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Stephan M. Feller - One of the best experts on this subject based on the ideXlab platform.

  • The multi-site Docking Protein Gab1 is constitutively phosphorylated independent from its recruitment to the plasma membrane in Jak2-V617F-positive cells and mediates proliferation of human erythroleukaemia cells
    Cellular signalling, 2017
    Co-Authors: Hannes Bongartz, Wiebke Hessenkemper, Iris Behrmann, Claude Haan, Christian D. Muller, Melissa Fensky, Johannes Fritsch, Katharina Mandel, Thomas Fischer, Stephan M. Feller
    Abstract:

    The constitutively active Janus kinase 2 mutant Jak2-V617F is responsible for cytokine-independent growth of hematopoietic cells and the development of myeloproliferative neoplasms, such as polycythaemia vera and essential thrombocythaemia. Cells expressing Jak2-V617F exhibit constitutive STAT, MAPK, and PI3K signalling, and constitutive association of the multi-site Docking Protein Gab1 to PIP3 at the plasma membrane. Here, we demonstrate the crucial role of Gab1 for the proliferation of Jak2-V617F-positive human erythroleukaemia (HEL) cells. In Jak2-V617F-expressing cells Gab1 is constitutively phosphorylated by Erk1/2 on serine residue 552, which regulates binding to PIP3. Additionally, Gab1 is constitutively phosphorylated on tyrosine residue 627. Tyrosine 627 is a SHP2 binding site and required for Gab1-dependent Erk1/2 activation. As previously shown, Jak2-V617F-dependent Erk1/2 and PI3K activation act synergistically on the proliferation of Jak2-V617F-positive cells. Here, we examined whether constitutive membrane association of Gab1 explains cytokine-independent Gab1 phosphorylation in Jak2-V617F-expressing cells. Although we could demonstrate Jak2-V617F-dependent constitutive serine 552 and tyrosine 627 phosphorylation of Gab1, interestingly, both phosphorylations do not require binding of Gab1 to PIP3 at the plasma membrane. Instead, we observed a constitutive interaction of Gab1 with the erythropoietin receptor in Jak2-V617F-expressing cells, which depends on Janus kinase activity. Thus, constitutive Gab1-dependent signalling in Jak2-V617F-expressing cells does not occur due to the constitutive association of Gab1 with PIP3 at the plasma membrane.

  • ID: 79: Constitutive membrane binding of the multi-site Docking Protein Gab1 in JAK2-V617F expressing cells
    Cytokine, 2015
    Co-Authors: Hannes Bongartz, Wiebke Hessenkemper, Alexandra Wolf, René Eulenfeld, Philip C. Simister, Iris Behrmann, Jan Tavernier, Stephan M. Feller, Claude Haan, Fred Schaper
    Abstract:

    The activating JAK2-V617F mutation is found in many patients suffering from Myeloproliferative neoplasms such as Polycythemia Vera and Essential Thrombocythemia. JAK2-V617F mutation confers cytokine hypersensitivity, constitutive activation of the JAK-STAT pathway, and cytokine-independent growth. We demonstrate uncontrolled membrane binding of the multi-site Docking Protein Gab1 in JAK2-V617F expressing cells. We elaborated the molecular mechanism and regulation of Gab1-membrane recruitment. In line with the regulatory function of Gab1 on STAT-independent signalling we show that PI3K signalling regulates MAPK activation in JAK2-V617F-positive cells. This cross-regulation of the MAPK pathway by PI3K affects JAK2-V617F-specific target gene induction, erythroid colony formation, and regulates proliferation of JAK2-V617F-positive patient cells in a synergistically manner. In summary, our study demands to consider cross-talk mechanisms as potential new targets. This may increase the specificity for intervention approaches compared to the interference in very upstream signalling elements such as receptor kinases or receptor-associated kinases.

  • signaling of hepatocyte growth factor scatter factor hgf to the small gtpase rap1 via the large Docking Protein gab1 and the adapter Protein crkl
    Journal of Biological Chemistry, 2000
    Co-Authors: Dima Sakkab, Ute Schaeper, Walter Birchmeier, Marc Lewitzky, Guido Posern, Martin M Sachs, Stephan M. Feller
    Abstract:

    Abstract Hepatocyte growth factor (HGF; scatter factor) is a multipotent Protein with mitogenic, motogenic, and developmental functions. Upon activation, the HGF-receptor c-Met binds and phosphorylates the multisite Docking Protein Gab1. Besides binding motifs for phosphatidylinositol 3-kinase and Grb2, Gab 1 contains multiple Tyr-X-X-Pro (YXXP) motifs which, when phosphorylated, are potential binding sites for the adapter Proteins c-Crk and Crk-like (CRKL). Stimulation of human embryonic kidney cells (HEK293) with HGF leads to Gab1 association with CRKL. The Gab1-CRKL interaction requires both, the SH2 domain of CRKL and the region containing the YXXP motifs in Gab1. CRKL binds via its first SH3 domain to several downstream signal transducers, including C3G an activator of the small GTPase Rap1. Indeed, Rap1 was rapidly activated after HGF stimulation of HEK293 cells. Rap1 activation through HGF was suppressed through transfection of a truncated C3G Protein which only contains the SH3-binding motifs of C3G. Transfection of nonmutated Gab1 led to a strong increase of Rap1·GTP in the absence of HGF. In contrast, transfection of the GabΔYXXP mutant abolished the elevation of Rap1·GTP by HGF. A replating assay indicated that HGF decreases the adhesion of HEK293 cells. The results presented here delineate a novel signaling pathway from HGF to the GTPase Rap1 which depends on the interaction of the adapter Protein CRKL with the exchange factor C3G and could be linked to cell migration.

  • Signaling of hepatocyte growth factor/scatter factor (HGF) to the small GTPase Rap1 via the large Docking Protein Gab1 and the adapter Protein CRKL.
    The Journal of biological chemistry, 2000
    Co-Authors: Dima Sakkab, Ute Schaeper, Walter Birchmeier, Marc Lewitzky, Guido Posern, Martin M Sachs, Stephan M. Feller
    Abstract:

    Abstract Hepatocyte growth factor (HGF; scatter factor) is a multipotent Protein with mitogenic, motogenic, and developmental functions. Upon activation, the HGF-receptor c-Met binds and phosphorylates the multisite Docking Protein Gab1. Besides binding motifs for phosphatidylinositol 3-kinase and Grb2, Gab 1 contains multiple Tyr-X-X-Pro (YXXP) motifs which, when phosphorylated, are potential binding sites for the adapter Proteins c-Crk and Crk-like (CRKL). Stimulation of human embryonic kidney cells (HEK293) with HGF leads to Gab1 association with CRKL. The Gab1-CRKL interaction requires both, the SH2 domain of CRKL and the region containing the YXXP motifs in Gab1. CRKL binds via its first SH3 domain to several downstream signal transducers, including C3G an activator of the small GTPase Rap1. Indeed, Rap1 was rapidly activated after HGF stimulation of HEK293 cells. Rap1 activation through HGF was suppressed through transfection of a truncated C3G Protein which only contains the SH3-binding motifs of C3G. Transfection of nonmutated Gab1 led to a strong increase of Rap1·GTP in the absence of HGF. In contrast, transfection of the GabΔYXXP mutant abolished the elevation of Rap1·GTP by HGF. A replating assay indicated that HGF decreases the adhesion of HEK293 cells. The results presented here delineate a novel signaling pathway from HGF to the GTPase Rap1 which depends on the interaction of the adapter Protein CRKL with the exchange factor C3G and could be linked to cell migration.

Tony Pawson - One of the best experts on this subject based on the ideXlab platform.

  • The ShcA Phosphotyrosine Docking Protein Uses Distinct Mechanisms to Regulate Myocyte and Global Heart Function
    Circulation research, 2010
    Co-Authors: Rachel D. Vanderlaan, W. Rod Hardy, M. Golam Kabir, Adrian Pasculescu, Nina Jones, Pieter P. Detombe, Peter H. Backx, Tony Pawson
    Abstract:

    Rationale:Although tyrosine kinases (TKs) are important for cardiac function, their relevant downstream targets in the adult heart are unknown. The ShcA Docking Protein binds specific phosphotyrosine (pTyr) sites on activated TKs through its N-terminal pTyr-binding (PTB) and C-terminal SH2 domains and stimulates downstream pathways through motifs such as pTyr sites in its central CH1 region. Therefore, ShcA could be a potential hub for downstream TK signaling in the myocardium. Objective:To define the role of ShcA, a TK scaffold, in the adult heart using a myocardial-specific knockout of murine ShcA (ShcA CKO) and domain knock-in models. Methods and Results:ShcA CKO mice developed a dilated cardiomyopathy phenotype involving impaired systolic function with enhanced cardiomyocyte contractility. This uncoupling of global heart and intrinsic myocyte functions was associated with altered collagen and extracellular matrix compliance properties, suggesting disruption of mechanical coupling. In vivo dissection o...

  • structural basis for specific binding of the gads sh3 domain to an rxxk motif containing slp 76 peptide a novel mode of peptide recognition
    Molecular Cell, 2003
    Co-Authors: Donna M Berry, Piers Nash, Jane C Mcglade, Tony Pawson, Shawn S C Li
    Abstract:

    Abstract The SH3 domain, which normally recognizes proline-rich sequences, has the potential to bind motifs with an RxxK consensus. To explore this novel specificity, we have determined the solution structure of the Gads T cell adaptor C-terminal SH3 domain in complex with an RSTK-containing peptide, representing its physiological binding site on the SLP-76 Docking Protein. The SLP-76 peptide engages four distinct binding pockets on the surface of the Gads SH3 domain and upon binding adopts a unique structure characterized by a right-handed 3 10 helix at the RSTK locus, in contrast to the left-handed polyproline type II helix formed by canonical proline-rich SH3 ligands. The structure, and supporting mutagenesis and peptide binding data, reveal a novel mode of ligand recognition by SH3 domains.

  • The ShcA phosphotyrosine Docking Protein sensitizes cardiovascular signaling in the mouse embryo
    Genes & development, 2000
    Co-Authors: Ka-man Venus Lai, Tony Pawson
    Abstract:

    The ShcA gene products have served as a model for the analysis of phosphotyrosine-recognition domains, and for the functions of Docking Proteins during tyrosine kinase signaling. Here we show that ShcA is primarily expressed in the cardiovascular system during early mouse embryogenesis and regulates both heart development and establishment of mature blood vessels. Targeted mutation suggests that the ShcA adaptor is a pivotal target of tyrosine kinases that selectively potentiates activation of the MAP kinase pathway in the remodeling vasculature. Biochemical analysis of mutant cells shows that ShcA sensitizes cells to growth factor-induced MAP kinase activation, and also organizes cytoskeletal rearrangement in response to the extracellular matrix. ShcA may therefore orchestrate complex interactions within the vascular compartment by rendering cells permissive to respond to soluble and adhesive external cues.

Joseph Schlessinger - One of the best experts on this subject based on the ideXlab platform.

  • The Docking Protein FRS2α is a critical regulator of VEGF receptors signaling
    Proceedings of the National Academy of Sciences of the United States of America, 2014
    Co-Authors: Pei-yu Chen, Lingfeng Qin, Zhen W. Zhuang, George Tellides, Irit Lax, Joseph Schlessinger, Michael Simons
    Abstract:

    Vascular endothelial growth factors (VEGFs) signal via their cognate receptor tyrosine kinases designated VEGFR1-3. We report that the Docking Protein fibroblast growth factor receptor substrate 2 (FRS2α) plays a critical role in cell signaling via these receptors. In vitro FRS2α regulates VEGF-A and VEGF-C–dependent activation of extracellular signal-regulated receptor kinase signaling and blood and lymphatic endothelial cells migration and proliferation. In vivo endothelial-specific deletion of FRS2α results in the profound impairment of postnatal vascular development and adult angiogenesis, lymphangiogenesis, and arteriogenesis. We conclude that FRS2α is a previously unidentified component of VEGF receptors signaling.

  • The Docking Protein Gab1 is the primary mediator of EGF-stimulated activation of the PI-3K/Akt cell survival pathway
    BMC biology, 2004
    Co-Authors: Dawn Mattoon, Irit Lax, Betty Lamothe, Joseph Schlessinger
    Abstract:

    Gab1 is a Docking Protein that recruits phosphatidylinositol-3 kinase (PI-3 kinase) and other effector Proteins in response to the activation of many receptor tyrosine kinases (RTKs). As the autophosphorylation sites on EGF-receptor (EGFR) do not include canonical PI-3 kinase binding sites, it is thought that EGF stimulation of PI-3 kinase and its downstream effector Akt is mediated by an indirect mechanism. We used fibroblasts isolated from Gab1-/- mouse embryos to explore the mechanism of EGF stimulation of the PI-3 kinase/Akt anti-apoptotic cell signaling pathway. We demonstrate that Gab1 is essential for EGF stimulation of PI-3 kinase and Akt in these cells and that these responses are mediated by complex formation between p85, the regulatory subunit of PI-3 kinase, and three canonical tyrosine phosphorylation sites on Gab1. Furthermore, complex formation between Gab1 and the Protein tyrosine phosphatase Shp2 negatively regulates Gab1 mediated PI-3 kinase and Akt activation following EGF-receptor stimulation. We also demonstrate that tyrosine phosphorylation of ErbB3 may lead to recruitment and activation of PI-3 kinase and Akt in Gab1-/- MEFs. The primary mechanism of EGF-induced stimulation of the PI-3 kinase/Akt anti-apoptotic pathway occurs via the Docking Protein Gab1. However, in cells expressing ErbB3, EGF and neuroregulin can stimulate PI-3 kinase and Akt activation in a Gab1-dependent or Gab1-independent manner.

  • The Docking Protein Gab1 is an essential component of an indirect mechanism for fibroblast growth factor stimulation of the phosphatidylinositol 3-kinase/Akt antiapoptotic pathway.
    Molecular and cellular biology, 2004
    Co-Authors: Betty Lamothe, Irit Lax, Masashi Yamada, Ute Schaeper, Walter Birchmeier, Joseph Schlessinger
    Abstract:

    The Docking Protein Gab1 has been implicated as a mediator of multiple signaling pathways that are activated by a variety of receptor tyrosine kinases and cytokines. We have previously proposed that fibroblast growth factor 1 (FGF1) stimulation of tyrosine phosphorylation of Gab1 and recruitment of phosphatidylinositol (PI) 3-kinase are mediated by an indirect mechanism in which the Docking Protein fibroblast receptor substrate 2α (FRS2α) plays a critical role. In this report, we explore the role of Gab1 in FGF1 signaling by using mouse embryo fibroblasts (MEFs) derived from Gab1−/− or FRS2α−/− mice. We demonstrate that Gab1 is essential for FGF1 stimulation of both PI 3-kinase and the antiapoptotic Protein kinase Akt, while FGF1-induced mitogen-activated Protein kinase (MAPK) stimulation is not affected by Gab1 deficiency. To test the indirect mechanism for FGF1 stimulation of PI 3-kinase and Akt, we use a chimeric Docking Protein composed of the membrane targeting signal and the phosphotyrosine-binding domain of FRS2α fused to the C-terminal portion of Gab1, the region including the binding sites for the complement of signaling Proteins that are recruited by Gab1. We demonstrate that expression of the chimeric Docking Protein in Gab1−/− MEFs rescues PI 3-kinase and the Akt responses, while expression of the chimeric Docking Protein in FRS2α−/− MEFs rescues stimulation of both Akt and MAPK. These experiments underscore the essential role of Gab1 in FGF1 stimulation of the PI 3-kinase/Akt signaling pathway and provide further support for the indirect mechanism for FGF1 stimulation of PI 3-kinase involving regulated assembly of a multiProtein complex.

  • the Docking Protein frs2α controls a map kinase mediated negative feedback mechanism for signaling by fgf receptors
    Molecular Cell, 2002
    Co-Authors: Andrew S Wong, Betty Lamothe, Adam Frost, Jessica J Hawes, Joseph Schlessinger
    Abstract:

    Abstract The Docking Protein FRS2α functions as a major mediator of signaling by FGF and NGF receptors. Here we demonstrate that, in addition to tyrosine phosphorylation, FRS2α is phosphorylated by MAP kinase on multiple threonine residues in response to FGF stimulation or by insulin, EGF, and PDGF, extracellular stimuli that do not induce tyrosine phosphorylation of FRS2α. Prevention of FRS2α threonine phosphorylation results in constitutive tyrosine phosphorylation of FRS2α in unstimulated cells and enhanced tyrosine phosphorylation of FRS2α, MAPK stimulation, cell migration, and proliferation in FGF-stimulated cells. Expression of an FRS2α mutant deficient in MAPK phosphorylation sites induces anchorage-independent cell growth and colony formation in soft agar. These experiments reveal a novel MAPK-mediated, negative feedback mechanism for control of signaling pathways that are dependent on FRS2 and a mechanism for heterologous control of signaling via FGF receptors.

  • Critical role for the Docking-Protein FRS2α in FGF receptor-mediated signal transduction pathways
    Proceedings of the National Academy of Sciences of the United States of America, 2001
    Co-Authors: Y. R. Hadari, Irit Lax, Noriko Gotoh, H. Kouhara, Joseph Schlessinger
    Abstract:

    The Docking Protein FRS2α has been implicated as a mediator of signaling via fibroblast growth factor receptors (FGFRs). We have demonstrated that targeted disruption of FRS2α gene causes severe impairment in mouse development resulting in embryonal lethality at E7.0–E7.5. Experiments with FRS2α-deficient fibroblasts demonstrate that FRS2α plays a critical role in FGF-induced mitogen-activated Protein (MAP) kinase stimulation, phosphatidylinositol-3 (PI-3) kinase activation, chemotactic response, and cell proliferation. Following FGF stimulation, tyrosine phosphorylated FRS2α functions as a site for coordinated assembly of a multiProtein complex that includes Gab1 and the effector Proteins that are recruited by this Docking Protein. Furthermore, we demonstrate that different tyrosine phosphorylation sites on FRS2α are responsible for mediating different FGF-induced biological responses. These experiments establish the central role of FRS2α in signaling via FGFRs and demonstrate that FRS2α mediates multiple FGFR-dependent signaling pathways critical for embryonic development.

Xiao Zhong Peng - One of the best experts on this subject based on the ideXlab platform.

Graham Warren - One of the best experts on this subject based on the ideXlab platform.

  • the vesicle Docking Protein p115 binds gm130 a cis golgi matrix Protein in a mitotically regulated manner
    Cell, 1997
    Co-Authors: Nobuhiro Nakamura, Timothy P. Levine, Catherine Rabouille, Martin Lowe, Graham Warren
    Abstract:

    The Docking of transport vesicles with their target membrane is thought to be mediated by p115. We show here that GM130, a cis-Golgi matrix Protein, interacts specifically with p115 and so could provide a membrane Docking site. Deletion analysis showed that the N-terminus binds to p115, whereas the C-terminus binds to Golgi membranes. Mitotic phosphorylation of GM130 or a peptide derived from the N-terminus prevented binding to p115. The peptide also inhibited the NSF- but not the p97-dependent reassembly of Golgi cisternae from mitotic fragments, unless it was mitotically phosphorylated. Together, these data provide a molecular explanation for the COPI-mediated fragmentation of the Golgi apparatus at the onset of mitosis.

  • Binding of the vesicle Docking Protein p115 to Golgi membranes is inhibited under mitotic conditions.
    The Journal of biological chemistry, 1996
    Co-Authors: Timothy P. Levine, Catherine Rabouille, Regina Kieckbusch, Graham Warren
    Abstract:

    The vesicle Docking Protein p115 showed saturable, high affinity binding to interphase Golgi membranes. The affinity of binding was up to 20-fold lower using membranes preincubated with mitotic cytosol. In contrast, binding was not affected by mitotic pretreatment of p115. The reduction in p115 binding was mediated by phosphorylation, could be induced by a cyclin-dependent kinase, and was fully reversible. A shift of p115 from membranes to cytosol was also found after fractionating mitotic cells. The functional significance of the decreased binding was addressed by in vitro mitotic incubations which disassemble Golgi cisternae, predominantly producing transport vesicles. The addition of excess p115 decreased loss of membrane from cisternae, indicating that p115's action is limiting while transport vesicles accumulate. The cessation of intra-Golgi traffic in mitosis has been hypothesized to result from an inhibition of membrane fusion while budding of transport vesicles continues. This process also contributes to mitotic Golgi disassembly. Our results imply that there is a mitotic modification to Golgi membranes leading to a reduction in the affinity of the p115 receptor. Reduced p115 binding may play a part in the inhibition of membrane fusion by preventing prior vesicle Docking.