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.     

M-CSF stimulated differentiation requires persistent MEK activity and MAPK phosphorylation independent of Grb2-Sos association and phosphatidylinositol 3-kinase activity

Macrophage colony-stimulating factor (M-CSF) is a physiological regulator of monocyte-macrophage lineage. Ectopic expression of the M-CSF receptor (M-CSFR, or Fms) in murine myeloid cell line FDC-P1 (FD/Fms cells) results in M-CSF-dependent macrophage differentiation. Previously, we observed that M-CSF induces two temporally distinct phases of mitogen-activated protein kinase (MAPK) phosphorylation. Here we show that levels of phosphorylated MAPK kinase MEK1 follow the same kinetics as MAPK phosphorylation, characterized by an early and transient phase (the first 30 min of M-CSF stimulation) and a late and persistent phase from 4 h of stimulation. The MEK inhibitor U0126 strongly inhibited both phases of MAPK phosphorylation as well as FD/Fms cell differentiation, indicating that MAPK may relay M-CSF differentiation signaling downstream of M-CSFR. Treatment of FD/Fms cells with U0126 during the first hour of M-CSF stimulation reversibly blocked the early phase of MAPK phosphorylation but did not affect differentiation. In contrast, U0126 still inhibited FD/Fms cell differentiation when its addition was delayed by 24 h. This demonstrated that late and persistent MEK activity is specifically required for macrophage differentiation to occur. Furthermore, disrupting Grb2-Sos complexes with a specific blocking peptide did not prevent FD/Fms cells differentiation in response to M-CSF, nor did it abolish MAPK phosphorylation. The role of phosphatidylinositol 3-kinase (PI 3-kinase), another potential regulator of the MAPK pathway, was examined using the specific inhibitor LY294002. This compound could not impede FD/Fms cell commitment to macrophage differentiation and did not significantly affect MAPK phosphorylation in response to M-CSF. Therefore, M-CSF differentiation signaling in myeloid progenitor cells is mediated through persistent MEK activity but it is not strictly dependent upon Grb2-Sos interaction or PI 3-kinase activity.     

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.     

G‐CSF‐dependent neutrophil differentiation requires downregulation of MAPK activities through the Gab2 signaling pathway

Granulocyte colony‐stimulating factor (G‐CSF) stimulation of myeloid cells induced tyrosine‐phosphorylation of cellular proteins. One of the tyrosine‐phosphorylated proteins was found to be a scaffold protein, Grb2‐associated binding protein 2 (Gab2). Another member of Gab family protein, Gab3, was exogenously overexpressed in neutrophil progenitor cells to make the Gab3 protein to compete with the endogenous Gab2 for the G‐CSF‐dependent signaling. In Gab3‐overexpressed cells, the level of tyrosine phosphorylation of endogenous Gab2 by G‐CSF stimulation was markedly downregulated, while the phosphorylation of Gab3 was significantly enhanced. The Gab3‐overexpressed cells continuously proliferated in the medium containing G‐CSF and lost the ability to differentiate to the mature neutrophil, characterized by the lobulated nucleus. The G‐CSF stimulation‐dependent tyrosine phosphorylation of Gab3, the association of SHP2 to Gab3 and the following mitogen‐activated protein kinase (MAPK) activation were prolonged in the Gab3‐overexpressed cells, compared to the parental cells, where the binding of SHP2 to Gab2 protein and thereby the activation of MAPK were not sustained after G‐CSF stimulation. Inhibition of MAPK by pharmaceutical inhibitor restored the Gab3‐overexpressed cells to the ability to differentiate to mature neutrophil. Therefore, G‐CSF‐dependent Gab2 phosphorylation and following its downregulation led the short‐term MAPK activation. The downregulation of MAPK after transient Gab2 phosphorylation was necessary for the consequent neutrophil differentiation induced by G‐CSF stimulation.     

Tyrosine 569 in the c-Fms juxtamembrane domain is essential for kinase activity and macrophage colony-stimulating factor-dependent internalization.

The receptor (Fms) for macrophage colony-stimulating factor (M-CSF) is a member of the tyrosine kinase class of growth factor receptors. It maintains survival, stimulates growth, and drives differentiation of the macrophage lineage of hematopoietic cells. Fms accumulates on the cell surface and becomes activated for signal transduction after M-CSF binding and is then internalized via endocytosis for eventual degradation in lysosomes. We have investigated the mechanism of endocytosis as part of the overall signaling process of this receptor and have identified an amino acid segment near the cytoplasmic juxtamembrane region surrounding tyrosine 569 that is important for internalization. Mutation of tyrosine 569 to alanine (Y569A) eliminates ligand-induced rapid endocytosis of receptor molecules. The mutant Fms Y569A also lacks tyrosine kinase activity; however, tyrosine kinase activity is not essential for endocytosis because the kinase inactive receptor Fms K614A does undergo ligand-induced endocytosis, albeit at a reduced rate. Mutation of tyrosine 569 to phenylalanine had no effect on the M-CSF-induced endocytosis of Fms, and a four-amino-acid sequence containing Y-569 could support endocytosis when transferred into the cytoplasmic juxtamembrane region of a glycophorin A construct. These results indicate that tyrosine 569 within the juxtamembrane region of Fms is part of a signal recognition sequence for endocytosis that does not require tyrosine phosphorylation at this site and that this domain also influences the kinase activity of the receptor. These results are consistent with a ligand-dependent step in recognition of the potential cryptic internalization signal.     

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.     

Shc, Grb2, Sos1, and a 150-kilodalton tyrosine-phosphorylated protein form complexes with Fms in hematopoietic cells.

Fms, the macrophage colony-stimulating factor (M-CSF) receptor, is normally expressed in myeloid cells and initiates signals for both growth and development along the monocyte/macrophage lineage. We have examined Fms signal transduction pathways in the murine myeloid progenitor cell line FDC-P1. M-CSF stimulation of FDC-P1 cells expressing exogenous Fms resulted in tyrosine phosphorylation of a variety of cellular proteins in addition to Fms. M-CSF stimulation also resulted in Fms association with two of these tyrosine-phosphorylated proteins, one of which was identified as the 55-kDa Shc, which is shown in other systems to be involved in growth stimulation, and the other was a previously uncharacterized 150-kDa protein (p150). Fms also formed complexes with Grb2 and Sos1, and neither contained phosphotyrosine. Whereas both Grb2 and Sos1 complexed with Fms only after M-CSF stimulation, the amount of Sos1 complexed with Grb2 was not M-CSF dependent. Shc coimmunoprecipitated Sos1, Grb2, and tyrosine-phosphorylated p150, while Grb2 immunoprecipitates contained mainly phosphorylated p150, Fms, Shc, and Sos1. Shc interacted with tyrosine-phosphorylated p150 via its SH2 domain, and the Grb2 SH2 domain likewise bound tyrosine-phosphorylated Fms and p150. Analysis of Fms mutated at each of four tyrosine autophosphorylation sites indicated that none of these sites dramatically affected p150 phosphorylation or its association with Shc and Grb2. M-CSF stimulation of fibroblast cell lines expressing exogenous murine Fms did not phosphorylate p150, and this protein was not detected either in cell lysates or in Grb2 or Shc immunoprecipitates. The p150 protein is not related to known signal transduction molecules and may be myeloid cell specific. These results suggest that M-CSF stimulation of myeloid cells could activate Ras through the nucleotide exchange factor Sos1 by Grb2 binding to either Fms, Shc, or p150 and that Fms signal transduction in myeloid cells differs from that in fibroblasts.     

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.     

Sequential activation of phoshatidylinositol 3-kinase and phospholipase C-gamma2 by the M-CSF receptor is necessary for differentiation signaling.

Binding of macrophage colony stimulating factor (M-CSF) to its receptor (Fms) induces dimerization and activation of the tyrosine kinase domain of the receptor, resulting in autophosphorylation of cytoplasmic tyrosine residues used as docking sites for SH2-containing signaling proteins that relay growth and development signals. To determine whether a distinct signaling pathway is responsible for the Fms differentiation signal versus the growth signal, we sought new molecules involved in Fms signaling by performing a two-hybrid screen in yeast using the autophosphorylated cytoplasmic domain of the wild-type Fms receptor as bait. Clones containing SH2 domains of phospholipase C-gamma2 (PLC-gamma2) were frequently isolated and shown to interact with phosphorylated Tyr721 of the Fms receptor, which is also the binding site of the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase). At variance with previous reports, M-CSF induced rapid and transient tyrosine phosphorylation of PLC-gamma2 in myeloid FDC-P1 cells and this activation required the activity of the PI3-kinase pathway. The Fms Y721F mutation strongly decreased this activation. Moreover, the Fms Y807F mutation decreased both binding and phosphorylation of PLC-gamma2 but not that of p85. Since the Fms Y807F mutation abrogates the differentiation signal when expressed in FDC-P1 cells and since this phenotype could be reproduced by a specific inhibitor of PLC-gamma, we propose that a balance between the activities of PLC-gamma2 and PI3-kinase in response to M-CSF is required for cell differentiation.     

Mona, a novel hematopoietic-specific adaptor interacting with the macrophage colony-stimulating factor receptor, is implicated in monocyte/macrophage development.

The production, survival and function of monocytes and macrophages are regulated by the macrophage colony-stimulating factor (M-CSF or CSF-1) through its tyrosine kinase receptor Fms. Binding of M-CSF results in Fms autophosphorylation on specific tyrosines that act as docking sites for intracellular signaling molecules containing SH2 domains. Using a yeast two-hybrid screen, we cloned a novel adaptor protein which we called 'Mona' for monocytic adaptor. Mona contains one SH2 domain and two SH3 domains related to the Grb2 adaptor. Accordingly, Mona interacts with activated Fms on phosphorylated Tyr697, which is also the Grb2-binding site. Furthermore, Mona contains a unique proline-rich region located between the SH2 domain and the C-terminal SH3 domain, and is apparently devoid of any catalytic domain. Mona expression is restricted to two hematopoietic tissues: the spleen and the peripheral blood mononuclear cells, and is induced rapidly during monocytic differentiation of the myeloid NFS-60 cell line in response to M-CSF. Strikingly, overexpression of Mona in bone marrow cells results in strong reduction of M-CSF-dependent macrophage production in vitro. Taken together, our results suggest an important role for Mona in the regulation of monocyte/macrophage development as controlled by M-CSF.     

Association of Cbl with Fms and p85 in response to macrophage colony-stimulating factor

Tyrosine phosphorylation of Cbl and its association with signal-transducing molecules in response to macrophage colony-stimulating factor (M-CSF) were analyzed by using cell lines which express the wild-type and a mutant M-CSF receptor, Fms. We found that in a clone, F723 TF-1 cells expressing mutant Fms in which tyrosine 723 had been substituted with phenylalanine, the M-CSF stimulation-dependent association between Cbl and Fms was markedly impaired. However, phosphorylation of Cbl and its association with the p85 subunit of phosphatidylinositol 3-kinase were induced in these mutant cells as seen in the wild-type fms transfectant. These results suggest that phosphorylation of tyrosine 723 is particularly important for the recruitment of Cbl to the M-CSF receptor, but is not required for the phosphorylation and binding of Cbl to signal-transducing molecules such as p85.     

Induced expression and association of the Mona/Gads adapter and Gab3 scaffolding protein during monocyte/macrophage differentiation

Mona/Gads is a Grb2-related, Src homology 3 (SH3) and SH2 domain-containing adapter protein whose expression is restricted to cells of hematopoietic lineage (i.e., monocytes and T lymphocytes). During monocyte/macrophage differentiation, Mona is induced and interacts with the macrophage colony-stimulating factor receptor, M-CSFR (also called Fms), suggesting that Mona could be involved in developmental signaling downstream of the M-CSFR by recruiting additional signaling proteins to the activated receptor. Our present results identify Mona as a specific partner protein for the DOS/Gab family member Gab3 in monocytic/macrophage development. Mona does not interact with Gab2; however, Gab3 also forms a complex with the Mona-related adapter Grb2. Glutathione S-transferase pull-down experiments demonstrate that the Mona and Gab3 interaction utilizes the carboxy-terminal SH3 domain of Mona and the atypical proline-rich domain of Gab3. Mona is known to interact with the phosphorylated Y697 site of the M-CSFR. The M-CSFR mutation Y697F exhibited qualitative and quantitative abnormalities in receptor and Gab3 tyrosine phosphorylation, and Mona induction was greatly reduced. The Y807F M-CSFR mutation is defective in differentiation signaling, but not growth signaling, and also fails to induce Mona protein expression. During M-CSF-stimulated macrophage differentiation of mouse bone marrow cells, Mona and Gab3 expression is coinduced, these proteins interact, and Mona engages in multimolecular complexes. These data suggest that association of Mona and Gab3 plays a specific role in mediating the M-CSFR differentiation signal.     

Growth and differentiation signals regulated by the M-CSF receptor

The normal proto-oncogene c-fms encodes the macrophage growth factor (M-CSF) receptor involved in growth, survival, and differentiation along the monocyte-macrophage lineage of hematopoietic cell development. A major portion of our research concerns unraveling the temporal, molecular, and structural features that determine and regulate these events. Previous results indicated that c-fms can transmit a growth signal as well as a signal for differentiation in the appropriate cells. To investigate the role of the Fms tyrosine autophosphorylation sites in proliferation vs. differentiation signaling, four of these sites were disrupted and the mutant receptors expressed in a clone derived from the myeloid FDC-P1 cell line. These analyses revealed that: (1) none of the four autophosphorylation sites studied (Y697, Y706, Y721, and Y807) are essential for M-CSF-dependent proliferation of the FDC-P1 clone; (2) Y697, Y706, and Y721 sites, located in the kinase insert region of Fms, are not necessary for differentiation but their presence augments this process; and (3) the Y807 site is essential for the Fms differentiation signal: its mutation totally abrogates the differentiation of the FDC-P1 clone and conversely increases the rate of M-CSF-dependent proliferation. This suggests that the Y807 site may control a switch between growth and differentiation. The assignment of Y807 as a critical site for the reciprocal regulation of growth and differentiation may provide a paradigm for Fms involvement in leukemogenesis, and we are currently investigating the downstream signals transmitted by the tyrosine-phosphorylated 807 site. In Fms-expressing FDC-P1 cells, M-CSF stimulation results in the rapid (30 sec) tyrosine phosphorylation of Fms on the five cytoplasmic tyrosine autophosphorylation sites, and subsequent tyrosine phosphorylation of several host cell proteins occurs within 1–2 min. Complexes are formed between Fms and other signal transduction proteins such as Grb2, Shc, Sos1, and p85. In addition, a new signal transduction protein of 150 kDa is detectable in the FDC-P1 cells. The p150 is phosphorylated on tyrosine, and forms a complex with Shc and Grb2. The interaction with Shc occurs via a protein tyrosine binding (PTB) domain at the N-terminus of Shc. The p150 is not detectable in Fms signaling within fibroblasts, yet the PDGF receptor induces the tyrosine phosphorylation of a similarly sized protein. In hematopoietic cells, this protein is involved in signaling by receptors for GM-CSF, IL-3, KL, MPO, and EPO. We have now cloned a cDNA for this protein and found at least one related family member. The related family member is a Fanconia Anemia gene product, and this suggests potential ways the p150 protein may function in Fms signaling. Mol Reprod Dev 46:96–103, 1997. © 1997 Wiley-Liss, Inc.     

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.     

Phosphotyrosines 627 and 659 of Gab1 constitute a bisphosphoryl tyrosine-based activation motif (BTAM) conferring binding and activation of SHP2

A major Grb2-associated binder-1 (Gab1) binding partner in epidermal growth factor (EGF)-stimulated cells is protein-tyrosine phosphatase (PTPase) SHP2, which contains tandem SH2 domains. The SHP2 PTPase activity is required for activation of the extracellular signal-regulated kinase (ERK) subfamily of mitogen-activated protein (MAP) kinase by EGF. To investigate the mechanism by which Gab1 and SHP2 mediate ERK activation, we characterized the Gab1-SHP2 interaction. We found that both Tyr-627 and Tyr-659 of Gab1 were required for SHP2 binding to Gab1 and for ERK2 activation by EGF. Far Western blot analysis suggested that the tandem SH2 domains of SHP2 bind to Gab1 in a specific orientation, in which the N-SH2 domain binds to phosphotyrosine (Tyr(P))-627 and the C-SH2 domain binds to Tyr(P)-659. When assayed with peptide substrates, SHP2 PTPase was activated by a bisphosphopeptide containing both Tyr(P)-627 and Tyr(P)-659, but not by monophosphopeptides containing Tyr(P)-627 or Tyr(P)-659 or a mixture of these monophosphopeptides. These results suggest that Tyr(P)-627 and Tyr(P)-659 of Gab1 constitute a bisphosphoryl tyrosine-based activation motif (BTAM) that binds and activates SHP2. Remarkably, while a constitutively active SHP2 (SHP2DeltaN) could not rescue the defect of a SHP2-binding defective Gab1 (Gab1FF) in ERK2 activation, expression of a Gab1FF-SHP2DeltaN chimera resulted in constitutive activation of ERK2 in transfected cells. Thus, physical association of activated SHP2 with Gab1 is necessary and sufficient to mediate the ERK mitogen-activated protein kinase activation. Phosphopeptides derived from Gab1 were dephosphorylated by active SHP2 in vitro. Consistently, substrate-trapping experiments with a SHP2 catalytic inactive mutant suggested that Gab1 was a SHP2 PTPase substrate in the cells. Therefore, Gab1 not only is a SHP2 activator but also is a target of its PTPase.     

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.     

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.     

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.     

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.     

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.