The Met receptor tyrosine kinase regulates the dispersal of epithelial sheets in culture and promotes the inherent morphogenic program of epithelia when grown in a collagen matrix. However, it is unclear how the Met receptor orchestrates the signaling pathways leading to its pleiotropic biological activities. We and colleagues have previously demonstrated that the multisubstrate binding protein Gab1 is required for the initiation of the morphogenic program (38
). In the absence of any catalytic activity, Gab1 functions as a docking protein that, when phosphorylated by Met or other receptors, recruits multiple signaling proteins (21
). Gab1 acts to recruit Pl3K downstream from the Met, EGF, and TrkA receptors (21
). This interaction has been shown to be essential for the survival of the neuronal PC12 cell line following stimulation of nerve growth factor (22
) but is not required for the induction of the morphogenic program by Gab1 (38
). While other proteins including PLCγ1, Crk, and SHP-2 also associate with Gab1, the contribution of these to the biological activities of the Met receptor is unknown (14
). We therefore undertook a structure-function approach to determine the contribution of SHP-2 in Met-mediated branching tubulogenesis. We show in this paper that both the interaction of Gab1 with SHP-2 and SHP-2 phosphatase activity are essential for epithelial tubulogenesis downstream from the Met receptor tyrosine kinase. Moreover, the recruitment of SHP-2 to Gab1 is required for the sustained activation of the Erk pathway observed following HGF stimulation of MDCK cells.
SHP-2 is one of the signaling proteins recruited to Gab1 following Met activation, yet the role of SHP-2 biological activity in epithelial cell morphogenesis and signaling downstream from the Met receptor has not been addressed. To investigate the role of this interaction in Met-mediated signaling pathways involved in epithelial tubulogenesis, we identified the SHP-2 binding site on Gab1 (Y637) that is phosphorylated following Met activation (Fig. ) and show that mutation of this site results in Gab1 proteins unable to recruit SHP-2. Although overexpression of SHP-2 results in dephosphorylation of Gab1 (Fig. ), mutating the SHP-2 binding site does not enhance Gab1 phosphorylation in stable cell lines in response to Met activations. Instead, the level of phosphorylation of the Gab1 ΔSHP-2 mutant protein is lower than that observed in wild-type Gab1 (Fig. ). This is consistent with the observation that the SHP-2 binding site (Y637) in the Gab1 carboxy terminus is the predominant site of phosphorylation of Gab1 in vitro by a recombinant EGF receptor kinase domain (33
). Importantly, the failure to recruit SHP-2 results in the inability of Gab1 to rescue tubulogenesis in MDCK cells that express mutant CSF-Met receptors, implying, for the first time, a role for SHP-2 in the morphogenic response of epithelial cells. In addition, the overexpression of the Gab1 ΔSHP-2 mutant abrogates tubulogenesis downstream from the endogenous Met receptor (Fig. and ), suggesting that overexpressed Gab1 ΔSHP-2 proteins function as dominant inhibitory proteins by competing with endogenous Gab1 proteins. Importantly, the overexpression of a catalytically inactive SHP-2 C/S mutant protein inhibits tubulogenesis by the Met receptor in the presence of wild-type Gab1, implicating SHP-2 catalytic activity in the morphogenic program (Fig. ).
The ability of HGF to promote branching tubulogenesis correlates with the sustained phosphorylation of Gab1 and the prolonged activation of signaling pathways such as Erk (28
). The inhibition of MEK with a pharmacological agent, PD98059, inhibits tubulogenesis following HGF stimulation in MDCK cells (28
), suggesting that the tubulogenic response in MDCK cells requires Erk activation. However, it remained to be determined how the Met receptor regulated sustained Erk activity. We have shown that the interaction of Gab1 with SHP-2 is required for the sustained Erk activity in response to HGF (180 min), whereas epithelial cells that express Gab1 proteins which fail to associate with SHP-2 display transient activation of Erk (15 min; Fig. ). Importantly, we show that the kinetics of phosphorylation of the Gab1 ΔSHP-2 mutant protein is similar to that observed with wild-type Gab1 (Fig. ). Moreover, the Gab1 ΔSHP-2 protein associated with the p85 subunit of Pl3K to a similar extent as wild-type Gab1, and activation of Akt is observed in cells expressing Gab1 ΔSHP-2 or wild-type Gab1 proteins with similar kinetics (Fig. ). Hence, mutation of the SHP-2 binding site of Gab1 did not affect the coupling of Gab1 with the Pl3K signaling pathway, yet resulted in attenuation of the Erk pathway downstream from Met.
While this paper was in preparation, a role for Gab1 in the activation of Erk downstream from the EGF receptor was suggested using SHP-2 exon 3−/−
). However, a Gab1 ΔSHP2 mutant protein was not evaluated (64
). Since mutation of the SHP-2 binding site within the Gab2 multisubstrate docking protein did not abrogate Erk activation stimulated through the IL-3 receptor (16
), this raised the possibility that Gab1-SHP-2 interactions were not required for Erk activity. Our data provide the first direct evidence that the recruitment of SHP-2 to Gab1 is important for Erk activation. This supports data from Drosophila
indicating that the interaction of corkscrew with DOS, a docking protein related to Gab1, is required for positive regulation of Erk (1
). Moreover, in a manner similar to that of Gab1, mutation of the SHP-2 binding site on FRS2 compromises activation of Erk and neurite outgrowth in PC12 cells following stimulation with FGF (16
), implying an important role for docking proteins in modulating Erk activity dependent on SHP-2.
A positive role for SHP-2 as a regulator of the Erk pathway has been proposed based on the observation that SHP-2 is phosphorylated on tyrosine residues in response to PDGF. This provides a binding site for the Grb2 adapter protein and hence the ability to form a SHP-2/Grb2/SOS complex with potential to activate the Ras pathway (5
). However, the overexpression of a SHP-2 mutant that fails to bind the Grb2 adapter protein has not been shown to alter Erk activation downstream from several receptor tyrosine kinases, suggesting that this is unlikely to be a significant mechanism through which SHP-2 can regulate Erk activity (4
). However, since SHP-2 is phosphorylated following activation of the Met receptor (Fig. ), and reduced Grb2 is recruited to the Gab1 ΔSHP2 mutant, we cannot exclude the possibility that SHP-2 links Met at least in part to Grb2/SOS/Ras.
The phosphatase activity of SHP-2 is required for Erk activation both in mammalian systems and in lower organisms, such as Drosophila
embryos, suggesting that the dephosphorylation of proteins by SHP-2 may be critical for the onset of signaling pathways leading to Erk activation (1
). Consistent with these observations, we show that the overexpression of a catalytically inactive SHP-2 mutant abrogates sustained Erk activation downstream from the Met receptor tyrosine kinase (Fig. ). Importantly, the overexpression of the catalytically inactive SHP-2 C/S mutant also inhibits branching tubulogenesis, indicating that the phosphatase activity of SHP-2 is required for the ability of Met-Gab1 to induce branching tubulogenesis in epithelial cells (Fig. ).
homologue of SHP-2, Csw, dephosphorylates a tyrosine residue on the Torso receptor tyrosine kinase that binds to p120 Ras GAP, thus uncoupling a negative regulator of Ras from the Torso receptor (7
). The dephosphorylation of the Gab1-related docking protein DOS by Csw has also been implicated in linking the receptor tyrosine kinase Sevenless to the Ras pathway (18
). Similarly, in cells overexpressing SHP-2, Gab1 can also act as a substrate for SHP-2 following phosphorylation by the Met receptor (Fig. ) and following activation of the EGF receptor (64
). This supports the observation that in response to IL-6, Gab1 is an in vitro substrate for GST fusion protein containing the SHP-2 phosphatase domain (45
). Since we have shown that Gab1 can associate with the SH2 domain of p120 RasGAP, it is possible that SHP-2 dephosphorylates tyrosine residues important for the interaction of p120 RasGAP with Gab1. However, either in transient overexpression assays (Fig. ) or in stable epithelial cell lines expressing Gab1 ΔSHP-2 mutant proteins (not shown), the ability of Gab1 to associate with a p120 RasGAP SH2 domain fusion protein is not altered. Thus, under these conditions, the binding site for p120 RasGAP on Gab1 is not dephosphorylated by SHP-2.
We have shown that either the failure to recruit the SHP-2 phosphatase to Gab1 or the overexpression of a catalytically inactive SHP-2 phosphatase results in improper epithelial organization in response to Met activation. This is consistent with studies of SHP-2 exon 3−/−
mutant mice, where gastrulation is interrupted due to inappropriate mesodermal cell migration and organization (59
). Moreover, expression of a dominant negative SHP-2 mutant inhibits elongation of Xenopus
animal caps in response to FGF, a process that involves the reorganization of existing cells (69
). Epithelial morphogenesis requires both cell proliferation and the remodeling of epithelial junctions (50
). MEK-Erk activity is necessary for the breakdown of adherens junctions in response to HGF (52
). Thus, the transient activation of Erk in cells expressing the Gab1 ΔSHP-2 or SHP-2 C/S mutant may be insufficient for the remodeling of adherens junctions and for the cell proliferation required for epithelial morphogenesis. Both Gab1 and the Met receptor are localized to the basolateral membranes of polarized epithelial cells in the vicinity of adherens junctions, and this localization of Gab1 is important for its ability to rescue branching tubulogenesis in cells expressing Met receptor mutants (38
). In a manner similar to that of wild-type Gab1, the Gab1 ΔSHP-2 protein is localized to sites of cell-cell contact (38
), demonstrating that association with SHP-2 is not essential for Gab1 subcellular localization (Fig. ). However, the inability of SHP-2 to be recruited to Gab1 may itself result in the failure of SHP-2 to colocalize with Gab1 in the proximity of critical membrane-associated substrates.
Fibroblasts isolated from SHP-2 exon 3−/−
mice have increased numbers of focal adhesions and actin aggregation at the cell periphery, suggesting that SHP-2 could also play a role in the regulation of cell spreading and migration on extracellular matrix (ECM) (46
). A class of adhesion proteins, the signal-regulatory proteins, including SHP substrate 1 and the distantly related PECAM and PIR-B/p91A proteins, are SHP-2 binding proteins and may serve as substrates for SHP-2 (13
). A role for SHP-2 in regulating SHP substrate 1 function and integrin signaling has been proposed, where the catalytic activity of SHP-2 is required for Erk activation downstream from cell-ECM interactions (46
). Hence SHP-2 may modify cell-ECM-dependent Erk signals required for branching morphogenesis. The determination of substrates dephosphorylated by SHP-2 that modify epithelial tubulogenesis downstream from the Met receptor will provide a better understanding of how these processes are normally controlled and which events lead to loss of epithelial organization during tumorigenesis.