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MAVS signaling pathway by targeting TRAF3 for degradation
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Abstracts / Cytokine 43 (2008) 326–332
either maintained their 2 h up-regulated expression levels or were further increased. In conclusion, Hyper-IL6 stimulated gp-130 SHP2/SOCS3 signaling in astrocytes not only moderates the gp-130 STAT3 pathway but also directly regulates the transcriptional activity of a number of genes. Support: NSW Spinal Cord Injury and Other Neurological Conditions Project Grant.
357 MGBP-2 inhibits RAC activation by platelet-derived growth factor or integrin engagement: Possible role of phosphatidylinositol 3-kinase Angela F. Messmer, Sujata Balasubramanian, Deborah J. Vestal, Angela F. Messmer, Deborah J. Vestal, Department of Biological Sciences, University of Toledo, Toledo, OH, USA
doi:10.1016/j.cyto.2008.07.439
355 Triad3A E3 ligase negatively regulates the RIG-I/MAVS signaling pathway by targeting TRAF3 for degradation Peyman Nakhaei 1,2, Thibault Mesplede 1,5, Qiang Sun 1, Mayra Solis 1,2, Long Yang 1,5, Rongtuan Lin 1,5, Tsung-Hsien Chuang 3, Carl F. Ware 4, John Hiscott 1,2,5, 1 Terry Fox Molecular Oncology Group, Lady Davis Institute for Medical Research, McGill University, Montreal, Que., Canada, 2 Department of Microbiology & Immunology, McGill University, Montreal, Que., Canada, 3 Department of Immunology, The Scripps Research Institute, La Jolla, CA, USA, 4 Division of Molecular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, CA, USA, 5 Department of Medicine, McGill University, Montreal, Que., Canada The primary role of the innate immune response is to limit the spread of infectious pathogens. Activation of Toll-like receptors (TLR) and TLR-independent pathways results in a pro-inflammatory response required to combat infection. Limiting the activation of these signaling pathways is likewise essential to prevent tissue injury in the host. Triad3A is an E3 ubiquitin–protein RING-domain containing ligase that interacts with several components of TLR signaling. In the present study, we demonstrate that Triad3A negatively regulates the RIG-I RNA sensing pathway through Lys48-linked, ubiquitin-mediated degradation of TRAF3. Triad3A is induced following dsRNA exposure and virus infection. Reduction of Triad3A expression by shRNA rendered cells hyperresponsive to Sendai virus infection; conversely, Triad3A expression blocked IRF-3 Ser-396 phosphorylation and enhanced vesicular stomatitis virus protein expression and replication. Interestingly, TRAF3 residues Y440 and Q442, previously shown to be important for association with MAVS adaptor, are critical for Triad3A interaction. Thus, Triad3A represents a versatile E3 ubiquitin–ligase that regulates the TLR-dependent and RIG-I-like receptor dependent signaling pathways. doi:10.1016/j.cyto.2008.07.440
356 Differential actions of GP130 cytokines: Mechanisms of oncostatin M functional specificity Nick Underhill-Day, John K. Heath, College of Life and Environmental Sciences, School of Biosciences, University of Birmingham, Birmingham, UK Oncostatin M (OSM), a member of the interleukin-6 (IL-6) cytokine family, exhibits diverse activities in inflammatory and wound healing processes and contributes to the pathology of certain diseases. IL-6 cytokines use the shared signal-transducing receptor, gp130. The majority of OSM-specific biological attributes require the specific type II gp130/ OSMb receptor and not the type I gp130/LIFa receptor. To address how this complex confers functional specificity we examined IL-6 cytokine-induced signalling, gene expression and cellular response in breast tumour cells. Through studies with mutant cytokines we determined that each of the representative receptor complexes generates a distinct signalling ‘portfolio’ leading to observable differences in biological response. Extrapolation of this data suggests that OSM-induced activities occur via coordinated integration of specific signalling networks. We used a proteomics approach to identify associations between PTB and SH2-domain containing effectors and the OSMb receptor intracellular region. Several novel associations between signalling effectors and OSMbR phosphopeptides were discovered extending the knowledge base of OSM signalling. The kinetics, intensity and composition of generated signals determine the downstream gene spectrum. To further characterise OSMb receptor specificity in relation to cellular response we looked at interleukin-6 and OSM-mediated gene expression. We generated a detailed timecourse of gene expression in response to IL-6 cytokines for multiple breast tumour cell lines. OSM gene regulation mediated via the gp130/OSMß receptor complex leads to expression of a ‘shared’ set of IL-6 regulated genes and a subset of ‘OSM-specific genes’ acting as candidate early-response genes involved in breast tumour cell morphological effects and cytostasis. At time-points subsequent to the initial wave of gene regulation, IL-6 and OSM gene expression diverges reflecting their difference in functional activity. doi:10.1016/j.cyto.2008.07.441
While it has been appreciated for some time that one growth factor or cytokine can modulate the activity of another, the molecular mechanisms responsible for many of these events are not well understood. Exposure of murine fibroblasts to interferonc (IFN-c) inhibits the ability of treatment with platelet-derived growth factor (PDGF) or integrin engagement during adhesion to fibronectin (FN) to activate the small GTPase, Rac. Rac activation during PDGF treatment and integrin engagement is also inhibited by the forced expression of a single IFN-c-induced protein, the large GTPase murine guanylate-binding protein-2 (mGBP-2). The inhibition of Rac activation during integrin engagement by either IFN-c treatment or mGBP-2 expression retards cell spreading on FN. Consistent with mGBP-2 being the IFN-c-induced protein responsible for the inhibition of cell spreading, siRNAs targeting mGBP-2 fully restore cell spreading on FN in the presence of IFN-c. Analysis of a truncated version of mGBP2 that lacks the N-terminal GTP-binding domain demonstrated that the C-terminal seven a-helices of mGBP-2 are sufficient to inhibit cell spreading. Therefore, we proposed that a direct interaction of mGBP-2 with one or more proteins involved in signal transduction from the integrin receptor to Rac would be involved. When no direct interaction was detected between mGBP-2 and either Rac or the Rac-specific guanine exchange factor, Tiam1, a possible inhibition of phosphatidylinositol 3-kinase (PI3-K) was examined. mGBP-2 inhibits PI3-K activation during cell spreading on FN, as measured by inhibition of activation of its substrate AKT. mGBP-2 appears to inhibit PI3-K activation during cell spreading by entering a molecular complex containing the catalytic subunit of PI3-K, p110. Work is currently underway to further delineate the mechanisms involved in mGBP-2-mediated inhibition of Rac activation. doi:10.1016/j.cyto.2008.07.442
358 Molecular cloning and functional studies of Atlantic salmon mitochondrial antiviral signaling protein Silje Lauksund, Tina Svingerud, Veronica Bergan, Øyvind Kileng, Børre Robertsen, Department of Marine Biotechnology, Norwegian College of Fishery Science, University of Tromsø, Norway Viral diseases are the cause of large economical losses in the Norwegian aquaculture industry. Understanding the responses to viral infection in Atlantic salmon, the predominant species in the Norwegian aquaculture industry, is important in order to elucidate potential new ways of controlling the threat of viral disease. Mitochondrial antiviral signaling protein (MAVS), also known as IPS-1/VISA/Cardif, is a central mediator in the signal transduction pathway from detection of viruses by the intracellular RNA helicases RIG-I/MDA5 to induction of interferon production. We have recently cloned an Atlantic salmon MAVS homologue (AsMAVS). Over-expression of AsMAVS leads to an up-regulation of Atlantic salmon interferon a1 promoter, an up-regulation of the NF-jB promoter, and also induces an antiviral state in CHSE214 cells against infectious pancreas necrosis virus (IPNV). This antiviral property is partly negated with interferon antibody, and is thus an effect of interferon induction. AsMAVS localizes to mitochondrial-like structures, and this localization is vital for retaining the antiviral properties of over-expressing the molecule. We want to investigate further whether fish pathogen viruses have the ability to inhibit the MAVSmediated signal transduction pathway in Atlantic salmon. doi:10.1016/j.cyto.2008.07.443
359 A novel mechanism for the regulation of GAB1 recruitment to the plasma membrane René Eulenfeld, Fred Schaper, Department of Biochemistry, RWTH Aachen University, Aachen, Germany Adapter proteins involved in signal transduction fulfil their cellular functions by bringing signalling molecules together and by targeting these signalling components to defined compartments within the cell. Furthermore, adapter proteins represent a molecular platform from which different signalling pathways are initiated. Gab1 is an adapter which recruits the p85 subunit of the phosphatidylinositol 3-kinase, the adapter Grb2, the adapter and phosphatase SHP2 and the GTPase-activating protein Ras-GAP. By this, Gab1 contributes to the activation of the PI3K cascade and the MAPK cascade by many growth factors and cytokines. The recruitment of Gab1 to phosphatidylinositol-3,4,5-tris-phosphate within the plasma membrane by its pleckstrin homology domain is regarded as a major regulatory step for the activation of Gab1.
Abstracts / Cytokine 43 (2008) 326–332
either maintained their 2 h up-regulated expression levels or were further increased. In conclusion, Hyper-IL6 stimulated gp-130 SHP2/SOCS3 signaling in astrocytes not only moderates the gp-130 STAT3 pathway but also directly regulates the transcriptional activity of a number of genes. Support: NSW Spinal Cord Injury and Other Neurological Conditions Project Grant.
357 MGBP-2 inhibits RAC activation by platelet-derived growth factor or integrin engagement: Possible role of phosphatidylinositol 3-kinase Angela F. Messmer, Sujata Balasubramanian, Deborah J. Vestal, Angela F. Messmer, Deborah J. Vestal, Department of Biological Sciences, University of Toledo, Toledo, OH, USA
doi:10.1016/j.cyto.2008.07.439
355 Triad3A E3 ligase negatively regulates the RIG-I/MAVS signaling pathway by targeting TRAF3 for degradation Peyman Nakhaei 1,2, Thibault Mesplede 1,5, Qiang Sun 1, Mayra Solis 1,2, Long Yang 1,5, Rongtuan Lin 1,5, Tsung-Hsien Chuang 3, Carl F. Ware 4, John Hiscott 1,2,5, 1 Terry Fox Molecular Oncology Group, Lady Davis Institute for Medical Research, McGill University, Montreal, Que., Canada, 2 Department of Microbiology & Immunology, McGill University, Montreal, Que., Canada, 3 Department of Immunology, The Scripps Research Institute, La Jolla, CA, USA, 4 Division of Molecular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, CA, USA, 5 Department of Medicine, McGill University, Montreal, Que., Canada The primary role of the innate immune response is to limit the spread of infectious pathogens. Activation of Toll-like receptors (TLR) and TLR-independent pathways results in a pro-inflammatory response required to combat infection. Limiting the activation of these signaling pathways is likewise essential to prevent tissue injury in the host. Triad3A is an E3 ubiquitin–protein RING-domain containing ligase that interacts with several components of TLR signaling. In the present study, we demonstrate that Triad3A negatively regulates the RIG-I RNA sensing pathway through Lys48-linked, ubiquitin-mediated degradation of TRAF3. Triad3A is induced following dsRNA exposure and virus infection. Reduction of Triad3A expression by shRNA rendered cells hyperresponsive to Sendai virus infection; conversely, Triad3A expression blocked IRF-3 Ser-396 phosphorylation and enhanced vesicular stomatitis virus protein expression and replication. Interestingly, TRAF3 residues Y440 and Q442, previously shown to be important for association with MAVS adaptor, are critical for Triad3A interaction. Thus, Triad3A represents a versatile E3 ubiquitin–ligase that regulates the TLR-dependent and RIG-I-like receptor dependent signaling pathways. doi:10.1016/j.cyto.2008.07.440
356 Differential actions of GP130 cytokines: Mechanisms of oncostatin M functional specificity Nick Underhill-Day, John K. Heath, College of Life and Environmental Sciences, School of Biosciences, University of Birmingham, Birmingham, UK Oncostatin M (OSM), a member of the interleukin-6 (IL-6) cytokine family, exhibits diverse activities in inflammatory and wound healing processes and contributes to the pathology of certain diseases. IL-6 cytokines use the shared signal-transducing receptor, gp130. The majority of OSM-specific biological attributes require the specific type II gp130/ OSMb receptor and not the type I gp130/LIFa receptor. To address how this complex confers functional specificity we examined IL-6 cytokine-induced signalling, gene expression and cellular response in breast tumour cells. Through studies with mutant cytokines we determined that each of the representative receptor complexes generates a distinct signalling ‘portfolio’ leading to observable differences in biological response. Extrapolation of this data suggests that OSM-induced activities occur via coordinated integration of specific signalling networks. We used a proteomics approach to identify associations between PTB and SH2-domain containing effectors and the OSMb receptor intracellular region. Several novel associations between signalling effectors and OSMbR phosphopeptides were discovered extending the knowledge base of OSM signalling. The kinetics, intensity and composition of generated signals determine the downstream gene spectrum. To further characterise OSMb receptor specificity in relation to cellular response we looked at interleukin-6 and OSM-mediated gene expression. We generated a detailed timecourse of gene expression in response to IL-6 cytokines for multiple breast tumour cell lines. OSM gene regulation mediated via the gp130/OSMß receptor complex leads to expression of a ‘shared’ set of IL-6 regulated genes and a subset of ‘OSM-specific genes’ acting as candidate early-response genes involved in breast tumour cell morphological effects and cytostasis. At time-points subsequent to the initial wave of gene regulation, IL-6 and OSM gene expression diverges reflecting their difference in functional activity. doi:10.1016/j.cyto.2008.07.441
While it has been appreciated for some time that one growth factor or cytokine can modulate the activity of another, the molecular mechanisms responsible for many of these events are not well understood. Exposure of murine fibroblasts to interferonc (IFN-c) inhibits the ability of treatment with platelet-derived growth factor (PDGF) or integrin engagement during adhesion to fibronectin (FN) to activate the small GTPase, Rac. Rac activation during PDGF treatment and integrin engagement is also inhibited by the forced expression of a single IFN-c-induced protein, the large GTPase murine guanylate-binding protein-2 (mGBP-2). The inhibition of Rac activation during integrin engagement by either IFN-c treatment or mGBP-2 expression retards cell spreading on FN. Consistent with mGBP-2 being the IFN-c-induced protein responsible for the inhibition of cell spreading, siRNAs targeting mGBP-2 fully restore cell spreading on FN in the presence of IFN-c. Analysis of a truncated version of mGBP2 that lacks the N-terminal GTP-binding domain demonstrated that the C-terminal seven a-helices of mGBP-2 are sufficient to inhibit cell spreading. Therefore, we proposed that a direct interaction of mGBP-2 with one or more proteins involved in signal transduction from the integrin receptor to Rac would be involved. When no direct interaction was detected between mGBP-2 and either Rac or the Rac-specific guanine exchange factor, Tiam1, a possible inhibition of phosphatidylinositol 3-kinase (PI3-K) was examined. mGBP-2 inhibits PI3-K activation during cell spreading on FN, as measured by inhibition of activation of its substrate AKT. mGBP-2 appears to inhibit PI3-K activation during cell spreading by entering a molecular complex containing the catalytic subunit of PI3-K, p110. Work is currently underway to further delineate the mechanisms involved in mGBP-2-mediated inhibition of Rac activation. doi:10.1016/j.cyto.2008.07.442
358 Molecular cloning and functional studies of Atlantic salmon mitochondrial antiviral signaling protein Silje Lauksund, Tina Svingerud, Veronica Bergan, Øyvind Kileng, Børre Robertsen, Department of Marine Biotechnology, Norwegian College of Fishery Science, University of Tromsø, Norway Viral diseases are the cause of large economical losses in the Norwegian aquaculture industry. Understanding the responses to viral infection in Atlantic salmon, the predominant species in the Norwegian aquaculture industry, is important in order to elucidate potential new ways of controlling the threat of viral disease. Mitochondrial antiviral signaling protein (MAVS), also known as IPS-1/VISA/Cardif, is a central mediator in the signal transduction pathway from detection of viruses by the intracellular RNA helicases RIG-I/MDA5 to induction of interferon production. We have recently cloned an Atlantic salmon MAVS homologue (AsMAVS). Over-expression of AsMAVS leads to an up-regulation of Atlantic salmon interferon a1 promoter, an up-regulation of the NF-jB promoter, and also induces an antiviral state in CHSE214 cells against infectious pancreas necrosis virus (IPNV). This antiviral property is partly negated with interferon antibody, and is thus an effect of interferon induction. AsMAVS localizes to mitochondrial-like structures, and this localization is vital for retaining the antiviral properties of over-expressing the molecule. We want to investigate further whether fish pathogen viruses have the ability to inhibit the MAVSmediated signal transduction pathway in Atlantic salmon. doi:10.1016/j.cyto.2008.07.443
359 A novel mechanism for the regulation of GAB1 recruitment to the plasma membrane René Eulenfeld, Fred Schaper, Department of Biochemistry, RWTH Aachen University, Aachen, Germany Adapter proteins involved in signal transduction fulfil their cellular functions by bringing signalling molecules together and by targeting these signalling components to defined compartments within the cell. Furthermore, adapter proteins represent a molecular platform from which different signalling pathways are initiated. Gab1 is an adapter which recruits the p85 subunit of the phosphatidylinositol 3-kinase, the adapter Grb2, the adapter and phosphatase SHP2 and the GTPase-activating protein Ras-GAP. By this, Gab1 contributes to the activation of the PI3K cascade and the MAPK cascade by many growth factors and cytokines. The recruitment of Gab1 to phosphatidylinositol-3,4,5-tris-phosphate within the plasma membrane by its pleckstrin homology domain is regarded as a major regulatory step for the activation of Gab1.