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.     

Membrane targeting of Grb2-associated binder-1 (Gab1) scaffolding protein through Src myristoylation sequence substitutes for Gab1 pleckstrin homology domain and switches an epidermal growth factor response to an invasive morphogenic program

The hepatocyte growth factor receptor tyrosine kinase Met promotes cell dissociation and the inherent morphogenic program of epithelial cells. In a search for substrates downstream from Met, we have previously identified the Grb2-associated binder-1 (Gab1) as critical for the morphogenic program. Gab1 is a scaffold protein that acts to diversify the signal downstream from the Met receptor through its ability to couple with multiple signal transduction pathways. Gab1 contains a pleckstrin homology (PH) domain with specificity for phosphatidylinositol 3,4,5-trisphosphate. The phospholipid binding capacity of the Gab1 PH domain is required for the localization of Gab1 at sites of cell-cell contact in colonies of epithelial cells and for epithelial morphogenesis, suggesting that PH domain-dependent subcellular localization of Gab1 is a prerequisite for function. We have investigated the requirement for membrane localization of Gab1 for biological activity. We show that substitution of the Gab1 PH domain with the myristoylation signal from the c-Src protein is sufficient to replace the Gab1 PH domain for epithelial morphogenesis. The membrane targeting of Gab1 enhances Rac activity in the absence of stimulation and switches a nonmorphogenic noninvasive response to epidermal growth factor to a morphogenic invasive program. These results suggest that the subcellular localization of Gab1 is a critical determinant for epithelial morphogenesis and invasiveness.     

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.     

Crk synergizes with epidermal growth factor for epithelial invasion and morphogenesis and is required for the met morphogenic program

Activation of the Met receptor tyrosine kinase through its ligand, hepatocyte growth factor, stimulates cell spreading, cell dispersal, and the inherent morphogenic program of various epithelial cell lines. Although both hepatocyte growth factor and epidermal growth factor (EGF) can activate downstream signaling pathways in Madin-Darby canine kidney epithelial cells, EGF fails to promote the breakdown of cell-cell junctional complexes and initiate an invasive morphogenic program. We have undertaken a strategy to identify signals that synergize with EGF in this process. We provide evidence that the overexpression of the CrkII adapter protein complements EGF-stimulated pathways to induce cell dispersal in two-dimensional cultures and cell invasion and branching morphogenesis in three-dimensional collagen gels. This finding correlates with the ability of CrkII to promote the breakdown of adherens junctions in stable cell lines and the ability of EGF to stimulate enhanced Rac activity in cells overexpressing CrkII. We have previously shown that the Gab1-docking protein is required for branching morphogenesis downstream of the Met receptor. Consistent with a role for CrkII in promoting EGF-dependent branching morphogenesis, the binding of Gab1 to CrkII is required for the branching morphogenic program downstream of Met. Together, our data support a role for the CrkII adapter protein in epithelial invasion and morphogenesis and underscores the importance of considering the synergistic actions of signaling pathways in cancer progression.     

How to make tubes: signaling by the Met receptor tyrosine kinase

Hepatocyte growth factor/scatter factor (HGF/SF), acting through the receptor tyrosine kinase Met, stimulates cells derived from a variety of different organs to form elongated hollow tubules when grown in three-dimensional gels. In vivo data also indicate a role for HGF/SF and Met in tubule formation during liver and kidney regeneration and mammary gland formation. Activation of Met results in the recruitment of a myriad of signal transducers that regulate dissociation of adherens junctions and the stimulation of cellular motility, survival, proliferation and morphogenesis during tubule formation. Among these many signal transducers, the Gab1 adaptor protein and its effector, the SHP2 tyrosine phosphatase, have been found to be crucial for tubulogenesis and for the sustained stimulation of the ERK/MAP kinase pathway. Here, we discuss the contribution of these and other signaling pathways and the role of HGF/SF and Met in the formation of epithelial cell tubules both in vitro in branching-morphogenesis assays and in vivo during organogenesis.     

Distinct recruitment and function of Gab1 and Gab2 in Met receptor-mediated epithelial morphogenesis

The Gab family of docking proteins (Gab1 and Gab2) are phosphorylated in response to various cytokines and growth factors. Gab1 acts to diversify the signal downstream from the Met receptor tyrosine kinase through the recruitment of multiple signaling proteins, and is essential for epithelial morphogenesis. To determine whether Gab1 and Gab2 are functionally redundant, we have examined the role of Gab2 in epithelial cells. Both Gab1 and Gab2 are expressed in epithelial cells and localize to cell-cell junctions. However, whereas overexpression of Gab1 promotes a morphogenic response, the overexpression of Gab2 fails to induce this response. We show that Gab2 recruitment to the Met receptor is dependent on the Grb2 adapter protein. In contrast, Gab1 recruitment to Met is both Grb2 dependent and Grb2 independent. The latter requires a novel amino acid sequence present in the Met-binding domain of Gab1 but not Gab2. Mutation of these residues in Gab1 impairs both association with the Met receptor and the ability of Gab1 to promote a morphogenic response, whereas their insertion into Gab2 increases Gab2 association with Met, but does not confer on Gab2 the ability to promote epithelial morphogenesis. We propose that the Grb2-independent recruitment of Gab proteins to Met is necessary but not sufficient to promote epithelial morphogenesis.     

Gab2 requires membrane targeting and the Met binding motif to promote lamellipodia, cell scatter, and epithelial morphogenesis downstream from the Met receptor

Gab1 and Gab2 are conserved scaffolding proteins that amplify and integrate signals stimulated by many growth factor receptors including the Met receptor. Gab1 acts to diversify the signal downstream from Met through the recruitment of multiple signaling proteins, and is essential for epithelial morphogenesis. However, whereas Gab1 and Gab2 are both expressed in epithelial cells, Gab2 fails to support a morphogenic response. We demonstrate that Gab1 and Gab2 are divergent in their function whereby Gab1, but not Gab2, promotes lamellipodia formation, and is localized to the membrane of lamellipodia upon Met activation. We have identified activation of ERK1/2 as a requirement for lamellipodia formation. Moreover, activated ERK1/2 are localized to lamellipodia in Gab1 expressing cells but not in cells that overexpress Gab2. By structure-function studies, we identify that enhanced membrane localization conferred through the addition of a myristoylation signal, together with the addition of the direct Met binding motif (MBM) from Gab1, are required to promote lamellipodia and confer a morphogenic signaling response to Gab2. Moreover, the morphogenesis competent myristoylated Gab2MBM promotes localization of activated ERK1/2 to the leading edge of lamellipodia in a similar manner to Gab1. Hence, subcellular localization of the Gab scaffold, as well as the ability of Gab to interact directly with the Met receptor, are both essential components of the morphogenic signaling response which involves lamellipodia formation and the localization of ERK1/2 activation in membrane ruffles.     

Recruitment of the protein-tyrosine phosphatase SHP-2 to the C-terminal tyrosine of the prolactin receptor and to the adaptor protein Gab2

The protein-tyrosine phosphatase SHP-2 modulates signaling events through receptor tyrosine kinases and cytokine receptors including the receptor for prolactin (PRLR). Here we investigated mechanisms of SHP-2 recruitment within the PRLR signaling complex. Using SHP-2 and PRLR immunoprecipitation studies in 293 cells and in the mouse mammary epithelial cell line HC11, we found that SHP-2 co-immunoprecipitates with the PRLR and that the C-terminal tyrosine of the PRLR plays a regulatory role in both the tyrosine phosphorylation and the recruitment of SHP-2. Our results further indicate that SHP-2 association to the PRLR occurs via the C-terminal SH2 domain of the phosphatase. In addition, we determined that the newly identified adaptor protein Gab2, but not Gab1, is specifically tyrosine phosphorylated and is able to recruit SHP-2 and phosphatidyinositol 3-kinase in response to PRLR activation. Together, these studies suggest the presence of dual recruitment sites for SHP-2; the first is to the C-terminal tyrosine of the PRLR and the second is to the adaptor protein Gab2.     

Receptor-type protein tyrosine phosphatase beta (RPTP-beta) directly dephosphorylates and regulates hepatocyte growth factor receptor (HGFR/Met) function

Protein tyrosine phosphorylation is a ubiquitous, fundamental biochemical mechanism that regulates essential eukaryotic cellular functions. The level of tyrosine phosphorylation of specific proteins is finely tuned by the dynamic balance between protein tyrosine kinase and protein tyrosine phosphatase activities. Hepatocyte growth factor receptor (also known as Met), a receptor protein tyrosine kinase, is a major regulator of proliferation, migration, and survival for many epithelial cell types. We report here that receptor-type protein tyrosine phosphatase β (RPTP-β) specifically dephosphorylates Met and thereby regulates its function. Expression of RPTP-β, but not other RPTP family members or catalytically inactive forms of RPTP-β, reduces hepatocyte growth factor (HGF)-stimulated Met tyrosine phosphorylation in HEK293 cells. Expression of RPTP-β in primary human keratinocytes reduces both basal and HGF-induced Met phosphorylation at tyrosine 1356 and inhibits downstream MEK1/2 and Erk activation. Furthermore, shRNA-mediated knockdown of endogenous RPTP-β increases basal and HGF-stimulated Met phosphorylation at tyrosine 1356 in primary human keratinocytes. Purified RPTP-β intracellular domain preferentially dephosphorylates purified Met at tyrosine 1356 in vitro. In addition, the substrate-trapping mutant of RPTP-β specifically interacts with Met in intact cells. Expression of RPTP-β in human primary keratinocytes reduces HGF induction of VEGF expression, proliferation, and motility. Taken together, the above data indicate that RPTP-β is a key regulator of Met function.     

Hepatocyte growth factor receptor tyrosine kinase met is a substrate of the receptor protein-tyrosine phosphatase DEP-1

The receptor protein-tyrosine phosphatase (PTP) DEP-1 (CD148/PTP-eta) has been implicated in the regulation of cell growth, differentiation, and transformation, and most recently has been identified as a potential tumor suppressor gene mutated in colon, lung, and breast cancers. We have generated constructs comprising the cytoplasmic segment of DEP-1 fused to the maltose-binding protein to identify potential substrates and thereby suggest a physiological function for DEP-1. We have shown that the substrate-trapping mutant form of DEP-1 interacted with a small subset of tyrosine-phosphorylated proteins from lysates of the human breast tumor cell lines MDA-MB-231, T-47D, and T-47D/Met and have identified the hepatocyte growth factor/scatter factor receptor Met, the adapter protein Gab1, and the junctional component p120 catenin as potential substrates. Following ligand stimulation, phosphorylation of specific tyrosyl residues in Met induces mitogenic, motogenic, and morphogenic responses. When co-expressed in 293 cells, the full-length substrate-trapping mutant form of DEP-1 formed a stable complex with the chimeric receptor colony stimulating factor 1 (CSF)-Met and wild type DEP-1 dephosphorylated CSF-Met. Furthermore, we observed that DEP-1 preferentially dephosphorylated a Gab1 binding site (Tyr(1349)) and a COOH-terminal tyrosine implicated in morphogenesis (Tyr(1365)), whereas tyrosine residues in the activation loop of Met (Tyr(1230), Tyr(1234), and Tyr(1235)) were not preferred targets of the PTP. The ability of DEP-1 preferentially to dephosphorylate particular tyrosine residues that are required for Met-induced signaling suggests that DEP-1 may function in controlling the specificity of signals induced by this PTK, rather than as a simple "off-switch" to counteract PTK activity.     

Grb2-independent recruitment of Gab1 requires the C-terminal lobe and structural integrity of the Met receptor kinase domain

The Gab1 docking protein forms a platform for the assembly of a multiprotein signaling complex downstream from receptor tyrosine kinases. In general, recruitment of Gab1 occurs indirectly, via the adapter protein Grb2. In addition, Gab1 interacts with the Met/hepatocyte growth factor receptor in a Grb2-independent manner. This interaction requires a Met binding domain (MBD) in Gab1 and is essential for Met-mediated epithelial morphogenesis. The Gab1 MBD has been proposed to act as a phosphotyrosine binding domain that binds Tyr-1349 in the Met receptor. We show that a 16-amino acid motif within the Gab1 MBD is sufficient for interaction with the Met receptor, suggesting that it is unlikely that the Gab1 MBD forms a structured domain. Alternatively, the structural integrity of the Met receptor, and residues upstream of Tyr-1349 located in the C-terminal lobe of the kinase domain, are required for Grb2-independent interaction with the Gab1 MBD. Moreover, the substitution of Tyr-1349 with an acidic residue allows for the recruitment of the Gab1 MBD and for phosphorylation of Gab1. We propose that Gab1 and the Met receptor interact in a novel manner, such that the activated kinase domain of Met and the negative charge of phosphotyrosine 1349 engage the Gab1 MBD as an extended peptide ligand.     

Signaling complexes and protein-protein interactions involved in the activation of the Ras and phosphatidylinositol 3-kinase pathways by the c-Ret receptor tyrosine kinase

Proximal signaling events and protein-protein interactions initiated after activation of the c-Ret receptor tyrosine kinase by its ligand, glial cell line-derived neurotrophic factor (GDNF), were investigated in cells carrying native and mutated forms of this receptor. Mutation of Tyr-1062 (Y1062F) in the cytoplasmic tail of c-Ret abolished receptor binding and phosphorylation of the adaptor Shc and eliminated activation of Ras by GDNF. Phosphorylation of Erk kinases was also greatly attenuated but not eliminated by this mutation. This residual wave of Erk phosphorylation was independent of the kinase activity of c-Ret. Mutation of Tyr-1096 (Y1096F), a binding site for the adaptor Grb2, had no effect on Erk activation by GDNF. Activation of phosphatidylinositol-3 kinase (PI3K) and its downstream effector Akt was also reduced in the Y1062F mutant but not completely abolished unless Tyr-1096 was also mutated. Ligand stimulation of neuronal cells induced the assembly of a large protein complex containing c-Ret, Grb2, and tyrosine-phosphorylated forms of Shc, p85(PI3K), the adaptor Gab2, and the protein-tyrosine phosphatase SHP-2. In agreement with Ras-independent activation of PI3K by GDNF in neuronal cells, survival of sympathetic neurons induced by GDNF was dependent on PI3K but was not affected by microinjection of blocking anti-Ras antibodies, which did compromise neuronal survival by nerve growth factor, suggesting that Ras is not required for GDNF-induced survival of sympathetic neurons. These results indicate that upon ligand stimulation, at least two distinct protein complexes assemble on phosphorylated Tyr-1062 of c-Ret via Shc, one leading to activation of the Ras/Erk pathway through recruitment of Grb2/Sos and another to the PI3K/Akt pathway through recruitment of Grb2/Gab2 followed by p85(PI3K) and SHP-2. This latter complex can also assemble directly onto phosphorylated Tyr-1096, offering an alternative route to PI3K activation by GDNF.     

Identification of an atypical Grb2 carboxyl-terminal SH3 domain binding site in Gab docking proteins reveals Grb2-dependent and -independent recruitment of Gab1 to receptor tyrosine kinases

The Gab family of docking proteins is phosphorylated in response to various growth factors and cytokines and serves to recruit multiple signaling proteins. Gab1 acts downstream from the Met-hepatocyte growth factor receptor, and Gab1 overexpression promotes Met-dependent morphogenesis of epithelial cells. Recruitment of Gab1 to Met or epidermal growth factor (EGF) receptors requires a receptor-binding site for the Grb2 adapter protein and a proline-rich domain in Gab1, defined as the Met-binding domain. To determine the requirement for Grb2 in Gab1 recruitment, we have mapped two Grb2 carboxyl-terminal SH3 domain binding sites conserved in Gab1 and related protein Gab2. One corresponds to a canonical Grb2-binding motif, whereas the second, located within the Gab1 Met-binding domain, requires the proline and arginine residues of an atypical PXXXR motif. The PXXXR motif is required but not sufficient for Grb2 binding, whereas an extended motif, PX3RX2KPX7PLD, conserved in Gab proteins as well as the Grb2/Gads-docking protein, Slp-76, efficiently competes binding of Grb2 or Gads adapter proteins. The association of Gab1 with Grb2 is required for Gab1 recruitment to the EGF receptor but not the Met receptor. Hence different mechanisms of Gab1 recruitment may reflect the distinct biological functions for Gab1 downstream from the EGF and Met receptors.     

Protein kinase C-alpha and protein kinase C-epsilon are required for Grb2-associated binder-1 tyrosine phosphorylation in response to platelet-derived growth factor

Grb2-associated binder-1 (Gab1) is an adapter protein related to the insulin receptor substrate family. It is a substrate for the insulin receptor as well as the epidermal growth factor (EGF) receptor and other receptor-tyrosine kinases. To investigate the role of Gab1 in signaling pathways downstream of growth factor receptors, we stimulated rat aortic vascular smooth muscle cells (VSMC) with EGF and platelet-derived growth factor (PDGF). Gab1 was tyrosine-phosphorylated by EGF and PDGF within 1 min. AG1478 (an EGF receptor kinase-specific inhibitor) failed to block PDGF-induced Gab1 tyrosine phosphorylation, suggesting that transactivated EGF receptor is not responsible for this signaling event. Because Gab1 associates with phospholipase Cgamma (PLCgamma), we studied the role of the PLCgamma pathway in Gab1 tyrosine phosphorylation. Gab1 tyrosine phosphorylation by PDGF was impaired in Chinese hamster ovary cells expressing mutant PDGFbeta receptor (Y977F/Y989F: lacking the binding site for PLCgamma). Pretreatment of VSMC with (a specific PLCgamma inhibitor) inhibited Gab1 tyrosine phosphorylation as well, indicating the importance of the PLCgamma pathway. Gab1 was tyrosine-phosphorylated by phorbol ester to the same extent as PDGF stimulation. Studies using antisense protein kinase C (PKC) oligonucleotides and specific inhibitors showed that PKCalpha and PKCepsilon are required for Gab1 tyrosine phosphorylation. Binding of Gab1 to the protein-tyrosine phosphatase SHP2 and phosphatidylinositol 3-kinase was significantly decreased by PLCgamma and/or PKC inhibition, suggesting the importance of the PLCgamma/PKC-dependent Gab1 tyrosine phosphorylation for the interaction with other signaling molecules. Because PDGF-mediated ERK activation is enhanced in Chinese hamster ovary cells that overexpress Gab1, Gab1 serves as an important link between PKC and ERK activation by PDGFbeta receptors in VSMC.     

Adaptor molecule Crk is required for sustained phosphorylation of Grb2-associated binder 1 and hepatocyte growth factor-induced cell motility of human synovial sarcoma cell lines

Activation of the c-Met receptor tyrosine kinase through its ligand, hepatocyte growth factor (HGF), promotes mitogenic, motogenic, and morphogenic cellular responses. Aberrant HGF/c-Met signaling has been strongly implicated in tumor cell invasion and metastasis. Both HGF and its receptor c-Met have been shown to be overexpressed in human synovial sarcoma, which often metastasizes to the lung; however, little is known about HGF-mediated biological effects in this sarcoma. Here, we provide evidence that Crk adaptor protein is required for the sustained phosphorylation of c-Met-docking protein Grb2-associated binder 1 (Gab1) in response to HGF, leading to the enhanced cell motility of human synovial sarcoma cell lines SYO-1, HS-SY-II, and Fuji. HGF stimulation induced the sustained phosphorylation on Y307 of Gab1 where Crk was recruited. Crk knockdown by RNA interference disturbed this HGF-induced tyrosine phosphorylation of Gab1. By mutational analysis, we identified that Src homology 2 domain of Crk is indispensable for the induction of the phosphorylation on multiple Tyr-X-X-Pro motifs containing Y307 in Gab1. HGF remarkably stimulated cell motility and scattering of synovial sarcoma cell lines, consistent with the prominent activation of Rac1, extreme filopodia formation, and membrane ruffling. Importantly, the elimination of Crk in these cells induced the disorganization of actin cytoskeleton and complete abolishment of HGF-mediated Rac1 activation and cell motility. Time-lapse microscopic analysis revealed the significant attenuation in scattering of Crk knockdown cells following HGF treatment. Furthermore, the depletion of Crk remarkably inhibited the tumor formation and its invasive growth in vivo. These results suggest that the sustained phosphorylation of Gab1 through Crk in response to HGF contributes to the prominent activation of Rac1 leading to enhanced cell motility, scattering, and cell invasion, which may support the crucial role of Crk in the aggressiveness of human synovial sarcoma.     

Biochemical and biological responses induced by coupling of Gab1 to phosphatidylinositol 3-kinase in RET-expressing cells

Grb2-associated binder-1 (Gab1) is a docking protein closely related to insulin receptor substrates. We previously reported that tyrosine 1062 in RET receptor tyrosine kinase activated by glial cell line-derived neurotrophic factor (GDNF) represents a binding site for the Shc-Grb2-Gab1 complex, and that the p85 subunit of phosphatidylinositol 3-kinase (PI3K) and SHP2 tyrosine phosphatase is associated with Gab1 in GDNF-treated cells. In the present study, we further analyzed the physiological roles of Gab1 downstream of RET, using Gab1 mutants that lack the binding sites for PI3K (Gab1 PI3K-m) or SHP-2 (Gab1 SHP2-m). Expression of Gab1 PI3K-m in SK-N-MC human primitive neuroectodermal tumor cells expressing wild-type RET markedly impaired Akt phosphorylation, Rac1 activation, and lamellipodia formation that were induced by GDNF whereas expression of Gab1 SHP2-m partially impaired Erk activation. Furthermore, expression of Gab1 PI3K-m, but not Gab1 SHP2-m, in TT human medullary thyroid carcinoma cells expressing RET with a multiple endocrine neoplasia 2A mutation enhanced cytochrome c release, and apoptosis induced by etoposide, suggesting that PI3K is involved in survival of TT cells via a mitochondrial pathway. These findings demonstrated that coupling of Gab1 to PI3K is important for biological responses in RET-expressing cells.     

The involvement of the docking protein Gab1 in mitogenic signalling induced by EGF and HGF in rat hepatocytes

Grb2-assosiated binder (Gab) family proteins are docking molecules that can interact with receptor tyrosine kinases (RTKs) and cytokine receptors and bind several downstream signalling proteins. Studies in several cell types have shown that Gab1 may have a role in signalling mediated by the two RTKs epidermal growth factor (EGF) receptor (EGFR) and Met, the receptor for hepatocyte growth factor (HGF), but the involvement of Gab1 in EGFR and Met signalling has not been directly compared in the same cell. We have studied mechanisms of activation and role in mitogenic signalling of Gab1 in response to EGF and HGF in cultured rat hepatocytes. Gab1, but not Gab2, was expressed in the hepatocytes and was phosphorylated upon stimulation with EGF or HGF. Depletion of Gab1, using siRNA, decreased the ERK and Akt activation, cyclin D1 expression, and DNA synthesis in response to both EGF and HGF. Studies of mechanisms of recruitment to the receptors showed that HGF induced co-precipitation of Gab1 and Met while EGF induced binding of Gab1 to Grb2 but not to EGFR. Gab1 activation in response to both EGF and HGF was dependent on PI3K. While EGF activated Gab1 and Shc equally, within the same concentration range, HGF very potently and almost exclusively activated Gab1, having only a minimal effect on Shc. Collectively, our results strongly suggest that although Gab1 interacts differently with EGFR and Met, it is involved in mitogenic signalling mediated by both these growth factor receptors in hepatocytes.     

Regulation of the mitogen-activated protein kinase signaling pathway by SHP2.

Gab1-SHP2 association is required for Erk mitogen-activated protein kinase activation by several growth factors. Gab1-SHP2 interaction activates SHP2. However, an activated SHP2 still needs to associate with Gab1 to mediate Erk activation. It was unclear whether SHP2 is required to dephosphorylate a negative phosphorylation site on Gab1 or whether SHP2 needs the Gab1 pleckstrin homology (PH) domain to target it to the plasma membrane. We found that expression of a fusion protein consisting of the Gab1 PH domain and an active SHP2 (Gab1PH-SHP2DeltaN) induced constitutive Mek1 and Erk2 activation. Linking the active SHP2DeltaN to the PDK1 PH domain or the FRS2beta myristoylation sequence also induced Mek1 activation. Mek1 activation by Gab1PH-SHP2DeltaN was inhibited by an Src inhibitor and by Csk. Significantly, Gab1PH-SHP2DeltaN induced Src activation. Gab1PH-SHP2DeltaN expression activated Ras, and the Gab1PH-SHP2DeltaN-induced Mek1 activation was blocked by RasN17. These findings suggest that Gab1PH-SHP2DeltaN activated a signaling step upstream of Src and Ras. The SHP2 tyrosine phosphatase activity is essential for the function of the fusion protein. Together, these data show that the Gab1 sequence, besides the PH domain and SHP2 binding sites, is dispensable for Erk activation, suggesting that the primary role of Gab1 association with an activated SHP2 is to target it to the membrane.     

Dorsal Ruffle Microdomains Potentiate Met Receptor Tyrosine Kinase Signaling and Down-regulation

Dorsal ruffles are apical protrusions induced in response to many growth factors, yet their function is poorly understood. Here we report that downstream from the hepatocyte growth factor (HGF) receptor tyrosine kinase (RTK), Met, dorsal ruffles function as both a localized signaling microdomain as well as a platform from which the Met RTK internalizes and traffics to a degradative compartment. In response to HGF, colonies of epithelial Madin-Darby canine kidney cells form dorsal ruffles for up to 20 min. Met is transcytosed from the basolateral membrane on Rab4 endosomes, to the apical surface where Met, as well as a Met substrate and scaffold protein, Gab1, localize to the dorsal ruffle membrane. This results in activation of downstream signaling proteins, as evidenced by localization of phospho-ERK1/2 to dorsal ruffles. As dorsal ruffles collapse, Met is internalized into EEA1- and Rab5-positive endosomes and is targeted for degradation through delivery to an Hrs-positive sorting compartment. Enhancing HGF-dependent dorsal ruffle formation, through overexpression of Gab1 or activated Pak1 kinase, promotes more efficient degradation of the Met RTK. Conversely, the ablation of dorsal ruffle formation, by pre-treatment with SITS (4-acetamido-4′-isothiocyabatostilbene-2′,2-disulfonic acid) or expression of a Gab1 mutant, impairs Met degradation. Taken together, these data support a function for dorsal ruffles as a biologically relevant signaling microenvironment and a mechanism for Met receptor internalization and degradation.