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.

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.

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.

Cbl-transforming variants trigger a cascade of molecular alterations that lead to epithelial mesenchymal conversion.

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. Structure-function 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.

Distinct roles for CD4 and CD8 as co-receptors in T cell receptor signalling.

We demonstrate that CD4 and CD8 modify signals induced through the T cell receptor for antigen (TCR alpha beta) in distinct fashions. Pretreatment of CD4+ lymph node T cells with CD4-specific monoclonal antibody results in a tenfold inhibition of DNA synthesis induced by anti-TCR alpha beta. In contrast, pretreatment of CD8+ T cells with CD8-specific mAb has no effect on DNA synthesis subsequently induced through TCR alpha beta. While inhibiting late activation signals, pretreatment with anti-CD4 does not detectably alter the pattern of anti-TCR alpha beta-induced tyrosine phosphorylation of cellular proteins, nor subsequent Ca2+ mobilization. The distinct biological consequences of anti-CD4 and anti-CD8 pretreatment correlate with the differential association of their respective ligands with the cellular protein tyrosine kinase, p56lck. While both T cell lineages contain similar levels of cellular p56lck, tenfold more is associated with CD4 than with CD8. This difference is associated with the differential effects of pretreatment with anti-CD4 and anti-CD8 on the distribution and activity of p56lck. Further, antibody-mediated aggregation of TCR alpha beta on CD4+ T cells induces the appearance of a p56lck species with decreased mobility in sodium dodecylsulfate-polyacrylamide gel electrophoresis. This effect is observed in CD4+ T cells exclusively and involves the fraction of p56lck which is not associated with CD4. The results presented here demonstrate that the signalling elements which couple the antigen receptor to second messenger-generating systems are under distinct physical and/or functional constraints in the two T cell lineages.

Distinct involvement of CD45 in antigen receptor signalling in CD4+ and CD8+ primary T cells.

We demonstrate that pretreatment of primary CD4+, but not CD8+ T cells with anti-CD45 inhibits activation signals induced through the T cell receptor for antigen (TCR alpha beta). Specifically, anti-TCR alpha beta-mediated tyrosine phosphorylation of phospholipase C-gamma 1 is inhibited, and this in turn correlates with the inhibition of subsequent Ca2+ mobilization and DNA synthesis. In marked contrast, none of these activation parameters are affected by anti-CD45 in CD8+ T cells. Perturbation of TCR alpha beta signalling in CD4+ cells is observed in conditions which do not detectably affect the level of CD45 expression, or its membrane distribution. Further, changes in the intrinsic phosphatase activity of CD45 are not detectable. While anti-CD45 ablates TCR alpha beta signalling, anti-CD3 epsilon-mediated activation is unaffected. This suggests that elements of the antigen receptor complex can be functionally uncoupled, and indicates that the requirements for CD45 in signalling through these two elements are different. The results demonstrate that the involvement of CD45 in coupling TCR alpha beta to second messenger-generating pathways is under distinct physical and/or functional constraints in primary CD4+ and CD8+ T cells.

Physical association of CD4 and CD45 in primary, resting CD4+ T cells.

CD45 is a family of transmembrane protein tyrosine phosphatases that are essential to T lymphocytes' responses to antigen-receptor stimulation. It is involved in the regulation of Src-family protein tyrosine kinases, Lck and Fyn. The object of this study was to determine how CD45 molecules are directed to such substrates at the antigen-receptor complex upon stimulation of resting T cells. We demonstrate that CD45 is physically associated with CD4 in resting, primary lymph node T cells. Further, CD4-dependent, antigen-mediated activation of primary CD4+ T cells results in disruption of CD4-CD45 complexes, suggesting a role for these complexes in the activation process. Moreover, CD45 coprecipitates with CD4 molecules which are associated with Lck, as well as with those which are not associated with Lck. Consistent with these observations and the role of CD45 in the regulation of Lck function, effects on CD4-associated membrane Lck are demonstrable. Since antigen presentation by MHC class II results in the coaggregation of CD4 with the antigen-receptor complex, the association described in this study provides a physical basis through which CD45 could be included.

Distinct roles for CD4 and CD8 as co-receptors in antigen receptor signalling.

The co-ordinated interactions of multiple membrane molecules with the T-cell receptor for antigen (the TCR-CD3) are prerequisite for T-cell activation. In this review we consider the involvement of CD4, CD8, and CD45 on the two lymphocyte lineages. Experiments from many laboratories have provided concordant results leading to the consensus that CD4 and CD8 are functional analogues, providing similar supplementary signals to those generated through the TCR-CD3 complex on MHC class-II- and MHC class-I-restricted T cells, respectively. However, recent results demonstrate striking differences in the coreceptor functions of CD4 and CD8. These differences reflect the distinct properties of the molecules themselves, which in turn are associated with CD45 involvement in the activation of CD4+ and CD8+ T cells.

Death of mature T cells by separate ligation of CD4 and the T-cell receptor for antigen.

Effector T cells are restricted to recognizing antigens associated with major histocompatibility complex (MHC) molecules. Specific recognition is mediated by the alpha beta heterodimer of the T-cell receptor (TCR)/CD3 complex, although other membrane components are involved in T-cell antigen recognition and functions. There has been much controversy in this regard over the part played by the CD4 glycoprotein. It is known that expression of CD4 correlates closely with the cell's ability to recognize antigens bound to class II MHC molecules and that CD4 can bind to class II molecules. Also monoclonal antibodies to CD4 can modify signals generated through the TCR/CD3 complex. It has therefore been proposed that CD4 binds to class II molecules, coaggregates with the TCR-CD3 complex and aids the activation of T cells. But given that TCR can itself impart restriction on the cell, it remains unclear whether the contribution of CD4-derived signals to those generated through the TCR alpha beta-CD3 complex is central to this activation. Here we report that when preceded by ligation of CD4, signalling through TCR alpha beta results in T cell unresponsiveness due to the induction of activation dependent cell death by apoptosis. These results imply that CD4 is critically involved in determining the outcome of signals generated through TCR, and could explain why the induction of effector T cells needs to be MHC-restricted.

A conserved inositol phospholipid binding site within the pleckstrin homology domain of the Gab1 docking protein is required for epithelial morphogenesis.

Stimulation of the hepatocyte growth factor receptor tyrosine kinase, Met, induces the inherent morphogenic program of epithelial cells. The multisubstrate binding protein Gab1 (Grb2-associated binder-1) is the major phosphorylated protein in epithelial cells following activation of Met. Gab1 contains a pleckstrin homology domain and multiple tyrosine residues that act to couple Met with multiple signaling proteins. Met receptor mutants that are impaired in their association with Gab1 fail to induce a morphogenic program in epithelial cells, which is rescued by overexpression of Gab1. The Gab1 pleckstrin homology domain binds to phosphatidylinositol 3,4, 5-trisphosphate and contains conserved residues, shown from studies of other pleckstrin homology domains to be crucial for phospholipid binding. Mutation of conserved phospholipid binding residues tryptophan 26 and arginine 29, generates Gab1 proteins with decreased phosphatidylinositol 3,4,5-trisphosphate binding, decreased localization at sites of cell-cell contact, and reduced ability to rescue Met-dependent morphogenesis. We conclude that the ability of the Gab1 pleckstrin homology domain to bind phosphatidylinositol 3,4,5-trisphosphate is critical for subcellular localization of Gab1 and for efficient morphogenesis downstream from the Met receptor.

The Gab1 docking protein links the b cell antigen receptor to the phosphatidylinositol 3-kinase/Akt signaling pathway and to the SHP2 tyrosine phosphatase.

B cell antigen receptor (BCR) signaling causes tyrosine phosphorylation of the Gab1 docking protein. This allows phosphatidylinositol 3-kinase (PI3K) and the SHP2 tyrosine phosphatase to bind to Gab1. In this report, we tested the hypothesis that Gab1 acts as an amplifier of PI3K- and SHP2-dependent signaling in B lymphocytes. By overexpressing Gab1 in the WEHI-231 B cell line, we found that Gab1 can potentiate BCR-induced phosphorylation of Akt, a PI3K-dependent response. Gab1 expression also increased BCR-induced tyrosine phosphorylation of SHP2 as well as the binding of Grb2 to SHP2. We show that the pleckstrin homology (PH) domain of Gab1 is required for BCR-induced phosphorylation of Gab1 and for Gab1 participation in BCR signaling. Moreover, using confocal microscopy, we show that BCR ligation can induce the translocation of Gab1 from the cytosol to the plasma membrane and that this requires the Gab1 PH domain as well as PI3K activity. These findings are consistent with a model in which the binding of the Gab1 PH domain to PI3K-derived lipids brings Gab1 to the plasma membrane, where it can be tyrosine-phosphorylated and then act as an amplifier of BCR signaling.