c-Cbl is involved in Met signaling in B cells and mediates hepatocyte growth factor-induced receptor ubiquitination

Hepatocyte growth factor/scatter factor (HGF) and its receptor tyrosine kinase Met are key regulators of epithelial motility and morphogenesis. Recent studies indicate that the HGF/Met pathway also plays a role in B cell differentiation, whereas uncontrolled Met signaling may lead to B cell neoplasia. These observations prompted us to explore HGF/Met signaling in B cells. In this study, we demonstrate that HGF induces strong tyrosine phosphorylation of the proto-oncogene product c-Cbl in B cells and increases Cbl association with the Src family tyrosine kinases Fyn and Lyn, as well as with phosphatidylinositol-3 kinase and CrkL. In addition, we demonstrate that c-Cbl mediates HGF-induced ubiquitination of Met. This requires the juxtamembrane tyrosine Y1001 (Y2) of Met, but not the multifunctional docking site (Y14/15) or any additional C-terminal tyrosine residues (Y13-16). In contrast to wild-type c-Cbl, the transforming mutants v-Cbl and 70Z/3 Cbl, which lack the ubiquitin ligase RING finger domain, suppress Met ubiquitination. Our findings identify c-Cbl as a negative regulator of HGF/Met signaling in B cells, mediating ubiquitination and, consequently, proteosomal degradation of Met, and suggest a role for Cbl in Met-mediated tumorigenesis.     

Compromised E-cadherin adhesion and epithelial barrier function with activation of G protein-coupled receptors is rescued by Y-to-F mutations in beta-catenin

Activation of the type 1 histamine (H1) or the type 2 protease-activated (PAR-2) G protein-coupled receptors interrupts E-cadherin adhesion and decreases the transepithelial resistance (TER) of epithelium. Several reports suggest that cadherin adhesive function depends on the association of cadherin with beta-catenin and that this association is regulated by phosphorylation of tyrosines in beta-catenin. We tested the hypothesis that loss of cadherin adhesion and compromise of TER on activation of the H1 or PAR-2 receptor is due to phosphorylation of tyrosines in beta-catenin. L cells were stably transfected to express E-cadherin (L-E-cad cells) and H1 (L-H1-E-cad cells). L cells and Madin-Darby canine kidney (MDCK) cells constitutively express PAR-2. Stably transfected L-E-cad, L-H1-E-cad, and MDCK cells were also stably transfected with FLAG-tagged wild-type (WT) or mutant beta-catenin, converting tyrosine 142, 489, or 654 to the nonphosphorylatable mimetic, phenylalanine (WT, Y142F, Y489F, or Y654F). Activation of H1 or PAR-2 interrupted adhesion to an immobilized E-cadherin-Fc fusion protein of L-H1-E-cad, L-E-cad, and MDCK cells expressing WT or Y142F beta-catenin but did not interrupt adhesion of L-H1-E-cad, L-E-cad, and MDCK cells expressing the Y489F or Y654F mutant beta-catenins. PAR-2 activation decreased the TER of monolayers of MDCK cells expressing WT or Y142F beta-catenin 40-45%. However, PAR-2 activation did not decrease the TER of monolayers of MDCK cells expressing Y489F or Y654F beta-catenin. The protein tyrosine phosphatase PTP1B binds to the cadherin cytoplasmic domain and dephosphorylates beta-catenin. Inhibition of PTP1B interrupted adhesion to E-cadherin-Fc of MDCK cells expressing WT beta-catenin but did not affect the adhesion of MDCK cells expressing Y489F or Y654F beta-catenin. Similarly, inhibition of PTP1B compromised the TER of MDCK cells expressing WT beta-catenin but did not affect the TER of MDCK cells expressing Y489F or Y654F beta-catenin. We conclude that phosphorylation of tyrosines 489 and 654 in beta-catenin is a necessary step in the process by which G protein-coupled H1 and PAR-2 receptors interrupt E-cadherin adhesion. We also conclude that activation of PAR-2 has no effect on the TER without first interrupting E-cadherin adhesion.     

Reactive oxygen species mediate Met receptor transactivation by G protein-coupled receptors and the epidermal growth factor receptor in human carcinoma cells

Cross-communication between the Met receptor tyrosine kinase and the epidermal growth factor receptor (EGFR) has been proposed to involve direct association of both receptors and EGFR kinase-dependent phosphorylation. Here, we demonstrate that in human hepatocellular and pancreatic carcinoma cells the Met receptor becomes tyrosine phosphorylated not only upon EGF stimulation but also in response to G protein-coupled receptor (GPCR) agonists. Whereas specific inhibition of the EGFR kinase activity blocked EGF- but not GPCR agonist-induced Met receptor transactivation, it was abrogated in the presence of a reducing agent or treatment of cells with a NADPH oxidase inhibitor. Both GPCR ligands and EGF are further shown to increase the level of reactive oxygen species within the cell. Interestingly, stimulation of the Met receptor by either GPCR agonists, EGF or its cognate ligand HGF, resulted in release of Met-associated beta-catenin and in its Met-dependent translocation into the nucleus, as analyzed by small interfering RNA-mediated knockdown of the Met receptor. Our results provide a new molecular explanation for cell surface receptor cross-talk involving the Met receptor and thereby link the wide diversity of GPCRs and the EGFR to the oncogenic potential of Met signaling in human carcinoma cells.     

The regulation of cadherin-mediated adhesion by tyrosine phosphorylation/dephosphorylation of beta-catenin

The formation of stable cell-cell adhesions by type I cadherins depends on the association of their cytoplasmic domain with beta-catenin, and of beta-catenin with alpha-catenin. The binding of beta-catenin to these partners is regulated by phosphorylation of at least three critical tyrosine residues. Each of these residues is targeted by one or more specific kinases: Y142 by Fyn, Fer and cMet; Y489 by Abl; and Y654 by Src and the epidermal growth factor receptor. Developmental and physiological signals have been identified that initiate the specific phosphorylation and dephosphorylation of these residues, regulating cadherin function during neurite outgrowth, permeability of airway epithelium and synapse remodeling, and possibly initiating epithelial cell migration during development and metastasis.     

P68 RNA helicase mediates PDGF-induced epithelial mesenchymal transition by displacing Axin from beta-catenin

The nuclear p68 RNA helicase (referred to as p68) is a prototypical member of the DEAD box family of RNA helicases. The protein plays a very important role in early organ development. In the present study, we characterized the tyrosine phosphorylation of p68 under platelet-derived growth factor (PDGF) stimulation. We demonstrated that tyrosine phosphorylation of p68 at Y593 mediated PDGF-stimulated epithelial-mesenchymal transition (EMT). We showed that PDGF treatment led to phosphorylation of p68 at Y593 in the cell nucleus. The Y593-phosphorylated p68 (referred to as phosphor-p68) promotes beta-catenin nuclear translocation via a Wnt-independent pathway. The phosphor-p68 facilitates beta-catenin nuclear translocation by blocking phosphorylation of beta-catenin by GSK-3beta and displacing Axin from beta-catenin. The beta-catenin nuclear translocation and subsequent interaction with the LEF/TCF was required for the EMT process. These data demonstrated a novel mechanism of phosphor-p68 in mediating the growth factor-induced EMT and uncovered a new pathway to promote beta-catenin nuclear translocation.     

Acetaldehyde dissociates the PTP1B-E-cadherin-beta-catenin complex in Caco-2 cell monolayers by a phosphorylation-dependent mechanism

Interactions between E-cadherin, beta-catenin and PTP1B (protein tyrosine phosphatase 1B) are crucial for the organization of AJs (adherens junctions) and epithelial cell-cell adhesion. In the present study, the effect of acetaldehyde on the AJs and on the interactions between E-cadherin, beta-catenin and PTP1B was determined in Caco-2 cell monolayers. Treatment of cell monolayers with acetaldehyde induced redistribution of E-cadherin and beta-catenin from the intercellular junctions by a tyrosine phosphorylation-dependent mechanism. The PTPase activity associated with E-cadherin and beta-catenin was significantly reduced and the interaction of PTP1B with E-cadherin and beta-catenin was attenuated by acetaldehyde. Acetaldehyde treatment resulted in phosphorylation of beta-catenin on tyrosine residues, and abolished the interaction of beta-catenin with E-cadherin by a tyrosine kinase-dependent mechanism. Protein binding studies showed that the treatment of cells with acetaldehyde reduced the binding of beta-catenin to the C-terminal region of E-cadherin. Pairwise binding studies using purified proteins indicated that the direct interaction between E-cadherin and beta-catenin was reduced by tyrosine phosphorylation of beta-catenin, but was unaffected by tyrosine phosphorylation of E-cadherin-C. Treatment of cells with acetaldehyde also reduced the binding of E-cadherin to GST (glutathione S-transferase)-PTP1B. The pairwise binding study showed that GST-E-cadherin-C binds to recombinant PTP1B, but this binding was significantly reduced by tyrosine phosphorylation of E-cadherin. Acetaldehyde increased the phosphorylation of beta-catenin on Tyr-331, Tyr-333, Tyr-654 and Tyr-670. These results show that acetaldehyde induces disruption of interactions between E-cadherin, beta-catenin and PTP1B by a phosphorylation-dependent mechanism.     

Depolarization drives beta-Catenin into neuronal spines promoting changes in synaptic structure and function

Activity-induced changes in adhesion molecules may coordinate presynaptic and postsynaptic plasticity. Here, we demonstrate that beta-catenin, which mediates interactions between cadherins and the actin cytoskeleton, moves from dendritic shafts into spines upon depolarization, increasing its association with cadherins. beta-catenin's redistribution was mimicked or prevented by a tyrosine kinase or phosphatase inhibitor, respectively. Point mutations of beta-catenin's tyrosine 654 altered the shaft/spine distribution: Y654F-beta-catenin-GFP (phosphorylation-prevented) was concentrated in spines, whereas Y654E-beta-catenin-GFP (phosphorylation-mimic) accumulated in dendritic shafts. In Y654F-expressing neurons, the PSD-95 or associated synapsin-I clusters were larger than those observed in either wild-type-beta-catenin or also Y654E-expressing neurons. Y654F-expressing neurons exhibited a higher minifrequency. Thus, neural activity induces beta-catenin's redistribution into spines, where it interacts with cadherin to influence synaptic size and strength.     

Cooperative effect of hepatocyte growth factor and fibronectin in anchorage-independent survival of mammary carcinoma cells: requirement for phosphatidylinositol 3-kinase activity.

Anchorage-independent survival and growth are critical characteristics of malignant cells. We showed previously that the addition of exogenous hepatocyte growth factor (HGF) and the presence of fibronectin fibrils stimulate anchorage-independent colony growth of a murine mammary carcinoma, SP1, which expresses both HGF and HGF receptor (Met; R. Saulnier et al., Exp. Cell Res., 222: 360-369, 1996). We now show that tyrosine phosphorylation of Met in carcinoma cells is augmented by cell adhesion and spreading on fibronectin substratum. In contrast, detached serum-starved cells exhibit reduced tyrosine phosphorylation of Met and undergo apoptotic cell death within 18-24 h. Under these conditions, the addition of HGF stimulates tyrosine phosphorylation of Met and restores survival of carcinoma cells. Soluble fibronectin also stimulates cell survival and shows a cooperative survival response with HGF but does not affect tyrosine phosphorylation of Met; these results indicate that fibronectin acts via a pathway independent of Met in detached cells. We demonstrated previously that inhibition of phosphatidylinositol (PI) 3-kinase activity blocks HGF-induced DNA synthesis of carcinoma cells (N. Rahimi et al., J. Biol. Chem., 271: 24850-24855, 1996). We now show in detached cells a cooperative effect of HGF and FN in the activation of PI 3-kinase and on the phosphorylation of PKB/Akt at serine 473. PI 3-kinase activity is also required for the HGF- and fibronectin-induced survival responses, as well as anchorage-independent colony growth. However, c-Src kinase or MEK1/2 activities are not required for the cell survival effect. Together, these results demonstrate that the PI 3-kinase/Akt pathway is a key effector of the HGF- and fibronectin-induced survival response of breast carcinoma cells under detached conditions and corroborate an interaction between integrin and HGF/ Met signalling pathways in the development of invasive breast cancer.     

Abl functions as a negative regulator of Met-induced cell motility via phosphorylation of the adapter protein CrkII

HGF, the ligand for the Met receptor tyrosine kinase, is a potent modulator of epithelial-mesenchymal transition and dispersal of epithelial cells, which are processes that play a crucial role in cell motility during normal development and malignant transformation. We and others have shown earlier that the adapter protein CrkII and its associated proteins positively regulate cell migratory events in response to both haptotactic and chemotactic stimuli, including HGF. Here, we demonstrate for the first time that phosphorylation of CrkII serves as a negative feedback loop to regulate motile responses upon Met stimulation. Thus, we found that the treatment of cells with HGF induces tyrosine phosphorylation of CrkII at Y221, which in turn results in inhibition of CrkII signaling via formation of an intramolecular pY221-SH2-domain interaction. Accordingly, expression of a mutant form of CrkII, CrkII-Y221F, which is resistant to phosphorylation at this negative regulatory site, enhanced Met-induced cell motility. Furthermore, we demonstrate here that the Met-induced CrkII phosphorylation depends on the Abl tyrosine kinase activity. As a corollary, we found that Abl inhibitors, such as the STI571 compound, significantly enhanced Met-induced cell motility, but failed to do so in cells that expressed the CrkII-Y221F mutant protein. Taken together, these results demonstrate that the Abl tyrosine kinase functions as a negative regulator of Met-induced cell migration, and that it does so by inducing CrkII phosphorylation at the site Y221.     

Regulation of E-cadherin/Catenin association by tyrosine phosphorylation

Alteration of cadherin-mediated cell-cell adhesion is frequently associated to tyrosine phosphorylation of p120- and beta-catenins. We have examined the role of this modification in these proteins in the control of beta-catenin/E-cadherin binding using in vitro assays with recombinant proteins. Recombinant pp60(c-src) efficiently phosphorylated both catenins in vitro, with stoichiometries of 1.5 and 2.0 mol of phosphate/mol of protein for beta-catenin and p120-catenin, respectively. pp60(c-src) phosphorylation had opposing effects on the affinities of beta-catenin and p120 for the cytosolic domain of E-cadherin; it decreased (in the case of beta-catenin) or increased (for p120) catenin/E-cadherin binding. However, a role for p120-catenin in the modulation of beta-catenin/E-cadherin binding was not observed, since addition of phosphorylated p120-catenin did not modify the affinity of phosphorylated (or unphosphorylated) beta-catenin for E-cadherin. The phosphorylated Tyr residues were identified as Tyr-86 and Tyr-654. Experiments using point mutants in these two residues indicated that, although Tyr-86 was a better substrate for pp60(c-src), only modification of Tyr-654 was relevant for the interaction with E-cadherin. Transient transfections of different mutants demonstrated that Tyr-654 is phosphorylated in conditions in which adherens junctions are disrupted and evidenced that binding of beta-catenin to E-cadherin in vivo is controlled by phosphorylation of beta-catenin Tyr-654.     

HDAC6 is required for epidermal growth factor-induced beta-catenin nuclear localization

Nuclear translocation of beta-catenin is a hallmark of Wnt signaling and is associated with various cancers. In addition to the canonical Wnt pathway activated by Wnt ligands, growth factors such as epidermal growth factor (EGF) also induce beta-catenin dissociation from the adherens junction complex, translocation into the nucleus, and activation of target genes such as c-myc. Here we report that EGF-induced beta-catenin nuclear localization and activation of c-myc are dependent on the deacetylase HDAC6. We show that EGF induces HDAC6 translocation to the caveolae membrane and association with beta-catenin. HDAC6 deacetylates beta-catenin at lysine 49, a site frequently mutated in anaplastic thyroid cancer, and inhibits beta-catenin phosphorylation at serine 45. HDAC6 inactivation blocks EGF-induced beta-catenin nuclear localization and decreases c-Myc expression, leading to inhibition of tumor cell proliferation. These results suggest that EGF-induced nuclear localization of beta-catenin is regulated by HDAC6-dependent deacetylation and provide a new mechanism by which HDAC inhibitors prevent tumor growth.     

Identification of two C-terminal autophosphorylation sites in the PDGF beta-receptor: involvement in the interaction with phospholipase C-gamma.

Two novel sites of autophosphorylation were localized to the C-terminal tail of the PDGF beta-receptor. To evaluate the importance of these phosphorylation sites, receptor mutants in which Tyr1009, Tyr1021 or both were replaced with phenylalanine residues, were expressed in porcine aortic endothelial (PAE) cells. These mutants were similar to the wild type receptor with regard to protein tyrosine kinase activity and ability to induce mitogenicity in response to PDGF-BB. However, both the Y1009F and Y1021F mutants showed a decreased ability to mediate association with and the tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma) compared to the wild type PDGF beta-receptor; in the case of the Y1009F/Y1021F double mutant, no association or phosphorylation of PLC-gamma could be detected. These data show that tyrosine phosphorylation of PLC-gamma is dependent on autophosphorylation of the PDGF beta-receptor at Tyr1009 and Tyr1021.     

The tyrosine phosphatase SHP-2 is required for sustained activation of extracellular signal-regulated kinase and epithelial morphogenesis downstream from the met receptor tyrosine kinase

Epithelial morphogenesis is critical during development and wound healing, and alterations in this program contribute to neoplasia. Met, the hepatocyte growth factor (HGF) receptor, promotes a morphogenic program in epithelial cell lines in matrix cultures. Previous studies have identified Gab1, the major phosphorylated protein following Met activation, as important for the morphogenic response. Gab1 is a docking protein that couples the Met receptor with multiple signaling proteins, including phosphatidylinositol-3 kinase, phospholipase Cgamma, the adapter protein Crk, and the tyrosine specific phosphatase SHP-2. HGF induces sustained phosphorylation of Gab1 and sustained activation of extracellular signal-regulated kinase (Erk) in epithelial Madin-Darby canine kidney cells. In contrast, epidermal growth factor fails to promote a morphogenic program and induces transient Gab1 phosphorylation and Erk activation. To elucidate the Gab1-dependent signals required for epithelial morphogenesis, we undertook a structure-function approach and demonstrate that association of Gab1 with the tyrosine phosphatase SHP-2 is required for sustained Erk activation and for epithelial morphogenesis downstream from the Met receptor. Epithelial cells expressing a Gab1 mutant protein unable to recruit SHP-2 elicit a transient activation of Erk in response to HGF. Moreover, SHP-2 catalytic activity is required, since the expression of a catalytically inactive SHP-2 mutant, C/S, abrogates sustained activation of Erk and epithelial morphogenesis by the Met receptor. These data identify SHP-2 as a positive modulator of Erk activity and epithelial morphogenesis downstream from the Met receptor.     

Regulation of the wild-type and Y1235D mutant Met kinase activation

Met receptor tyrosine kinase plays a crucial role in the regulation of a large number of cellular processes and, when deregulated by overexpression or mutations, leads to tumor growth and invasion. The Y1235D mutation identified in metastases was shown to induce constitutive activation and a motile-invasive phenotype on transduced carcinoma cells. Wild-type Met activation requires phosphorylation of both Y1234 and Y1235 in the activation loop. We mapped the major phosphorylation sites in the kinase domain of a recombinant Met protein and identified the known residues Y1234 and Y1235 as well as a new phosphorylation site at Y1194 in the hinge region. Combining activating and silencing mutations at these sites, we characterized in depth the mechanism of activation of wild-type and mutant Met proteins. We found that the phosphotyrosine mimetic mutation Y1235D is sufficient to confer constitutive kinase activity, which is not influenced by phosphorylation at Y1234. However, the specific activity of this mutant was lower than that observed for fully activated wild-type Met and induced less phosphorylation of Y1349 in the signaling site, indicating that this mutation cannot entirely compensate for a phosphorylated tyrosine at this position. The Y1194F silencing mutation yielded an enzyme that could be activated to a similar extent as the wild type but with significantly slower activation kinetics, underlying the importance of this residue, which is conserved among different tyrosine kinase receptors. Finally, we observed different interactions of wild-type and mutant Met with the inhibitor K252a that may have therapeutic implications for the selective inhibition of this kinase.     

Dissociation of c‐Met phosphotyrosine sites in human cells in response to mouse hepatocyte growth factor but not human hepatocyte growth factor: the possible roles of different amino acids in different species

Hepatocyte growth factor (HGF) is essential for embryogenesis, tissue regeneration and tumour malignancy through the activation of its receptor, c‐Met. We previously demonstrated that HGF α‐chain hairpin–loop, K1 domain and β‐chain are required for c‐Met signalling. The sequential phosphorylation of tyrosine residues, from c‐Met kinase domain to multidocking regions, is required for HGF‐signalling transduction. Herein, we provide evidence that the disconcerted activation of c‐Met tyrosine regions fails to induce biological functions. When human cells were incubated with ‘mouse HGF’, kinase domain activation (i.e. phospho‐Tyr‐1230/34/35) became evident, but the multidocking site (i.e. Tyr‐1349) was not phosphorylated, resulting in unsuccessful induction of migration and mitogenesis. The binding ability of mouse HGF α‐chain, or of β‐chain, to human c‐Met was lower than that of human HGF, as evidenced by HGF–chimera assay. Notably, only four amino acid positions in HGF α‐chain hairpin–loop and K1 domain and six positions in β‐chain differed between human HGF and mouse HGF. The human‐specific amino acids (such as Gln‐95 in hairpin–loop, Arg‐134 in K1 domain and Cys‐561 in β‐chain) may be important for accurate c‐Met assembly and signalling transduction. Copyright © 2012 John Wiley & Sons, Ltd.     

Growth factors stimulate kidney proximal tubule cell migration independent of augmented tyrosine phosphorylation of focal adhesion kinase

Migration of human proximal tubule cells (HKC-5) was stimulated by epidermal growth factor (EGF), hepatocyte growth factor (HGF), and insulin-like growth factor-1 (IGF-1). Integrin signaling via phosphorylation of focal adhesion kinase (FAK) appears to play a central role in cell migration. Once stimulated, FAK undergoes autophosphorylation at tyrosine (Y) 397, followed by phosphorylation of several sites including Y576/Y577 which increases FAK's kinase activity, as well as at Y407, Y861, and Y925. EGF, HGF, and IGF-1 stimulate FAK phosphorylation in various cells. We showed that endothelin stimulated phosphorylation of Y397 in fibroblasts but not HKC-5 cells. After EGF stimulation, HKC-5 cells showed no change in tyrosine phosphorylation at FAK Y397, 407, 576, 861, or 925. Similarly, HGF and IGF-1 did not stimulate the phosphorylation of FAK Y397 in HKC-5 cells. Further, after inhibition of FAK expression by siRNA, cell migration was similar to cells treated with non-target siRNA and responded to EGF with increased migration. Thus, in proximal tubule cells, stimulation of cell migration by growth factors was independent of augmented FAK tyrosine phosphorylation.