Scaffolding protein Gab2 mediates differentiation signaling downstream of Fms receptor tyrosine kinase

Fms is the receptor for macrophage colony-stimulating factor (M-CSF) and contains intrinsic tyrosine kinase activity. Expression of exogenous Fms in a murine myeloid progenitor cell line, FDC-P1 (FD-Fms), results in M-CSF-dependent growth and macrophage differentiation. Previously, we described a 100-kDa protein that was tyrosine phosphorylated upon M-CSF stimulation of FD-Fms cells. In this report, we identify this 100-kDa protein as the recently cloned scaffolding protein Gab2, and we demonstrate that Gab2 associates with several molecules involved in M-CSF signaling, including Grb2, SHP2, the p85 subunit of phosphatidylinositol 3'-kinase, SHIP, and SHC. Tyrosine phosphorylation of Gab2 in response to M-CSF requires the kinase activity of Fms, but not that of Src. Overexpression of Gab2 in FD-Fms cells enhanced both mitogen-activated protein kinase (MAPK) activity and macrophage differentiation, but reduced proliferation, in response to M-CSF. In contrast, a mutant of Gab2 that is unable to bind SHP2 did not potentiate MAPK activity. Furthermore, overexpression of this mutant in FD-Fms cells inhibited macrophage differentiation and resulted in a concomitant increase in growth potential in response to M-CSF. These data indicate that Gab2 is involved in the activation of the MAPK pathway and that the interaction between Gab2 and SHP2 is essential for the differentiation signal triggered by M-CSF.     

A function for phosphoinositide 3-kinase beta lipid products in coupling beta gamma to Ras activation in response to lysophosphatidic acid

Although Gbetagamma is thought to mediate mitogen-activated protein kinase (MAPK) activation in response to G protein-coupled receptor stimulation, the mechanisms involved in this pathway have not been clearly defined. Phosphoinositide 3-kinase (PI3K) has been proposed as an early intermediate in this process, but its role has remained elusive. We have observed that dominant negative mutants of p110beta, but not of p110gamma, inhibited MAPK stimulation in response to lysophosphatidic acid (LPA). The role of p110beta was located upstream from Ras. To determine which of the lipid or protein kinase activities of p110beta were important for Ras activation, we produced a mutant p110beta lacking the lipid but not the protein kinase activity. This protein displayed a dominant negative activity similar to a kinase-dead mutant, indicating that p110beta lipid kinase activity was essentially involved in Ras activation. In agreement, overexpression of the lipid phosphatase PTEN was found to specifically inhibit Ras stimulation induced by LPA. In addition, we have observed that the PH domain-containing adapter protein Gab1, which is involved in p110beta activation during LPA stimulation, is also implicated in this pathway downstream of p110beta. Indeed, both membrane redistribution and phosphorylation of Gab1 were reduced in the presence of PI3K inhibitors or dominant negative p110beta. Downstream of Gab1, the tyrosine phosphatase SHP2 was found to mediate Ras activation in response to LPA and to be recruited through PI3K and Gab1, because transfection of Gab1 mutant deficient for SHP2 binding inhibited Ras activation without interfering with PI3K activation. We conclude that LPA-induced Ras activation is mediated by a p110beta/Gab1/SHP2 pathway. Moreover, we present data indicating that p110beta is effectively the target of betagamma in this pathway, suggesting that the p110beta/Gab1/SHP2 pathway provides a novel link between betagamma and Ras by integrating two early events of LPA signaling, i.e. Gbetagamma release and tyrosine kinase receptor transactivation.     

A critical role for phosphoinositide 3-kinase upstream of Gab1 and SHP2 in the activation of ras and mitogen-activated protein kinases by epidermal growth factor

Although the mechanisms involved in the activation of mitogen-activated protein kinases (MAPK) by receptor tyrosine kinases do not display an obvious role for phosphoinositide 3-kinases (PI3Ks), we have observed in the nontransformed cell line Vero stimulated with epidermal growth factor (EGF) that wortmannin and LY294002 nearly abolished MAPK activation. The effect was observed under strong stimulation and was independent of EGF concentration. In addition, three mutants of class Ia PI3Ks were found to inhibit MAPK activation to an extent similar to their effect on Akt/protein kinase B activation. To determine the importance of PI3K lipid kinase activity in MAPK activation, we have used the phosphatase PTEN and the pleckstrin homology domain of Tec kinase. Overexpression of these proteins, but not control mutants, was found to inhibit MAPK activation, suggesting that the lipid products of class Ia PI3K are necessary for MAPK signaling. We next investigated the location of PI3K in the MAPK cascade. Pharmacological inhibitors and dominant negative forms of PI3K were found to block the activation of Ras induced by EGF. Upstream from Ras, although association of Grb2 with its conventional effectors was independent of PI3K, we have observed that the recruitment of the tyrosine phosphatase SHP2 required PI3K. Because SHP2 was also essential for Ras activation, this suggested the existence of a PI3K/SHP2 pathway leading to the activation of Ras. In addition, we have observed that the docking protein Gab1, which is involved in PI3K activation during EGF stimulation, is also implicated in this pathway downstream of PI3K. Indeed, the association of Gab1 with SHP2 was blocked by PI3K inhibitors, and expression of Gab1 mutant deficient for binding to SHP2 was found to inhibit Ras stimulation without interfering with PI3K activation. These results show that, in addition to Shc and Grb2, a PI3K-dependent pathway involving Gab1 and SHP2 is essential for Ras activation under EGF stimulation.     

ERK negatively regulates the epidermal growth factor-mediated interaction of Gab1 and the phosphatidylinositol 3-kinase

We have examined the ability of epidermal growth factor (EGF)-stimulated ERK activation to regulate Grb2-associated binder-1 (Gab1)/phosphatidylinositol 3-kinase (PI3K) interactions. Inhibiting ERK activation with the MEK inhibitor U0126 increased the EGF-stimulated association of Gab1 with either full-length glutathione S-transferase-p85 or the p85 C-terminal Src homology 2 (SH2) domain, a result reproduced by co-immunoprecipitation of the native proteins from intact cells. This increased association of Gab1 and the PI3K correlates with an increase in PI3K activity and greater phosphorylation of Akt. This result is in direct contrast to what we have previously reported following HGF stimulation where MEK inhibition decreased the HGF-stimulated association of Gab1 and p85. In support of this divergent effect of ERK on Gab1/PI3K association following HGF and EGF stimulation, U0126 decreased the HGF-stimulated association of p85 and the Gab1 c-Met binding domain but did not alter the EGF-stimulated association of p85 and the c-Met binding domain. An examination of the mechanism of this effect revealed that the treatment of cells with EGF + U0126 increased the tyrosine phosphorylation of Gab1 as well as its association with another SH2-containing protein, SHP2. Furthermore, overexpression of a catalytically inactive form of SHP2 or pretreatment with pervanadate markedly increased EGF-stimulated Gab1 tyrosine phosphorylation. These experiments demonstrate that EGF and HGF-mediated ERK activation result in divergent effects on Gab1/PI3K signaling. HGF-stimulated ERK activation increases the Gab1/PI3K association, whereas EGF-stimulated ERK activation results in a decrease in the tyrosine phosphorylation of Gab1 and a decreased association with the PI3K. SHP2 is shown to associate with and dephosphorylate Gab1, suggesting that EGF-stimulated ERK might act through the regulation of SHP2.     

Requirement of SHP2 binding to Grb2-associated binder-1 for mitogen-activated protein kinase activation in response to lysophosphatidic acid and epidermal growth factor

Grb2-associated binder-1 (Gab1) is a multisite docking protein containing a pleckstrin homology (PH) domain, multiple potential tyrosine phosphorylation sites, and several proline-rich sequences. Gab1 becomes tyrosine-phosphorylated in cells stimulated with growth factors, cytokines, and ligands for G protein-coupled receptors. A major Gab1-binding protein detected in cells treated with extracellular stimuli is the tyrosine phosphatase, SHP2. Although the role of SHP2-Gab1 interaction in cell signaling has not yet been characterized, SHP2 is known to mediate mitogen-activated protein (MAP) kinase activation induced by the epidermal growth factor (EGF). However, the mechanism by which the SHP2 phosphatase exerts a positive signaling role remains obscure. In this study, we prepared Gab1 mutants lacking the SHP2 binding site (Gab1Y627F), the phosphatidylinositol 3-kinase (PI3K) binding sites (Gab1DeltaPI3K), and the PH domain (Gab1DeltaPH). Expression of Gab1Y627F blocked the extracellular signal-regulated kinase-2 (ERK2) activation by lysophosphatidic acid (LPA) and EGF. Conversely, expression of the wild-type Gab1 in HEK293 cells augmented the LPA receptor Edg2-mediated ERK2 activation. Whereas the PH domain was required for Gab1 mediation of ERK2 activation by LPA, it was not essential for EGF-induced ERK2 activation. Expression of Gab1DeltaPI3K had no apparent effect on ERK2 activation by LPA and EGF in the cells that we have examined. These results establish a role for Gab1 in the LPA-induced MAP kinase pathway and clearly demonstrate that Gab1-SHP2 interaction is essential for ERK2 activation by LPA and EGF. These findings also suggest that the positive role of SHP2 in the MAP kinase pathway depends on its interaction with Gab1.     

Gab3, a New DOS/Gab Family Member, Facilitates Macrophage Differentiation

Using the FDC-P1 cell line expressing the exogenous macrophage colony-stimulating factor (M-CSF) receptor, Fms, we have analyzed the role of a new mammalian DOS/Gab-related signaling protein, called Gab3, in macrophage cell development of the mouse. Gab3 contains an amino-terminal pleckstrin homology domain, multiple potential sites for tyrosine phosphorylation and SH2 domain binding, and two major polyproline motifs potentially interacting with SH3 domains. Among the growing family of Gab proteins, Gab3 exhibits a unique and overlapping pattern of expression in tissues of the mouse compared with Gab1 and Gab2. Gab3 is more restricted to the hematopoietic tissues such as spleen and thymus but is detectable at progressively lower levels within heart, kidney, uterus, and brain. Like Gab2, Gab3 is tyrosine phosphorylated after M-CSF receptor stimulation and associates transiently with the SH2 domain-containing proteins p85 and SHP2. Overexpression of exogenous Gab3 in FD-Fms cells dramatically accelerates macrophage differentiation upon M-CSF stimulation. Unlike Gab2, which shows a constant mRNA expression level after M-CSF stimulation, Gab3 expression is initially absent or low in abundance in FD cells expressing the wild-type Fms, but Gab3 mRNA levels are increased upon M-CSF stimulation. Moreover, M-CSF stimulation of FD-FmsY807F cells (which grow but do not differentiate) fails to increase Gab3 expression. These results suggest that Gab3 is important for macrophage differentiation and that differentiation requires the early phosphorylation of Gab2 followed by induction and subsequent phosphorylation of Gab3.     

Phosphoinositide 3-kinase-dependent regulation of interleukin-3-induced proliferation: involvement of mitogen-activated protein kinases, SHP2 and Gab2

We have demonstrated previously that class I(A) phosphoinositide 3-kinases play a major role in regulation of interleukin-3 (IL)-3-dependent proliferation. Investigations into the downstream targets involved have identified the MAPK cascade as a target. Expression of Deltap85 and incubation with LY294002 both inhibited IL-3-induced activation of Mek, Erk1, and Erk2. This was most pronounced during the initial phase of Erk activation. The Mek inhibitor, PD98059, blocked IL-3-driven proliferation, an effect enhanced by Deltap85 expression, suggesting that inhibition of Mek and Erks by Deltap85 contributes to the decrease in IL-3-induced proliferation in these cells but that additional pathways may also be involved. To investigate the mechanism leading to decreased activation of Erks, we investigated effects on SHP2 and Gab2, both implicated in IL-3 regulation of Erk activation. Expression of Deltap85 led to a reduction in SHP2 tyrosine phosphorylation and its ability to interact with Grb2 and Gab2 but increased overall tyrosine phosphorylation of Gab2. LY294002 did not perturb SHP2 interactions, potentially related to differences in the effects of these inhibitors on levels of phosphoinositides. These results imply that the regulation of Erks by class I(A) phosphoinositide 3-kinase may contribute to IL-3-driven proliferation and that both SHP2 and Gab2 are possibly involved in this regulation.     

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.     

Roles of Gab1 and SHP2 in paxillin tyrosine dephosphorylation and Src activation in response to epidermal growth factor

Epidermal growth factor (EGF) induces paxillin tyrosine dephosphorylation and Src activation, but the signaling pathways that mediate these responses were largely undefined. We found that Gab1, a docking protein for the SHP2 protein-tyrosine phosphatase in EGF-stimulated cells, was associated with paxillin. SHP2 dephosphorylated paxillin and caused dissociation of Csk, a negative regulator of Src, from paxillin but had no effect on paxillin-Src association. A lower level of Src Tyr-530 phosphorylation was detected in paxillin-associated Src in EGF-stimulated cells. Expression of an SHP2 binding defective mutant of Gab1 (Gab1FF) or a catalytically inactive mutant of SHP2 (SHP2DN) prevented paxillin tyrosine dephosphorylation and Src activation induced by EGF. Importantly, Gab1FF blocked paxillin-SHP2 complex formation, Src Tyr-530 dephosphorylation, Erk activation, and cell migration induced by EGF. Inhibition of Src tyrosine kinase activity abrogated EGF-stimulated Erk activation and cell migration. Together, these results reveal that Gab1 recruits SHP2 to dephosphorylate paxillin, leading to dissociation of Csk from the paxillin-Src complex and Src activation and that Src is an SHP2 effector involved in EGF-stimulated Erk activation and cell migration.     

Signal characteristics of G protein-transactivated EGF receptor.

The epidermal growth factor receptor (EGFR) tyrosine kinase recently was identified as providing a link to mitogen-activated protein kinase (MAPK) in response to G protein-coupled receptor (GPCR) agonists in Rat-1 fibroblasts. This cross-talk pathway is also established in other cell types such as HaCaT keratinocytes, primary mouse astrocytes and COS-7 cells. Transient expression of either Gq- or Gi-coupled receptors in COS-7 cells allowed GPCR agonist-induced EGFR transactivation, and lysophosphatidic acid (LPA)-generated signals involved the docking protein Gab1. The increase in SHC tyrosine phosphorylation and MAPK stimulation through both Gq- and Gi-coupled receptors was reduced strongly upon selective inhibition of EGFR function. Inhibition of phosphoinositide 3-kinase did not affect GPCR-induced stimulation of EGFR tyrosine phosphorylation, but inhibited MAPK stimulation, upon treatment with both GPCR agonists and low doses of EGF. Furthermore, the Src tyrosine kinase inhibitor PP1 strongly interfered with LPA- and EGF-induced tyrosine phosphorylation and MAPK activation downstream of EGFR. Our results demonstrate an essential role for EGFR function in signaling through both Gq- and Gi-coupled receptors and provide novel insights into signal transmission downstream of EGFR for efficient activation of the Ras/MAPK pathway.     

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.     

Epidermal growth factor-induced DNA synthesis. Key role for Src phosphorylation of the docking protein Gab2

We have previously demonstrated that phosphatidylinositol 3-kinase (PI3-kinase) is necessary and sufficient to account for epidermal growth factor (EGF)-induced mitogenesis in rat primary hepatocytes. A cytosolic Gab2-containing complex accounts for >80% of the total EGF-induced PI3-kinase activity (Kong, M., Mounier, C., Wu, J., and Posner, B. I. (2000) J. Biol. Chem. 275, 36035-36042), suggesting a key role for Gab2 in EGF-induced mitogenesis. Here, we demonstrate that PP1, a selective inhibitor of Src family kinases, blocks the EGF-induced Gab2 tyrosine phosphorylation without inhibiting EGF-induced phosphorylation of the EGF receptor, ErbB3, or Shc. We also show that Gab2 phosphorylation is increased in Csk knockout cells in which Src family kinases are constitutively activated. Furthermore, PP1 blocks Gab2-associated downstream events including EGF-induced PI3-kinase activation, Akt phosphorylation, and DNA synthesis. We demonstrate that Gab2 and Src are constitutively associated. Since this association involves the proline-rich sequences of Gab2, it probably involves the Src homology 3 domain of Src kinase. Mutation of the proline-rich sequences in Gab2 prevented EGF-induced Gab2 phosphorylation, PI3-kinase/Akt activation, and DNA synthesis, demonstrating that Gab2 phosphorylation is critical for EGF-induced mitogenesis and is not complemented by ErbB3 or Shc phosphorylation. We also found that overexpression of a Gab2 mutant lacking SHP2 binding sites increased EGF-induced Gab2 phosphorylation and the activation of PI3-kinase but blocked activation of MAPK. In addition, we demonstrated that the Src-induced response was down-regulated by Gab2-associated SHP2. In summary, our results have defined the role for Src activation in EGF-induced hepatic mitogenesis through the phosphorylation of Gab2 and the activation of the PI3-kinase cascade.     

A novel role for Gab1 and SHP2 in epidermal growth factor-induced Ras activation

SHP2 was recently found to down-regulate PI3K activation by dephosphorylating Gab1 but the mechanisms explaining the positive role of the Gab1/SHP2 pathway in EGF-induced Ras activation remain ill defined. Substrate trapping experiments now suggest that SHP2 dephosphorylates other Gab1 phosphotyrosines located within a central region displaying four YXXP motifs. Because these sites are potential docking motifs for Ras-GAP, we tested whether SHP2 dephosphorylates them to facilitate Ras activation. We observed that a Gab1 construct preventing SHP2 recruitment promoted membrane relocation of RasGAP. Moreover, a RasGAP-inactive mutant restored the activation of Ras in cells transfected with SHP2-inactivating Gab1 mutant or in SHP2-deficient fibroblasts, supporting the hypothesis that RasGAP is a downstream target of SHP2. To determine whether Gab1 is a RasGAP-binding partner, a Gab1 mutant deleted of four YXXP motifs was produced. The deletion suppressed RasGAP redistribution and restored the defective Ras activation caused by SHP2-inactivating mutations. Moreover, Gab1 was found to interact with RasGAP SH2 domains, only under conditions where SHP2 is not activated. To identify Ras-GAP-binding sites, Tyr to Phe mutants of Gab1 YXXP motifs were produced. Gab1 constructs mutated on Tyr(317) were severely affected in RasGAP binding and were the most active in compensating for Ras-defective activation and blocking RasGAP redistribution induced by SHP2 inactivation. We have thus localized on Gab1 a Ras-negative regulatory tyrosine phosphorylation site involved in RasGAP binding and showed that an important SHP2 function is to down-regulate its phosphorylation to disengage RasGAP and sustain Ras activation.     

The docking molecule gab2 is induced by lymphocyte activation and is involved in signaling by interleukin-2 and interleukin-15 but not other common gamma chain-using cytokines

Interleukin (IL)-2, a critical cytokine with indispensable functions in regulating lymphoid homeostasis, induces the activation of several biochemical pathways. Precisely how these pathways are linked and how they relate to the biological action of IL-2 is incompletely understood. We previously identified SHP-2 (Src homology 2 domain containing phosphatase 2) as an important intermediate in IL-2-dependent MAPK activation and showed its association with a 98-kDa phosphoprotein in response to IL-2. Here, we demonstrate that Gab2, a recently identified adapter molecule, is the major SHP-2 and phosphatidylinositol 3'-kinase-associated 98-kDa protein in normal, IL-2-activated lymphocytes. We further demonstrate that phosphorylation of both Gab2 and SHP-2 is largely dependent upon tyrosine 338 of the IL-2 receptor beta chain. Gab2 can be a substrate of all the three major classes of non-receptor tyrosine kinases associated with the IL-2R, but in terms of IL-2 signaling, JAK3 but not Lck or Syk is essential for Gab2 phosphorylation. We also demonstrate that only IL-2 and IL-15, but not other gammac cytokines induce Gab2 phosphorylation; the ability to phosphorylate Gab2 correlates with Shc phosphorylation and ERK1/ERK2 activation. Finally, we also show that Gab2 levels are regulated by T cell activation, and resting T cells express little Gab2. Therefore, up-regulation and activation of Gab2 may be important in linking the IL-2 receptor to activation of MAPK and may be an important means of achieving specificity in cytokine signaling.     

Gab2, via PI-3K, regulates ARF1 in FcεRI-mediated granule translocation and mast cell degranulation

Mast cells are major players in allergic responses. IgE-dependent activation through FcεR leads to degranulation and cytokine production, both of which require Gab2. To clarify how the signals diverge at Gab2, we established Gab2 knock-in mice that express Gab2 mutated at either the PI3K or SH2 domain-containing protein tyrosine phosphatase-2 (SHP2) binding sites. Examination of these mutants showed that both binding sites were required for the degranulation and anaphylaxis response but not for cytokine production or contact hypersensitivity. Furthermore, the PI3K, but not the SHP2, binding site was important for granule translocation during degranulation. We also identified a small GTPase, ADP-ribosylation factor (ARF)1, as the downstream target of PI3K that regulates granule translocation. FcεRI stimulation induced ARF1 activation, and this response was dependent on Fyn and the PI3K binding site of Gab2. ARF1 activity was required for FcεRI-mediated granule translocation. These data indicated that Fyn/Gab2/PI3K/ARF1-mediated signaling is specifically involved in granule translocation and the anaphylaxis response.     

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.     

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.     

Phosphorylation of Grb2-associated binder 2 on serine 623 by ERK MAPK regulates its association with the phosphatase SHP-2 and decreases STAT5 activation

IL-2 stimulation of T lymphocytes induces the tyrosine phosphorylation and adaptor function of the insulin receptor substrate/Grb2-associated binder (Gab) family member, Gab2. In addition, Gab2 undergoes a marked decrease in its mobility in SDS-PAGE, characteristic of migration shifts induced by serine/threonine phosphorylations in many proteins. This migration shift was strongly diminished by treating cells with the MEK inhibitor U0126, indicating a possible role for ERK in Gab2 phosphorylation. Indeed, ERK phosphorylated Gab2 on a consensus phosphorylation site at serine 623, a residue located between tyrosine 614 and tyrosine 643 that are responsible for Gab2/Src homology 2 domain-containing tyrosine phosphatase (SHP)-2 interaction. We report that pretreatment of Kit 225 cells with U0126 increased Gab2/SHP-2 association and tyrosine phosphorylation of SHP-2 in response to IL-2, suggesting that ERK phosphorylation of serine 623 regulates the interaction between Gab2 and SHP-2, and consequently the activity of SHP-2. This hypothesis was confirmed by biochemical analysis of cells expressing Gab2 WT, Gab2 serine 623A or Gab2 tyrosine 614F, a mutant that cannot interact with SHP-2 in response to IL-2. Activation of the ERK pathway was indeed blocked by Gab2 tyrosine 614F and slightly increased by Gab2 serine 623A. In contrast, STAT5 activation was strongly enhanced by Gab2 tyrosine 614F, slightly reduced by Gab2 WT and strongly inhibited by Gab2 serine 623A. Analysis of the rate of proliferation of cells expressing these mutants of Gab2 demonstrated that tyrosine 614F mutation enhanced proliferation whereas serine 623A diminished it. These results demonstrate that ERK-mediated phosphorylation of Gab2 serine 623 is involved in fine tuning the proliferative response of T lymphocytes to IL-2.     

SHP2 regulates IL-2 induced MAPK activation,but not Stat3 or Stat5 tyrosine phosphorylation,in cutaneous T cell lymphoma cells

The phosphotyrosine phosphatase SHP2 has been suggested to regulate activation of MAPK, Stat3, and Stat5 in several experimental models. In this study we investigated the role of SHP2 in IL-2 induced activation of MAPK and the Stat proteins using the human CTCL cell line MyLa2059 derived from a cutaneous T cell lymphoma (CTCL). For this purpose, MyLa2059 cells were stably transfected with wild-type SHP2 or inactive SHP2. The cells transfected with inactive SHP2 showed reduced MAPK activation upon IL-2 stimulation, suggesting that SHP2 upregulates IL-2 induced MAPK activation in T cells. However, the constitutive tyrosine phosphorylation of Stat3 as well as IL-2 induced Stat5 tyrosine phosphorylation and DNA binding were unaffected by the stably transfected wild-type SHP2 as well as the inactive SHP2. In conclusion, we show for the first time that SHP2 positively regulates IL-2 induced MAPK activation in malignant T cells. Furthermore, the results indicate that SHP2 may not be involved in the activation of Stat3 or Stat5 in CTCL cells.     

Gab1 is an integrator of cell death versus cell survival signals in oxidative stress

Upon the addition of different growth factors and cytokines, the Gab1 docking protein is tyrosine phosphorylated and in turn activates different signaling pathways. On the basis of the large body of evidence concerning cross talk between the signaling pathways activated by growth factors and oxidative stress, we decided to investigate the role of Gab1 in oxidative injury. We stimulated wild-type mouse embryo fibroblasts (MEF) or MEF with a homozygous deletion of the Gab1 gene (-/- MEF) with H(2)O(2). Our results show that Gab1 is phosphorylated in a dose- and time-dependent manner after H(2)O(2) triggering. Gab1 then recruits molecules such as SHP2, phosphatidylinositol 3-kinase (PI3K), and Shc. Gab1 phosphorylation is sensitive to the Src family kinase inhibitor PP2. Furthermore, we demonstrate that Gab1 is required for H(2)O(2)-induced c-Jun N-terminal kinase (JNK) activation but not for ERK2 or p38 activation. Reconstitution of Gab1 in -/- MEF rescues JNK activation, and we find that this is dependent on the SHP2 binding site in Gab1. Cell viability assays reveal that Gab1 has a dual role in cell survival: a positive one through its interaction with PI3K and a negative one through its interaction with SHP2. This is the first report identifying Gab1 as a component in oxidative stress signaling and one that is required for JNK activation.