines in culture. The receptor for hepatocyte growth factor has been identified as the Met tyrosine kinase. To investigate the signaling pathways that are involved in these events, we have generated chimeric receptors containing the colony stimulating factor-1 receptor fused to the transmembrane and intracellular domains of the Met receptor. Madin-Darby canine kidney epithelial cells expressing the Met chimera dissociate scatter and form branching tubules in response to colony stimulating factor-1. From structure-function analyses, tyrosine residue 1356 within the carboxyl terminus of the Met receptor is critical for these events. The amino acid sequence downstream from tyrosine 1356 represents a consensus binding site for the Grb2 adaptor protein and forms a multisubstrate binding site for the p85 subunit of phosphatidylinositol 3-kinase, phospholipase Cgamma, and the Shc adaptor protein. To distinguish which of these signaling pathways are required, we generated a mutant receptor that selectively fails to associate with the Grb2 adaptor protein. Cells expressing this mutant receptor scattered but were unable to form branching tubules, indicating that a Grb2 binding site in the Met receptor is critical for morphogenic responses.

Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional cytokine that induces mitogenesis, motility, invasion, and morphogenesis of several epithelial and endothelial cell lines in culture. The receptor for HGF/SF has been identified as the Met tyrosine kinase. To investigate the signaling pathways that are involved in these events, we have generated chimeric receptors containing the extracellular domain of the colony stimulating factor-1 (CSF-1) receptor fused to the transmembrane and intracellular domains of the Met receptor (MET). Madin-Darby canine kidney (MDCK) epithelial cells, expressing the CSF-MET chimera dissociate, scatter and form branching tubules in response to CSF-1. However, cells expressing a mutant CSF-MET receptor containing a phenylalanine substitution for tyrosine 1356 (Y1356F) are unable to scatter or form branching tubules following stimulation with CSF-1. Tyrosine 1356 is essential for the recruitment of multiple substrates including Grb2, the p85 subunit of PI3-kinase, and PLC gamma. To investigate the role of these signaling pathways, we have generated a mutant receptor that selectively fails to associate with Grb2, and have treated MDCK cells with potent inhibitors of PLC gamma, PI3-kinase, and p70S6K, a downstream target of PI3-kinase. Our results implicate pathways downstream from PI3-kinase in cell dissociation and scatter, whereas pathways downstream from Grb2 are required for branching tubulogenesis in MDCK cells.

Dispersal of epithelial cells is an important aspect of tumorigenesis, and invasion. Factors such as hepatocyte growth factor induce the breakdown of cell junctions and promote cell spreading and the dispersal of colonies of epithelial cells, providing a model system to investigate the biochemical signals that regulate these events. Multiple signaling proteins are phosphorylated in epithelial cells during hepatocyte growth factor-induced cell dispersal, including c-Cbl, a protooncogene docking protein with ubiquitin ligase activity. We have examined the role of c-Cbl and a transforming variant (70z-Cbl) in epithelial cell dispersal. We show that the expression of 70z-Cbl in Madin-Darby canine kidney epithelial cells resulted in the breakdown of cell-cell contacts and alterations in cell morphology characteristic of epithelial-mesenchymal transition. Structurefunction studies revealed that the amino-terminal portion of c-Cbl, which corresponds to the Cbl phosphotyrosine-binding/Src homology domain 2 , is sufficient to promote the morphological changes in cell shape. Moreover, a point mutation at Gly-306 abrogates the ability of the Cbl Src homology domain 2 to induce these morphological changes. Our results identify a role for Cbl in the regulation of epithelial-mesenchymal transition, including loss of adherens junctions, cell spreading, and the initiation of cell dispersal. INTRODUCTIONThe dissociation and migration of epithelial cell sheets are required during normal embryonic development and during pathological situations such as the dispersal of tumor cells (Gherardi, 1991). Epithelial cell dispersal is a complex process that requires the breakdown of cell-cell junctions in addition to the remodeling of the actin cytoskeleton and cell adhesion complexes. These changes contribute to a transition from an epithelial morphology toward a more mesenchymal fibroblastic phenotype, referred to as epithelial-mesenchymal transition (Boyer et al., 1996). Hepatocyte growth factor (HGF) and its receptor, the Met tyrosine kinase, is one of the predominant modulators of epithelial-mesenchymal transition described to date; it provides a model to examine the molecular signals involved in the regulation of epithelial cell dispersal .Little is known about the signaling pathways that regulate the dissociation and dispersal of epithelial cell sheets or colonies. To identify signal transduction pathways that are involved in the dispersal and invasion of epithelial cell sheets, we and others have performed structure-function analyses of the Met receptor tyrosine kinase with the use of an epithelial Madin-Darby canine kidney (MDCK) cell model (Komada and Kitamura, 1993;Weidner et al., 1993;Zhu et al., 1994a). Downstream from the Met receptor, the most highly tyrosine-phosphorylated proteins correspond to the Cbl and Gab1 docking proteins Nguyen et al., 1997;Maroun et al., 1999). Gab1 acts as a docking protein (Holgado-Madruga et al., 1996) that promotes a morphogenic program in response to Met activation in epithelial cells (Weidner...