Philip Cohen

University of Dundee, Dundee, SCT, United Kingdom

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Publications (102)728.35 Total impact

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    ABSTRACT: The siRNA knockdown of IFN Regulatory Factor 5 (IRF5) in the human plasmacytoid dendritic cell line Gen2.2 prevented IFNβ production induced by compound CL097, a ligand for Toll-like receptor 7 (TLR7). CL097 also stimulated the phosphorylation of IRF5 at Ser462 and stimulated the nuclear translocation of wild-type IRF5, but not the IRF5[Ser462Ala] mutant. The CL097-stimulated phosphorylation of IRF5 at Ser462 and its nuclear translocation was prevented by the pharmacological inhibition of protein kinase IKKβ or the siRNA knockdown of IKKβ or its "upstream" activator, the protein kinase TAK1. Similar results were obtained in a murine macrophage cell line stimulated with the TLR7 agonist compound R848 or the nucleotide oligomerization domain 1 (NOD1) agonist KF-1B. IKKβ phosphorylated IRF5 at Ser462 in vitro and induced the dimerization of wild-type IRF5 but not the IRF5[S462A] mutant. These findings demonstrate that IKKβ activates two "master" transcription factors of the innate immune system, IRF5 and NF-κB.
    Proceedings of the National Academy of Sciences of the United States of America. 10/2014;
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    ABSTRACT: We developed a pharmacophore model for type II inhibitors that was used to guide the construction of a library of kinase inhibitors. Kinome-wide selectivity profiling of the library resulted in the identification of a series of 4-substituted 1H-pyrrolo[2,3-b]pyridines that exhibited potent inhibitory activity against two mitogen-activated protein kinases (MAPKs), TAK1 (MAP3K7) and MAP4K2, as well as pharmacologically well interrogated kinases such as p38α (MAPK14) and ABL. Further investigation of the structure-activity relationship (SAR) resulted in the identification of potent dual TAK1 and MAP4K2 inhibitors such as 1 (NG25) and 2 as well as MAP4K2 selective inhibitors such as 16 and 17. Some of these inhibitors possess good pharmacokinetic properties that will enable their use in pharmacological studies in vivo. A 2.4 Å cocrystal structure of TAK1 in complex with 1 confirms that the activation loop of TAK1 assumes the DFG-out conformation characteristic of type II inhibitors.
    Journal of medicinal chemistry. 07/2014;
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    ABSTRACT: The IκB kinase β (IKKβ) is required to activate the transcription factor NF-κB, but how IKKβ itself is activated in vivo is still unclear. It was found to require phosphorylation by one or more "upstream" protein kinases in some reports but by auto-phosphorylation in others. Here, we resolve this controversy by demonstrating that the activation of IKKβ induced by IL-1 or TNF in embryonic fibroblasts, or by ligands that activate Toll-Like receptors in macrophages, requires two distinct phosphorylation events; first, the TAK1-catalysed phosphorylation of Ser177 and second the IKKβ-catalyzed auto-phosphorylation of Ser181. The phosphorylation of Ser177 by TAK1 is a priming event required for the subsequent autophosphorylation of Ser181, which enables IKKβ to phosphorylate exogenous substrates. We also provide genetic evidence which indicates that the IL-1-stimulated, LUBAC-catalysed formation of linear ubiquitin chains and their interaction with the NEMO component of the canonical IKK complex permits the TAK1-catalysed priming phosphorylation of IKKβ at Ser177 and IKKα at Ser176. These findings may be of general significance for the activation of other protein kinases.
    The Biochemical journal. 06/2014;
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    ABSTRACT: Pathogenic infections and tissue injuries trigger the assembly of inflammasomes, cytosolic protein complexes that activate caspase-1, leading to cleavage of pro-IL-1β and pro-IL-18 and to pyroptosis, a proinflammatory cell death program. Although microbial recognition by Toll-like receptors (TLRs) is known to induce the synthesis of the major caspase-1 substrate pro-IL-1β, the role of TLRs has been considered limited to up-regulation of the inflammasome components. During infection with a virulent microbe, TLRs and nucleotide-binding oligomerization domain-like receptors (NLRs) are likely activated simultaneously. To examine the requirements and outcomes of combined activation, we stimulated TLRs and a specific NLR, nucleotide binding and oligomerization, leucine-rich repeat, pyrin domain-containing 3 (NLRP3), simultaneously and discovered that such activation triggers rapid caspase-1 cleavage, leading to secretion of presynthesized inflammatory molecules and pyroptosis. This acute caspase-1 activation is independent of new protein synthesis and depends on the TLR-signaling molecule IL-1 receptor-associated kinase (IRAK-1) and its kinase activity. Importantly, Listeria monocytogenes induces NLRP3-dependent rapid caspase-1 activation and pyroptosis, both of which are compromised in IRAK-1-deficient macrophages. Our results reveal that simultaneous sensing of microbial ligands and virulence factors by TLRs and NLRP3, respectively, leads to a rapid TLR- and IRAK-1-dependent assembly of the NLRP3 inflammasome complex, and that such activation is important for release of alarmins, pyroptosis, and early IFN-γ production by memory CD8 T cells, all of which could be critical for early host defense.
    Proceedings of the National Academy of Sciences 12/2013; · 9.81 Impact Factor
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    ABSTRACT: Research over the past decade has revealed how NF-κB essential modulator (NEMO; also known as IKKγ) regulates the IKKα-IKKβ signalling axis in the innate immune system. The discovery that NEMO is a polyubiquitin-binding protein and that the IKK complex is modulated by other protein kinases that are themselves controlled by polyubiquitin chains has provided a deeper molecular understanding of the non-degradative roles of ubiquitylation. New mechanistic insights of NEMO and related polyubiquitin-binding proteins have become a paradigm for how the interplay between phosphorylation and ubiquitylation controls cell signalling networks in health and disease.
    Nature Reviews Molecular Cell Biology 08/2013; · 37.16 Impact Factor
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    ABSTRACT: Polyubiquitin (pUb) chains formed between the C terminus of ubiquitin and lysine 63 (K63) or methionine 1 (M1) of another ubiquitin have been implicated in the activation of the canonical IκB kinase (IKK) complex. Here, we demonstrate that nearly all of the M1-pUb chains formed in response to interleukin-1, or the Toll-Like Receptors 1/2 agonist Pam3CSK4, are covalently attached to K63-pUb chains either directly as K63-pUb/M1-pUb hybrids or indirectly by attachment to the same protein. Interleukin-1 receptor (IL-1R)-associated kinase (IRAK) 1 is modified first by K63-pUb chains to which M1-pUb linkages are added subsequently, and myeloid differentiation primary response gene 88 (MyD88) and IRAK4 are also modified by both K63-pUb and M1-pUb chains. We show that the heme-oxidized IRP2 ubiquitin ligase 1 interacting protein (HOIP) component of the linear ubiquitin assembly complex catalyzes the formation of M1-pUb chains in response to interleukin-1, that the formation of K63-pUb chains is a prerequisite for the formation of M1-pUb chains, and that HOIP interacts with K63-pUb but not M1-pUb linkages. These findings identify K63-Ub oligomers as a major substrate of HOIP in cells where the MyD88-dependent signaling network is activated. The TGF-beta-activated kinase 1 (TAK1)-binding protein (TAB) 2 and TAB3 components of the TAK1 complex and the NFκB Essential Modifier (NEMO) component of the canonical IKK complex bind to K63-pUb chains and M1-pUb chains, respectively. The formation of K63/M1-pUb hybrids may therefore provide an elegant mechanism for colocalizing both complexes to the same pUb chain, facilitating the TAK1-catalyzed activation of IKKα and IKKβ. Our study may help to resolve the debate about the relative importance of K63-pUb and M1-pUb chains in activating the canonical IKK complex.
    Proceedings of the National Academy of Sciences 08/2013; · 9.81 Impact Factor
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    ABSTRACT: The genetic factors underlying the pathogenesis of lupus nephritis associated with systemic lupus erythematosus are largely unknown, although animal studies indicate that nuclear factor (NF)-κB is involved. We reported previously that a knockin mouse expressing an inactive form of ABIN1 (ABIN1[D485N]) develops lupus-like autoimmune disease and demonstrates enhanced activation of NF-κB and mitogen-activated protein kinases in immune cells after toll-like receptor stimulation. In the current study, we show that ABIN1[D485N] mice develop progressive GN similar to class III and IV lupus nephritis in humans. To investigate the clinical relevance of ABIN1 dysfunction, we genotyped five single-nucleotide polymorphisms in the gene encoding ABIN1, TNIP1, in samples from European-American, African American, Asian, Gullah, and Hispanic participants in the Large Lupus Association Study 2. Comparing cases of systemic lupus erythematosus with nephritis and cases of systemic lupus erythematosus without nephritis revealed strong associations with lupus nephritis at rs7708392 in European Americans and rs4958881 in African Americans. Comparing cases of systemic lupus erythematosus with nephritis and healthy controls revealed a stronger association at rs7708392 in European Americans but not at rs4958881 in African Americans. Our data suggest that variants in the TNIP1 gene are associated with the risk for lupus nephritis and could be mechanistically involved in disease development via aberrant regulation of NF-κB and mitogen-activated protein kinase activity.
    Journal of the American Society of Nephrology 08/2013; · 8.99 Impact Factor
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    ABSTRACT: The roles of IL-1R-associated kinase (IRAK)2 and IRAK1 in cytokine production were investigated using immune cells from knock-in mice expressing the TNFR-associated factor 6 (TRAF6) binding-defective mutant IRAK2[E525A] or the catalytically inactive IRAK1[D359A] mutant. In bone marrow-derived macrophages (BMDMs), the IRAK2-TRAF6 interaction was required for the late (2-8 h) but not the early phase (0-2 h) of il6 and tnfa mRNA production, and hence for IL-6 and TNF-α secretion by TLR agonists that signal via MyD88. Loss of the IRAK2-TRAF6 interaction had little effect on the MyD88-dependent production of anti-inflammatory molecules produced during the early phase, such as Dual Specificity Phosphatase 1, and a modest effect on IL-10 secretion. The LPS/TLR4-stimulated production of il6 and tnfa mRNA and IL-6 and TNF-α secretion was hardly affected, because the Toll/IL-1R domain-containing adapter-inducing IFN-β (TRIF) signaling pathway was used instead of the IRAK2-TRAF6 interaction to sustain late-phase mRNA production. IRAK1 catalytic activity was not rate limiting for il6, tnfa, or il10 mRNA production or the secretion of these cytokines by BMDMs, but IFN-β mRNA induction by TLR7 and TLR9 agonists was greatly delayed in plasmacytoid dendritic cells (pDCs) from IRAK1[D359A] mice. In contrast, IFN-β mRNA production was little affected in pDCs from IRAK2[E525A] mice, but subsequent IFN-α mRNA production and IFN-α secretion were reduced. IFN-β and IFN-α production were abolished in pDCs from IRAK1[D359A] × IRAK2[E525A] double knock-in mice. Our results establish that the IRAK2-TRAF6 interaction is rate limiting for the late, but not the early phase of cytokine production in BMDM and pDCs, and that the IRAK2-TRAF6 interaction is needed to sustain IκB-inducing kinase β activity during prolonged activation of the MyD88 signaling.
    The Journal of Immunology 08/2013; · 5.52 Impact Factor
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    ABSTRACT: Double-stranded (ds) RNA of viral origin, a ligand for Melanoma Differentiation Associated gene 5 (MDA5) and Toll-Like Receptor 3 (TLR3), induces the TANK-Binding Kinase 1 (TBK1) -dependent phosphorylation and activation of Interferon Regulatory Factor 3 (IRF3) and the E3 ubiquitin ligase Pellino1, which are required for interferon β (IFNβ) gene transcription. Here, we report that Pellino1 interacts with the transcription factor Deformed Epidermal Autoregulatory Factor 1 (DEAF1) The interaction is independent of the E3 ligase activity of Pellino1, but weakened by the phosphorylation of Pellino1. We show that DEAF1 binds to the IFNβ promoter and to IRF3 and IRF7, that it is required for the transcription of the IFNβ gene and IFNβ secretion in MEFs infected with Sendai virus or transfected with poly(I:C). DEAF1 is also needed for TLR3-dependent IFNβ production. Taken together, our results identify DEAF1 as a novel component of the signal transduction network by which dsRNA of viral origin stimulates IFNβ production.
    Journal of Biological Chemistry 07/2013; · 4.65 Impact Factor
  • Philip Cohen, Dario R. Alessi
    ACS Chemical Biology 02/2013; 8(2):464–464. · 5.44 Impact Factor
  • Philip Cohen, Dario R Alessi
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    ABSTRACT: Over the past 15 years protein kinases have become the pharmaceutical industry's most important class of drug target in the field of cancer. Some 20 drugs that target kinases have been approved for clinical use over the past decade, and hundreds more are undergoing clinical trials. However, the recent approval of the first protein kinase inhibitors for the treatment of inflammatory diseases, coupled with an enhanced understanding of the signaling networks that control the immune system, suggests that there will be a surge of interest in this area over the next 10 years. In this connection, we discuss opportunities for targeting protein kinases in the MyD88 signaling network for the development of drugs to treat chronic inflammatory and autoimmune diseases. Activating mutations in protein kinases underlie many other diseases and conditions, and we also discuss why the protein kinases SPAK/OSR1 and LRRK2 have recently become interesting targets for the treatment of hypertension and Parkinson's disease, respectively, and the progress that has been made in developing LRRK2 inhibitors. Finally we suggest that more focus on the identification of inhibitors of kinase activation, rather than kinase activity, may pay dividends in identifying exquisitely specific inhibitors of signal transduction cascades, and we also highlight "pseudo-kinases" as an attractive and unexplored area for drug development that merits much more attention in the years to come.
    ACS Chemical Biology 12/2012; · 5.44 Impact Factor
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    ABSTRACT: Macrophages acquire strikingly different properties that enable them to play key roles during the initiation, propagation, and resolution of inflammation. Classically activated (M1) macrophages produce proinflammatory mediators to combat invading pathogens and respond to tissue damage in the host, whereas regulatory macrophages (M2b) produce high levels of anti-inflammatory molecules, such as IL-10, and low levels of proinflammatory cytokines, like IL-12, and are important for the resolution of inflammatory responses. A central problem in this area is to understand how the formation of regulatory macrophages can be promoted at sites of inflammation to prevent and/or alleviate chronic inflammatory and autoimmune diseases. Here, we demonstrate that the salt-inducible kinases (SIKs) restrict the formation of regulatory macrophages and that their inhibition induces striking increases in many of the characteristic markers of regulatory macrophages, greatly stimulating the production of IL-10 and other anti-inflammatory molecules. We show that SIK inhibitors elevate IL-10 production by inducing the dephosphorylation of cAMP response element-binding protein (CREB)-regulated transcriptional coactivator (CRTC) 3, its dissociation from 14-3-3 proteins and its translocation to the nucleus where it enhances a gene transcription program controlled by CREB. Importantly, the effects of SIK inhibitors on IL-10 production are lost in macrophages that express a drug-resistant mutant of SIK2. These findings identify SIKs as a key molecular switch whose inhibition reprograms macrophages to an anti-inflammatory phenotype. The remarkable effects of SIK inhibitors on macrophage function suggest that drugs that target these protein kinases may have therapeutic potential for the treatment of inflammatory and autoimmune diseases.
    Proceedings of the National Academy of Sciences 10/2012; 109(42):16986-16991. · 9.81 Impact Factor
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    ABSTRACT: The design, synthesis and structure-activity relationships of a novel series of 2,4-diamino-5-cyclopropyl pyrimidines is described. Starting from BX795, originally reported to be a potent inhibitor of PDK1, we have developed compounds with improved selectivity and drug-like properties. These compounds have been evaluated in a range of cellular and in vivo assays, enabling us to probe the putative role of the TBK1/IKKε pathway in inflammatory diseases.
    Bioorganic & medicinal chemistry letters 09/2012; · 2.65 Impact Factor
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    ABSTRACT: Viral double-stranded RNA, a ligand for Toll-like Receptor 3 (TLR3) and the cytoplasmic RNA receptors RIG1 and MDA5, activate a signaling network in which the IKK-related protein kinase TBK1 phosphorylates the transcription factor Interferon Regulatory Factor 3 (IRF3) and the E3 ubiquitin ligase Pellino1. IRF3 then translocates to the nucleus where it stimulates transcription of the interferonβ (IFNβ) gene, but the function of Pellino1 in this pathway is unknown. Here, we report that myeloid cells and embryonic fibroblasts from knock-in mice expressing an E3 ligase-deficient mutant of Pellino1 produce reduced levels of IFNβ mRNA and secrete much less IFNβ in response to viral double-stranded RNA because the interaction of IRF3 with the IFNβ promoter is impaired. These results identify Pellino1 as a novel component of the signal transduction network by which viral double-stranded RNA stimulates IFNβ gene transcription.
    Journal of Biological Chemistry 08/2012; 287(41):34825-35. · 4.65 Impact Factor
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    ABSTRACT: Plasmacytoid dendritic cells (pDCs) are characterized by their ability to produce high levels of type 1 interferons in response to ligands that activate TLR7 and TLR9, but the signaling pathways required for IFN production are incompletely understood. Here we exploit the human pDC cell line Gen2.2 and improved pharmacological inhibitors of protein kinases to address this issue. We demonstrate that ligands that activate TLR7 and TLR9 require the TAK1-IKKβ signaling pathway to induce the production of IFNβ via a pathway that is independent of the degradation of IκBα. We also show that IKKβ activity, as well as the subsequent IFNβ-stimulated activation of the JAK-STAT1/2 signaling pathway, are essential for the production of IFNα by TLR9 ligands. We further show that TLR7 ligands CL097 and R848 fail to produce significant amounts of IFNα because the activation of IKKβ is not sustained for a sufficient length of time. The TLR7/9-stimulated production of type 1 IFNs is inhibited by much lower concentrations of IKKβ inhibitors than those needed to suppress the production of NFκB-dependent proinflammatory cytokines, such as IL-6, suggesting that drugs that inhibit IKKβ may have a potential for the treatment of forms of lupus that are driven by self-RNA and self-DNA-induced activation of TLR7 and TLR9, respectively.
    Journal of Biological Chemistry 04/2012; 287(23):19216-28. · 4.65 Impact Factor
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    ABSTRACT: The E3 ubiquitin ligase Pellino 1 can be interconverted between inactive and active forms by a reversible phosphorylation mechanism. In vitro, phosphorylation and activation can be catalysed by either the IRAKs [IL (interleukin)-1-receptor-associated kinases] IRAK1 and IRAK4, or the IKK {IκB [inhibitor of NF-κB (nuclear factor κB)] kinase}-related kinases [IKKϵ and TBK1 (TANK {TRAF [TNF (tumour-necrosis-factor)-receptor-associated factor]-associated NF-κB activator}-binding kinase 1)]. In the present study we establish that IRAK1 is the major protein kinase that mediates the IL-1-stimulated activation of Pellino 1 in MEFs (mouse embryonic fibroblasts) or HEK (human embryonic kidney)-293 cells, whereas the IKK-related kinases activate Pellino 1 in TNFα-stimulated MEFs. The IKK-related kinases are also the major protein kinases that activate Pellino 1 in response to TLR (Toll-like receptor) ligands that signal via the adaptors MyD88 (myeloid differentiation primary response gene 88) and/or TRIF [TIR (Toll/IL-1 receptor) domain-containing adaptor protein inducing interferon β]. The present studies demonstrate that, surprisingly, the ligands that signal via MyD88 do not always employ the same protein kinase to activate Pellino 1. Our results also establish that neither the catalytic activity of IRAK1 nor the activation of Pellino 1 is required for the initial transient activation of NF-κB and MAPKs (mitogen-activated protein kinases) that is triggered by IL-1 or TNFα in MEFs, or by TLR ligands in macrophages. The activation of Pellino 1 provides the first direct readout for IRAK1 catalytic activity in cells.
    Biochemical Journal 01/2012; 441(1):339-46. · 4.65 Impact Factor
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    ABSTRACT: Mutations in leucine-rich repeat kinase 2 (LRRK2) are strongly associated with late-onset autosomal dominant Parkinson's disease. LRRK2 is highly expressed in immune cells and recent work points towards a link between LRRK2 and innate immunity. Here we demonstrate that stimulation of the Toll-Like Receptor (TLR) pathway by MyD88-dependent agonists in bone marrow-derived macrophages (BMDMs) or RAW264.7 macrophages induces marked phosphorylation of LRRK2 at Ser910 and Ser935, the phosphorylation sites that regulate the binding of 14-3-3 to LRRK2. Phosphorylation of these residues is prevented by knock-out of MyD88 in BMDMs, but not the alternative TLR adaptor protein TRIF. Utilising both pharmacological inhibitors, including a new TAK1 inhibitor, NG25, and genetic models, we provide evidence that both the canonical (IKKα and IKKβ) and IKK-related (IKKε and TBK1) kinases mediate TLR agonist induced phosphorylation of LRRK2 in vivo. Moreover, all four IKK members directly phosphorylate LRRK2 at Ser910 and Ser935 in vitro. Consistent with previous work describing Ser910 and Ser935 as pharmacodynamic biomarkers of LRRK2 activity, we find that the TLR independent basal phosphorylation of LRRK2 at Ser910 and Ser935 is abolished following treatment of macrophages with LRRK2 kinase inhibitors. However, the increased phosphorylation of Ser910 and Ser935 induced by activation of the MyD88 pathway is insensitive to LRRK2 kinase inhibitors. Finally, employing LRRK2-deficient BMDMs, we present data indicating that LRRK2 does not play a major role in regulating the secretion of inflammatory cytokines induced by activation of the MyD88 pathway. Our findings provide the first direct link between LRRK2 and the IKKs that mediate many immune responses. Further work is required to uncover the physiological roles that phosphorylation of LRRK2 by IKKs play in controlling macrophage biology and to determine how phosphorylation of LRRK2 by IKKs impacts upon the use of Ser910 and Ser935 as pharmacodynamic biomarkers.
    PLoS ONE 01/2012; 7(6):e39132. · 3.53 Impact Factor
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    ABSTRACT: Toll-like receptor (TLR) ligands that signal via TIR-domain-containing adapter-inducing IFNβ (TRIF) activate the IκB kinase (IKK)-related kinases, TRAF associated NFκB activator (TANK)-binding kinase-1 (TBK1) and IKKε, which then phosphorylate IRF3 and induce the production of IFNβ. Here we show that TBK1 and IKKε are also activated by TLR ligands that signal via MyD88. Notably, the activation of IKKε is rapid, transient, and it precedes a more prolonged activation of TBK1. The MyD88- and TRIF-dependent signaling pathways activate the IKK-related kinases by two signaling pathways. One is mediated by the canonical IKKs, whereas the other culminates in the autoactivation of the IKK-related kinases. Once activated, TBK1/IKKε then phosphorylate and inhibit the canonical IKKs. The negative regulation of the canonical IKKs by the IKK-related kinases occurs in both the TRIF- and MyD88-dependent TLR pathways, whereas IRF3 phosphorylation is restricted to the TRIF-dependent signaling pathway. We have discovered that the activation of IKKε is abolished, the activation of TBK1 is reduced, and the interaction between the IKK-related kinases and the canonical IKKs is suppressed in TANK(-/-) macrophages, preventing the IKK-related kinases from negatively regulating the canonical IKKs. In contrast, IRF3 phosphorylation and IFNβ production was normal in TANK(-/-) macrophages. Our results demonstrate a key role for TANK in enabling the canonical IKKs and the IKK-related kinases to regulate each other, which is required to limit the strength of TLR signaling and ultimately, prevent autoimmunity.
    Proceedings of the National Academy of Sciences 09/2011; 108(41):17093-8. · 9.81 Impact Factor
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    ABSTRACT: TANK-binding kinase (TBK1) is essential for transcription of the interferon (IFN) β gene in response to lipopolysaccharide (LPS) and double-stranded RNA, but the molecular mechanisms that underlie the activation of TBK1 are incompletely understood. Previously, we identified the NF-κB essential modulator (NEMO)-related polyubiquitin-binding protein, optineurin (OPTN), as a novel binding partner of TBK1. To determine whether the ubiquitin-binding function of OPTN is involved in regulating TBK1 and IFNβ production, we generated a mouse in which wild-type optineurin was replaced by the polyubiquitin binding-defective mutant, OPTN(D477N/D477N). In this study, we found that LPS or poly(I:C)-induced TBK1 activity was significantly reduced in bone marrow-derived macrophage (BMDM) from OPTN(D477N/D477N) mice. Consistent with this, the phosphorylation of IFN regulatory factor 3 (IRF3) and the production of IFNβ mRNA and secretion were reduced. Stimulation of BMDMs with LPS triggered the phosphorylation of OPTN, which was reversed by phosphatase treatment and prevented by pharmacological inhibition of both the canonical IκB kinases (IKKα/β) and the IKK-related kinases (TBK1/IKKε). In contrast, LPS-stimulated phosphorylation of OPTN(D477N) was markedly reduced in BMDMs from OPTN(D477N/D477N) mice, and inhibition of the canonical IKKs alone prevented phosphorylation, providing further evidence that ubiquitin binding to OPTN contributes to LPS-induced TBK1 activation. TBK1 and IKKβ phosphorylated OPTN preferentially at Ser-177 and Ser-513, respectively, in vitro. In conclusion, our results suggest that OPTN binds to polyubiquitylated species formed in response to LPS and poly(I:C), enhancing the activation of TBK1 that is required for optimal phosphorylation of IRF3 and production of IFNβ.
    Journal of Biological Chemistry 08/2011; 286(41):35663-74. · 4.65 Impact Factor
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    ABSTRACT: The protein ABIN1 possesses a polyubiquitin-binding domain homologous to that present in nuclear factor κB (NF-κB) essential modulator (NEMO), a component of the inhibitor of NF-κB (IκB) kinase (IKK) complex. To address the physiological significance of polyubiquitin binding, we generated knockin mice expressing the ABIN1[D485N] mutant instead of the wild-type (WT) protein. These mice developed all the hallmarks of autoimmunity, including spontaneous formation of germinal centers, isotype switching, and production of autoreactive antibodies. Autoimmunity was suppressed by crossing to MyD88(-/-) mice, demonstrating that toll-like receptor (TLR)-MyD88 signaling pathways are needed for the phenotype to develop. The B cells and myeloid cells of the ABIN1[D485N] mice showed enhanced activation of the protein kinases TAK, IKK-α/β, c-Jun N-terminal kinases, and p38α mitogen-activated protein kinase and produced more IL-6 and IL-12 than WT. The mutant B cells also proliferated more rapidly in response to TLR ligands. Our results indicate that the interaction of ABIN1 with polyubiquitin is required to limit the activation of TLR-MyD88 pathways and prevent autoimmunity.
    Journal of Experimental Medicine 06/2011; 208(6):1215-28. · 13.21 Impact Factor

Publication Stats

7k Citations
728.35 Total Impact Points

Institutions

  • 1992–2013
    • University of Dundee
      • • College of Life Sciences
      • • Division of Signal Transduction Therapy (DSTT)
      • • MRC Protein Phosphorylation Unit
      Dundee, SCT, United Kingdom
  • 2012
    • MRC Technology
      Londinium, England, United Kingdom
  • 2004
    • University of Cambridge
      • MRC Laboratory of Molecular Biology
      Cambridge, ENG, United Kingdom
  • 2003–2004
    • University of Tuebingen
      • Institute for Physiology
      Tübingen, Baden-Wuerttemberg, Germany
    • Emory University
      • Department of Internal Medicine
      Atlanta, GA, United States