Phosphorylation-dependent binding of 14-3-3 terminates signalling by the Gab2 docking protein (original) (raw)
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Journal of Biological Chemistry, 2000
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. Receptor tyrosine kinases regulate diverse biological processes ranging from cell proliferation and survival to cell motility, metabolism, and differentiation. The initiation of receptor signaling involves ligand-induced activation of the catalytic domain of the enzyme and the phosphorylation of specific tyrosine residues on the receptor. These provide binding sites for proteins containing Src homology 2 (SH2) 1 and phosphoty-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.This work was supported in part by operating grants from the National Cancer Institute of Canada (to M. P.), with money from the Canadian Cancer Society. § Recipient of a studentship from the Natural Sciences and Engineering Research Council.
Unique Phosphorylation Mechanism of Gab1 Using PI 3-Kinase as an Adaptor Protein
Biochemical and Biophysical Research Communications, 2001
Grb2-associated binder-1 (Gab1) undergoes tyrosine phosphorylation in response to stimulation by growth factors and hormones including insulin, epidermal growth factor (EGF), nerve growth factor (NGF), and hepatocyte growth factor (HGF). However, the HGF receptor is the only one known to associate directly with Gab1. Herein, we explore the mechanism of Gab1 phosphorylation by other receptor protein-tyrosine kinases unable to bind to Gab1 directly. The Src homology 2 (SH2) domain of the phosphatidylinositol 3-kinase (PI3K) regulatory subunit binds Gab1 in a phosphorylation-independent manner. Moreover, the regulatory subunit of PI3K can mediate the association of Gab1 and receptor protein-tyrosine kinases including the insulin, EGF, and NGF receptors, all of which phosphorylate Gab1. Thus, it appears that the PI3K regulatory subunit acts as an adaptor protein via a phosphotyrosyl-independent SH2 interaction, allowing Gab1 to serve as a substrate for several tyrosine kinases. This is a new role for the PI3K regulatory subunit.
In primary rat hepatocyte cultures, activation of phosphatidylinositol 3-kinase is both necessary and sufficient to account for epidermal growth factor (EGF)-induced DNA synthesis. In these cells, three major p85-containing complexes were formed after EGF treatment: ErbB3-p85, Shc-p85, and a multimeric Gab2-Grb2-SHP2-p85, which accounted for more than 80% of total EGF-induced PI3K activity (Kong, M., C. Mounier, J. Wu, and B. I. Posner, J Biol Chem, 2000, 275:36035-36042). More recently, we found that EGF-dependent tyrosine phosphorylation of endogenous Gab2 is essential for EGF-induced DNA synthesis in rat hepatocytes. Here we show that, after EGF treatment, ErbB3-p85 and Shc-p85 complexes were localized to plasma membrane and endosomes, whereas the multimeric Gab2-Grb2-SHP2-p85 complex was formed rapidly (peak at 30 sec) and exclusively in cytosol. Western blotting of subcellular fractions from intact liver and immunofluorescence analyses in cultured hepatocytes demonstrated that EGF did not promote the association of cytosolic Gab2 with cell membranes. These observations prompted us to evaluate the role of the PH domain of Gab2 in regulating its function. Overexpression of the PH domain of Gab2 did not affect EGF-induced Gab2 phosphorylation, PI3K activation, and DNA synthesis. Overexpressed Gab2 lacking the PH domain (⌬PHGab2) was comparable to wild-type Gab2 in respect to EGF-induced tyrosine phosphorylation, recruitment of p85, and DNA synthesis. In summary, after EGF stimulation, ErbB3, Shc, and Gab2 are differentially compartmentalized in rat liver, where they associate with and activate PI3K. Our data demonstrate that Gab2 mediates EGF-induced PI3K activation and DNA synthesis in a PH domain-independent manner. (Molecular Endocrinology 17: 935-944, 2003)
Cancers
GRB2-associated binder 1 (GAB1) is the inaugural member of the GAB/DOS family of pleckstrin homology (PH) domain-containing proteins. Upon receiving various stimuli, GAB1 transitions from the cytoplasm to the membrane where it is phosphorylated by a range of kinases. This event recruits SH2 domain-containing proteins like SHP2, PI3K’s p85 subunit, CRK, and others, thereby activating distinct signaling pathways, including MAPK, PI3K/AKT, and JNK. GAB1-deficient embryos succumb in utero, presenting with developmental abnormalities in the heart, placenta, liver, skin, limb, and diaphragm myocytes. Oncogenic mutations have been identified in the context of cancer. GAB1 expression levels are disrupted in various tumors, and elevated levels in patients often portend a worse prognosis in multiple cancer types. This review focuses on GAB1’s influence on cellular transformation particularly in proliferation, evasion of apoptosis, metastasis, and angiogenesis—each of these processes being a c...
Critical Role for Gab2 In Transformation by BCR/ABL
Cancer Cell, 2002
The BCR/ABL oncogene causes chronic myelogenous leukemia (CML) in humans and a CML-like disease, as well as lymphoid leukemia, in mice. p210 BCR/ABL is an activated tyrosine kinase that phosphorylates itself and several cellular signaling proteins. The autophosphorylation site tyrosine 177 binds the adaptor Grb2 and helps determine the lineage and severity of BCR/ABL disease: Tyr177 mutation (BCR/ABL-Y177F) dramatically impairs myeloid leukemogenesis, while diminishing lymphoid leukemogenesis. The critical signal(s) from Tyr177 has remained unclear. We report that Tyr177 recruits the scaffolding adaptor Gab2 via a Grb2/Gab2 complex. Compared to BCR/ABL-expressing Ba/F3 cells, BCR/ABL-Y177F cells exhibit markedly reduced Gab2 tyrosine phosphorylation and association of phosphatidylinositol-3 kinase (PI3K) and Shp2 with Gab2 and BCR/ABL, and decreased PI3K/Akt and Ras/Erk activation, cell proliferation, and spontaneous migration. Remarkably, bone marrow myeloid progenitors from Gab2 (Ϫ/Ϫ) mice are resistant to transformation by BCR/ABL, whereas lymphoid transformation is diminished as a consequence of markedly increased apoptosis. BCR/ABLevoked PI3K/Akt and Ras/Erk activation also are impaired in Gab2 (Ϫ/Ϫ) primary myeloid and lymphoid cells. Our results identify Gab2 and its associated proteins as key determinants of the lineage and severity of BCR/ABL transformation.
A novel positive feedback loop mediated by the docking protein Gab1 and …
… and Cellular Biology
The Gab1 protein is tyrosine phosphorylated in response to various growth factors and serves as a docking protein that recruits a number of downstream signaling proteins, including phosphatidylinositol 3-kinase (PI-3 kinase). To determine the role of Gab1 in signaling via the epidermal ...
Journal of Biological Chemistry, 2006
Phosphoinositide 3-kinase (PI3K) mediates essential functions of vascular endothelial growth factor (VEGF), including the stimulation of endothelial cell proliferation and migration. Nevertheless, the mechanisms coupling the receptor VEGFR-2 to PI3K remain obscure. We observed that the Grb2-bound adapter Gab1 is tyrosine-phosphorylated and relocated to membrane fractions upon VEGF stimulation of endothelial cells. We could detect the PI3K regulatory subunit p85 in immunoprecipitates of endogenous Gab1, and vice versa, and measure a Gab1-associated lipid kinase activity upon VEGF stimulation. Furthermore, transfection of the Gab1-YF3 mutant lacking all p85-binding sites strongly repressed PI3K activation measured in vitro. Moreover, Gab1-YF3 severely decreased the cellular amount of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) generated in response to VEGF. Furthermore, adenoviral expression of Gab1-YF3 suppressed both Akt phosphorylation and recovery of wounded human umbilical vein endothelial cell monolayers, a VEGF-dependent process involving cell migration and proliferation under PI3K control. Transfection of other Gab1 mutants, lacking Grb2-binding sites or the pleckstrin homology (PH) domain, also prevented Akt activation, further demonstrating Gab1 involvement in PI3K activation. These mutants were also used to show that interactions with both Grb2 and PtdIns(3,4,5)P 3 mediate Gab1 recruitment by VEGFR-2. Importantly, Gab1 mobilization was impaired by (i) PI3K inhibitors, (ii) deletion of Gab1 PH domain, (iii) PTEN (phosphatase and tensin homolog deleted on chromosome 10) overexpression to repress PtdIns(3,4,5)P 3 production, and (iv) overexpression of a competitor PH domain for PtdIns(3,4,5)P 3 binding, which altogether demonstrated that PI3K is also an upstream regulator of Gab1. Gab1 thus appears as a primary actor in coupling VEGFR-2 to PI3K/Akt, recruited through an amplification loop involving PtdIns(3,4,5)P 3 and its PH domain.
Gab2 is Essential for Transformation by FLT3-ITD in Acute Myeloid Leukemia
HemaSphere, 2019
About 30% of acute myeloid leukemias (AML) are driven by constitutive activation of the FMS-like tyrosine kinase 3 (FLT3), either due to an internal tandem duplication (ITD) in the juxtamembrane domain or mutations in the tyrosine kinase domain (TKD). FLT3-ITD is associated with poor outcomes, whereas FLT3-TKD mutations usually confer a less aggressive course of the disease. 1 Targeting FLT3 has been the objective of multiple clinical trials resulting in the recent approval of midostaurin, the first multikinase inhibitor for the treatment of FLT3-mutant AML, by the Food and Drug Administration. 2 However, in single agent studies with FLT3 inhibitors clinical responses are characterized by an only transient reduction in peripheral blood and/or bone marrow blasts. 3 Thus, despite being an important key player in AML, FLT3 might cooperate with other signaling molecules in promoting leukemogenesis. The docking protein GRB2-associated binder 2 (Gab2) serves as an amplifier in the signaling network of growth factor and cytokine receptors. 4,5 Gab2 works as an assembly platform by binding to FLT3 via the adaptor Grb2, 5,6 thereby amplifying signaling into SHP2/Ras/ ERK, PI3K/AKT, and STAT5 pathways leading to survival, proliferation, and migration. 5 However, it is not known whether Gab2 is similarly critical in FLT3-ITD-driven transformation as described for other oncogenic tyrosine kinases like Bcr-Abl. 7,8 Based on previous studies, we started to analyze the interplay between FLT3 and Gab2 by treating the human FLT3-ITDpositive AML cell line MOLM-13 with the FLT3-selective inhibitor quizartinib (QZ) and analyzed Gab2 phosphorylation on Western Blot (Figs. 1A and S1, Supplemental Digital Content, http://links.lww.com/HS/A30), as well as the binding of Gab2 to known interactors like Grb2, SHP2, and p85 (PI3K) in a Gab2 immunoprecipitation (Fig. 1B and C). FLT3 inhibition reduces Gab2 phosphorylation on various sites, for example, the PI3K binding site Y452 and the SHP2 binding site Y643 (Figs. 1A and S1, Supplemental Digital Content, http://links.lww.com/HS/ A30). Consequently and in line with these results, we observed less binding of SHP2 and of the p85 subunit of PI3K to Gab2 upon treatment with the FLT3 inhibitors QZ and sorafenib (SF) (Fig. 1B and C). Next, we established a Gab2 knockdown in MOLM-13 cells (Figs. 1D, S2, and S3A and B, Supplemental Digital Content, http://links.lww.com/HS/A30) using a vector allowing the doxycycline (dox) inducible expression of an shRNA together with turbo RFP (tRFP) from the same transcript. The knockdown of Gab2 lowers the activity of the Raf/MEK/ ERK and PI3K/AKT/mTOR signaling pathways as shown by less phosphorylation of MEK, ERK, and their downstream target c-Fos, as well as less phosphorylation of the mTOR substrate S6K
Engagement of Gab1 and Gab2 in Erythropoietin Signaling
Journal of Biological Chemistry, 1999
Several signaling cascades are activated during engagement of the erythropoietin receptor to mediate the biological effects of erythropoietin. The members of the insulin receptor substrate (IRS) family of proteins play a central role in signaling for various growth factor receptors and cytokines by acting as docking proteins for the SH2 domains of signaling elements, linking cytokine receptors to diverse downstream pathways. In the present study we provide evidence that the recently cloned IRSrelated proteins, Gab1 and Gab2, of the Gab family of proteins, are rapidly phosphorylated on tyrosine during erythropoietin treatment of erythropoietin-responsive cells and provide docking sites for the engagement of the SHP2 phosphatase and the p85 subunit of the phosphatidylinositol 3-kinase. Furthermore, our data show that Gab1 is the primary IRS-related protein activated by erythropoietin in primary erythroid progenitor cells. In studies to identify the erythropoietin receptor domains required for activation of Gab proteins, we found that tyrosines 425 and 367 in the cytoplasmic domain of the erythropoietin receptor are required for the phosphorylation of Gab2. Taken together, our data demonstrate that Gab proteins are engaged in erythropoietin signaling to mediate downstream activation of the SHP2 and phosphatidylinositol 3-kinase pathways and possibly participate in the generation of the erythropoietininduced mitogenic responses.
Grb2 associated binder 2 couples B-cell receptor to cell survival
Cellular Signalling, 2009
B-cell fate during maturation and the germinal center reaction is regulated through the strength and the duration of the B-cell receptor signal. Signaling pathways discriminating between apoptosis and survival in B cells are keys in understanding adaptive immunity. Gab2 is a member of the Gab/Dos adaptor protein family. It has been shown in several model systems that Gab/Dos family members may regulate both the anti-apoptotic PI3-K/Akt and the mitogenic Ras/MAPK pathways, still their role in B-cells have not been investigated in detail. Here we studied the role of Gab2 in B-cell receptor mediated signaling. We have shown that BCR crosslinking induces the marked phosphorylation of Gab2 through both Lyn and Syk kinases. Subsequently Gab2 recruits p85 regulatory subunit of PI3-K, and SHP-2. Our results revealed that Ig-alpha/Igbeta, signal transducing unit of the B-cell receptor, may function as scaffold recruiting Gab2 to the signalosome. Overexpression of Gab2 in A20 cells demonstrated that Gab2 is a regulator of the PI3-K/Akt but not that of the Ras/MAPK pathway in B cells. Accordingly to the elevated Akt phosphorylation, overexpression of wild-type Gab2 in A20 cells suppressed Fas-mediated apoptosis, and enhanced BCR-mediated rescue from Fas-induced cell death. Although PH-domain has only a stabilizing effect on membrane recruitment of Gab2, it is indispensable in mediating its anti-apoptotic effect.