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Philip Cohen - One of the best experts on this subject based on the ideXlab platform.
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dimeric structure of the pseudokinase irak3 suggests an allosteric mechanism for negative regulation
Structure, 2021Co-Authors: Sven M Lange, Philip Cohen, Marina I Nelen, Yogesh KulathuAbstract:Summary Interleukin-1 receptor associated kinases (IRAKs) are key players in innate immune signaling that mediate the host response to pathogens. In contrast to the active kinases IRAK1 and IRAK4, IRAK2 and IRAK3 are pseudokinases lacking catalytic activity and their functions are poorly understood. IRAK3 is thought to be a negative regulator of innate immune signaling and mutations in IRAK3 are associated with asthma and cancer. Here, we report the crystal structure of the human IRAK3 pseudokinase domain in a closed, pseudoactive conformation. IRAK3 dimerizes in a unique way through a head-to-head arrangement not observed in any other kinases. Multiple conserved cysteine residues imply a potential redox control of IRAK3 conformation and dimerization. By analyzing asthma-associated mutations, we identify an evolutionarily conserved surface on IRAK3 that could form an interaction interface with IRAK4, suggesting a model for the negative regulation of IRAK4 by IRAK3.
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the e3 ligase hoil 1 catalyses ester bond formation between ubiquitin and components of the myddosome in mammalian cells
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Ian R Kelsall, Jiazhen Zhang, Simon J C Arthur, Axel Knebel, Philip CohenAbstract:The linear ubiquitin assembly complex (LUBAC) comprises 3 components: HOIP, HOIL-1, and Sharpin, of which HOIP and HOIL-1 are both members of the RBR subfamily of E3 ubiquitin ligases. HOIP catalyses the formation of Met1-linked ubiquitin oligomers (also called linear ubiquitin), but the function of the E3 ligase activity of HOIL-1 is unknown. Here, we report that HOIL-1 is an atypical E3 ligase that forms oxyester bonds between the C terminus of ubiquitin and serine and threonine residues in its substrates. Exploiting the sensitivity of HOIL-1–generated oxyester bonds to cleavage by hydroxylamine, and macrophages from knock-in mice expressing the E3 ligase-inactive HOIL-1[C458S] mutant, we identify IRAK1, IRAK2, and MyD88 as physiological substrates of the HOIL-1 E3 ligase during Toll-like receptor signaling. HOIL-1 is a monoubiquitylating E3 ubiquitin ligase that initiates the de novo synthesis of polyubiquitin chains that are attached to these proteins in macrophages. HOIL-1 also catalyses its own monoubiquitylation in cells and most probably the monoubiquitylation of Sharpin, in which ubiquitin is also attached by an oxyester bond. Our study establishes that oxyester-linked ubiquitylation is used as an intracellular signaling mechanism.
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the mechanism of activation of irak1 and irak4 by interleukin 1 and toll like receptor agonists
Biochemical Journal, 2017Co-Authors: Stefan Vollmer, Nathanael S. Gray, Sam Strickson, Tinghu Zhang, Katherine L Lee, Vikram R Rao, Philip CohenAbstract:We have developed the first assays that measure the protein kinase activities of interleukin-1 receptor-associated kinase 1 (IRAK1) and IRAK4 reliably in human cell extracts, by employing Pellino1 as a substrate in conjunction with specific pharmacological inhibitors of IRAK1 and IRAK4. We exploited these assays to show that IRAK4 was constitutively active and that its intrinsic activity towards Pellino1 was not increased significantly by stimulation with interleukin-1 (IL-1) in IL-1R-expressing HEK293 cells, Pam3CSK4-stimulated human THP1 monocytes or primary human macrophages. Our results, in conjunction with those of other investigators, suggest that the IL-1-stimulated trans-autophosphorylation of IRAK4 is initiated by the myeloid differentiation primary response gene 88-induced dimerization of IRAK4 and is not caused by an increase in the intrinsic catalytic activity of IRAK4. In contrast with IRAK4, we found that IRAK1 was inactive in unstimulated cells and converted into an active protein kinase in response to IL-1 or Pam3CSK4 in human cells. Surprisingly, the IL-1-stimulated activation of IRAK1 was not affected by pharmacological inhibition of IRAK4 and not reversed by dephosphorylation and/or deubiquitylation, suggesting that IRAK1 catalytic activity is not triggered by a covalent modification but by an allosteric mechanism induced by its interaction with IRAK4.
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suppression of irak1 or irak4 catalytic activity but not type 1 ifn signaling prevents lupus nephritis in mice expressing a ubiquitin binding defective mutant of abin1
Journal of Immunology, 2016Co-Authors: Sambit Nanda, Simon J C Arthur, Francesco Marchesi, Marta Lopezpelaez, Philip CohenAbstract:Polymorphisms in the TNIP1 gene encoding A20-binding inhibitor of NF-κB1 (ABIN1) predispose to lupus and other autoimmune diseases in at least eight human populations. We found previously that knock-in mice expressing a ubiquitin-binding–defective mutant of ABIN1 (ABIN1[D485N]) develop autoimmunity as they age and succumb to a disease resembling lupus nephritis in humans. In this article, we report that Flt3-derived dendritic cells from these mice overproduced type 1 IFNs upon stimulation with ligands that activate TLR7 or TLR9. However, crossing ABIN1[D485N] mice to IFNAR1-knockout mice that do not express the α-subunit of the type 1 IFNR did not prevent splenomegaly, the appearance of high serum levels of autoantibodies and other Igs, or liver inflammation and only reduced kidney inflammation modestly. In contrast, crossing ABIN1[D485N] mice to knock-in mice expressing catalytically inactive mutants of IRAK1 or IRAK4 prevented splenomegaly, autoimmunity, and liver and kidney inflammation. Our results support the notion that IRAK1 and/or IRAK4 are attractive targets for the development of drugs to prevent, and perhaps treat, lupus nephritis and other autoinflammatory diseases caused by the decreased ability of ABIN1 or other proteins to restrict the strength of MyD88 signaling.
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targeting irak1 irak4 signaling in waldenstrom s macroglobulinemia
Blood, 2015Co-Authors: Guang Yang, Xia Liu, Jie Chen, Nicholas Tsakmaklis, Jiaji Chen, Christopher J. Patterson, Jorge J. Castillo, Philip Cohen, Li Tan, Sara J. BuhrlageAbstract:Background: MYD88 L265P somatic mutations are highly prevalent in Waldenstrom9s macroglobulinemia (WM) (NEJM 367(9):826-33). MYD88 L265P activates multiple downstream signaling pathways including BTK and IRAK1/IRAK4 that support malignant cell growth and survival (Nature 470(7332):115-9; Blood 122(7):1222-32). Ibrutinib targets BTK, and shows high overall and major clinical response rates, though no complete responses are observed indicating alternative survival signaling. Methodology: Phospho-flow analysis of IRAK1, IRAK4, and BTK was performed in primary WM cells taken from untreated WM patients, and those on ibrutinib therapy. IRAK1 or IRAK4 knockdown experiments were performed using lentiviral shRNA transduction. Immunoprecipitation, western blot and phospho-flow studies were used to detect protein expression and phosphorylation in WM cells. Cell survival following IRAK4 or IRAK1 knockdown, ibrutinib and/or IRAK4/IRAK1 inhibitor (EMD Millipore) treatment was assessed by Annexin V staining, AlamarBlue® Cell Viability Assay or CellTiter - Glo ® Luminescent Cell Viability Assays. Results: Phospho-flow analysis of bone marrow lymphoplasmacytic cells taken from WM patients following > 6 months of continued ibrutinib treatment demonstrated highly active IRAK1 and IRAK4, but not BTK. These findings prompted us to dissect the relative impact of IRAK1 and IRAK4 in supporting WM cell survival. Using lentiviral transduction, we identified shRNAs that produced similar levels of protein reduction by western blot analysis for both IRAK1 and IRAK4. Compared to scrambled control vector, knockdown of IRAK1 or IRAK4 both produced decreased tumor cell survival in MYD88 mutated BCWM.1 and MWCL-1 cells. More pronounced apoptosis, as well as sustained reduction in tumor cell growth occurred following knockdown of IRAK1 versus IRAK4. Treatment of primary WM cells taken from untreated patients, patients on ibrutinib therapy, as well as MYD88 mutated WM cells lines with ibrutinib and a toolbox IRAK4/IRAK1 inhibitor resulted in more robust reductions in NFkB signaling, and at least additive tumor cell killing versus either agent alone. Conclusions: MYD88 L265P mutated WM cells show greater dependence on IRAK1 versus IRAK4 directed signaling. IRAK1/IRAK4 signaling may contribute to persistent WM cell survival following ibrutinib treatment. Combined BTK and IRAK inhibition leads to augmented blockade of NFKB signaling and enhanced WM cell killing. The studies provide a framework for the development and investigation of IRAK inhibitors, alone and in combination with ibrutinib in WM patients. Disclosures No relevant conflicts of interest to declare.
Anne Puel - One of the best experts on this subject based on the ideXlab platform.
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mechanism of dysfunction of human variants of the irak4 kinase and a role for its kinase activity in interleukin 1 receptor signaling
Journal of Biological Chemistry, 2018Co-Authors: Fawziya Karim, Leah Cushing, Jeanlaurent Casanova, Anne Puel, Ezechielle Kiessu, Lih Ling Lin, Pegah Ghandil, Cyrille HoarauAbstract:Interleukin-1 receptor (IL1R)-associated kinase 4 (IRAK4) is a central regulator of innate immune signaling, controlling IL1R and Toll-like receptor (TLR)-mediated responses and containing both scaffolding and kinase activities. Humans deficient in IRAK4 activity have autosomal recessive primary immune deficiency (PID). Here, we characterized the molecular mechanism of dysfunction of two IRAK4 PID variants, G298D and the compound variant R12C (R12C/R391H/T458I). Using these variants and the kinase-inactive D329A variant to delineate the contributions of IRAK4's scaffolding and kinase activities to IL1R signaling, we found that the G298D variant is kinase-inactive and expressed at extremely low levels, acting functionally as a null mutation. The R12C compound variant possessed WT kinase activity, but could not interact with myeloid differentiation primary response 88 (MyD88) and IRAK1, causing impairment of IL-1–induced signaling and cytokine production. Quantitation of IL-1 signaling in IRAK4-deficient cells complemented with either WT or the R12C or D329A variant indicated that the loss of MyD88 interaction had a greater impact on IL-1–induced signaling and cytokine expression than the loss of IRAK4 kinase activity. Importantly, kinase-inactive IRAK4 exhibited a greater association with MyD88 and a weaker association with IRAK1 in IRAK4-deficient cells expressing kinase-inactive IRAK4 and in primary cells treated with a selective IRAK4 inhibitor. Loss of IRAK4 kinase activity only partially inhibited IL-1–induced cytokine and NF-κB signaling. Therefore, the IRAK4–MyD88 scaffolding function is essential for IL-1 signaling, but IRAK4 kinase activity can control IL-1 signal strength by modulating the association of IRAK4, MyD88, and IRAK1.
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interleukin 1 toll like receptor induced autophosphorylation activates interleukin 1 receptor associated kinase 4 and controls cytokine induction in a cell type specific manner
Journal of Biological Chemistry, 2014Co-Authors: Leah Cushing, Wayne Stochaj, Marshall M Siegel, Robert M Czerwinski, Ken Dower, Quentin G Wright, Margaret Hirschfield, Jeanlaurent Casanova, Capucine Picard, Anne PuelAbstract:IRAK4 is a central kinase in innate immunity, but the role of its kinase activity is controversial. The mechanism of activation for IRAK4 is currently unknown, and little is known about the role of IRAK4 kinase in cytokine production, particularly in different human cell types. We show IRAK4 autophosphorylation occurs by an intermolecular reaction and that autophosphorylation is required for full catalytic activity of the kinase. Phosphorylation of any two of the residues Thr-342, Thr-345, and Ser-346 is required for full activity, and the death domain regulates the activation of IRAK4. Using antibodies against activated IRAK4, we demonstrate that IRAK4 becomes phosphorylated in human cells following stimulation by IL-1R and Toll-like receptor agonists, which can be blocked pharmacologically by a dual inhibitor of IRAK4 and IRAK1. Interestingly, in dermal fibroblasts, although complete inhibition of IRAK4 kinase activity does not inhibit IL-1-induced IL-6 production, NF-κB, or MAPK activation, there is complete ablation of these processes in IRAK4-deficient cells. In contrast, the inhibition of IRAK kinase activity in primary human monocytes reduces R848-induced IL-6 production with minimal effect on NF-κB or MAPK activation. Taken together, these studies define the mechanism of IRAK4 activation and highlight the differential role of IRAK4 kinase activity in different human cell types as well as the distinct roles IRAK4 scaffolding and kinase functions play.
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the transmembrane activator taci triggers immunoglobulin class switching by activating b cells through the adaptor myd88
Nature Immunology, 2010Co-Authors: Raul Santamaria, April Chiu, Meimei Shan, Anne Puel, Montserrat Cols, Kang Chen, Irene Puga, Huabao Xiong, James B Bussel, Jeanine ReichenbachAbstract:BAFF and APRIL are innate immune mediators that trigger immunoglobulin G (IgG) and IgA class-switch recombination (CSR) in B cells by engaging the receptor TACI. The mechanism that underlies CSR signaling by TACI remains unknown. Here we found that the cytoplasmic domain of TACI encompasses a conserved motif that bound MyD88, an adaptor that activates transcription factor NF-kappaB signaling pathways via a Toll-interleukin 1 (IL-1) receptor (TIR) domain. TACI lacks a TIR domain, yet triggered CSR via the DNA-editing enzyme AID by activating NF-kappaB through a Toll-like receptor (TLR)-like MyD88-IRAK1-IRAK4-TRAF6-TAK1 pathway. TACI-induced CSR was impaired in mice and humans lacking MyD88 or the kinase IRAK4, which indicates that MyD88 controls a B cell-intrinsic, TIR-independent, TACI-dependent pathway for immunoglobulin diversification.
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irak 4 mutation q293x rapid detection and characterization of defective post transcriptional tlr il 1r responses in human myeloid and non myeloid cells
Journal of Immunology, 2006Co-Authors: Donald J Davidson, Anne Puel, Andrew Currie, Dawn M E Bowdish, Kelly L Brown, Carrie M Rosenberger, Rebecca C, Johan Bylund, Paul A Campsall, Capucine PicardAbstract:Innate immunodeficiency has recently been reported as resulting from the Q293X IRAK-4 mutation with consequent defective TLR/IL-1R signaling. In this study we report a method for the rapid allele-specific detection of this mutation and demonstrate both cell type specificity and ligand specificity in defective IL-1R-associated kinase (IRAK)-4-deficient cellular responses, indicating differential roles for this protein in human PBMCs and primary dermal fibroblasts and in LPS, IL-1beta, and TNF-alpha signaling. We demonstrate transcriptional and post-transcriptional defects despite NF-kappaB signaling and intact MyD88-independent signaling and propose that dysfunctional complex 1 (IRAK1/TRAF6/TAK1) signaling, as a consequence of IRAK-4 deficiency, generates specific defects in MAPK activation that could underpin this patient's innate immunodeficiency. These studies demonstrate the importance of studying primary human cells bearing a clinically relevant mutation; they underscore the complexity of innate immune signaling and illuminate novel roles for IRAK-4 and the fundamental importance of accessory proinflammatory signaling to normal human innate immune responses and immunodeficiencies.
Xiaoxia Li - One of the best experts on this subject based on the ideXlab platform.
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pellino 1 is required for interleukin 1 il 1 mediated signaling through its interaction with the il 1 receptor associated kinase 4 irak4 irak tumor necrosis factor receptor associated factor 6 traf6 complex
Journal of Biological Chemistry, 2003Co-Authors: Zhengfan Jiang, Jan H Johnson, Timothy A Bird, Xiaoxia LiAbstract:Abstract The signaling pathway downstream of the mammalian interleukin-1 receptor (IL-1R)/Toll-like receptor (TLR) is evolutionally conserved with that mediated by theDrosophila Toll protein. Toll initiates its signal through the adapter molecule Tube and the serine-threonine kinase Pelle. Pelle is highly homologous to members of the IL-1R-associated kinase (IRAK) family in mammals. Recently, a novel Pelle-interacting protein called Pellino was identified in Drosophila. We now report a mammalian counterpart of Pellino, termed Pellino 1, which is required for NFκB activation and IL-8 gene expression in response to IL-1, probably through its signal-dependent interaction with IRAK4, IRAK, and the tumor necrosis factor receptor-associated factor 6 (TRAF6). The Pellino 1-IRAK-IRAK4-TRAF6 signaling complex is likely to be intermediate, located between the IL-1 receptor complex and the TAK1 complex in the IL-1 pathway.
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interleukin 1 il 1 receptor associated kinase dependent il 1 induced signaling complexes phosphorylate tak1 and tab2 at the plasma membrane and activate tak1 in the cytosol
Molecular and Cellular Biology, 2002Co-Authors: Zhengfan Jiang, Jun Ninomiyatsuji, Kunihiro Matsumoto, Youcun Qian, Xiaoxia LiAbstract:Interleukin-1 (IL-1), a major inflammatory cytokine, exerts its biological effects by activating the transcription of various responsive genes (7). The transcription factors activated by IL-1 include NF-κB, AP1, and ATF (2, 19, 20). The IL-1 receptor complex is composed of the type 1 receptor (IL-1R) and the receptor accessory protein (IL-1RAcp) (8-10). Upon IL-1 stimulation, the cytosolic proteins MyD88 (1, 15, 28) and Tollip (3) are recruited to this receptor complex, where they function as adaptors, recruiting IL-1 receptor-associated kinase (IRAK) in turn. IRAK, a serine-threonine kinase, is phosphorylated at the receptor complex and then interacts with TRAF6 (4, 5, 12-14). Phosphorylated IRAK is eventually ubiquitinated and degraded (31). IRAK4 has recently been shown to be an essential component for the IL-1 signaling pathway and proposed to function as an IRAK kinase (11, 24). IRAK and TRAF6 interact with TAK1, a member of the MAP kinase kinase kinase (MAPKKK) family, and two proteins that bind to it, TAB1 and TAB2 (18, 25). The kinase activity of TAK1 is thus activated upon IL-1 stimulation. While genetic studies show that IRAK is required for the activation of TAK1 (26), in vitro biochemical analyses reveal that TRAF6-mediated ubiquitination may also play an important role in TAK1 activation (27). The activation of TAK1 eventually leads to the activation of IκB kinase (IKK) by an unknown mechanism. Activated IKK phosphorylates the inhibitory IκB proteins, which are then degraded, releasing NF-κB to activate transcription in the nucleus (17, 22, 29, 35). Activated TAK1 has also been implicated in the IL-1-induced activation of MKK6 and JNK (18), leading to the phosphorylation and activation of ATF and AP1, thereby also activating transcription. We have previously taken a genetic approach to study IL-1-dependent signaling pathways; through random mutagenesis, we generated IL-1-unresponsive cell lines lacking specific components of the pathways. Mutant cell line I1A, which lacks both IRAK protein and mRNA (12, 13), has been used effectively to study structure-function relationships of IRAK in IL-1-dependent signaling (12, 13). Neither NF-κB nor JNK is activated in IL-1-treated I1A cells, but these responses are restored in I1A-IRAK cells, indicating that IRAK is required for both. However, the kinase activity of IRAK is not required for IL-1-dependent signaling (12, 13), since kinase-dead IRAK mutants were still able to restore IL-1 responsiveness in mutant I1A cells. On the other hand, IL-1-induced phosphorylation of IRAK probably plays a critical role in its interaction with TRAF6, TAK1, TAB1, and TAB2 and in the activation of TAK1 and IKK. The IRAK phosphorylated in response to IL-1 is membrane-bound, whereas TRAF6 and TAB2 are localized on the membrane ever before stimulation. Previously, Qian et al. (21) reported that IRAK is required for the IL-1-induced translocation of TRAF6 and TAB2 from the membrane to the cytosol, probably through their signal-dependent interaction on the membrane, and proposed that the translocation of both TAB2 and TRAF6 are required to form a TRAF6-TAK1-TAB1-TAB2 complex in the cytosol, leading to the activation of NF-κB and JNK. Although much progress has been made in understanding IL-1-mediated signaling, many questions still remain. For example, we do not know exactly how TAK1 is activated, nor do we understand the precise role of IRAK in TAK1 activation. It is also unknown how activated TAK1 leads to the activation of IKK and JNK. We now attempt to elucidate the details of the molecular mechanism of IL-1-dependent signal transduction by investigating the sequential formation and activation of signaling complexes upon IL-1 stimulation. We find that TRAF6 is recruited to the IL-1 receptor complex (complex I) through IRAK upon IL-1 stimulation. IRAK-TRAF6 then leaves the receptor to form complex II with TAK1, TAB1, and TAB2, which are preassociated on the membrane before stimulation. Importantly, formation of this complex II leads to the phosphorylation of TAK1 and TAB2 but not the activation of TAK1 on the membrane. TRAF6, TAB2, TAK1, and TAB1 (complex III) subsequently dissociate from IRAK and translocate from the membrane to the cytosol, where the TAK1 is activated.
Zhengfan Jiang - One of the best experts on this subject based on the ideXlab platform.
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irak4 kinase activity is redundant for interleukin 1 il 1 receptor associated kinase phosphorylation and il 1 responsiveness
Journal of Biological Chemistry, 2004Co-Authors: Zhengfan Jiang, Youcun Qian, Jinzhong Qin, Jeanlaurent CasanovaAbstract:Interleukin-1 (IL-1) stimulation leads to the recruitment of interleukin-1 receptor-associated kinase (IRAK) to the IL-1 receptor, where IRAK is phosphorylated, ubiquitinated, and eventually degraded. Kinase-inactive mutant IRAK is still phosphorylated in response to IL-1 stimulation when it is transfected into IRAK-deficient cells, suggesting that there must be an IRAK kinase in the pathway. The fact that IRAK4, another IRAK family member necessary for the IL-1 pathway, is able to phosphorylate IRAK in vitro suggests that IRAK4 might be the IRAK kinase. However, we now found that the IRAK4 kinase-inactive mutant had the same ability as the wild-type IRAK4 in restoring IL-1-mediated signaling in human IRAK4-deficient cells, including NFκB-dependent reporter gene expression, the activation of NFκB and JNK, and endogenous IL-8 gene expression. These results strongly indicate that the kinase activity of human IRAK4 is not necessary for IL-1 signaling. Furthermore, we showed that the kinase activity of IRAK4 was not necessary for IL-1-induced IRAK phosphorylation, suggesting that IRAK phosphorylation can probably be achieved either by autophosphorylation or by trans-phosphorylation through IRAK4. In support of this, only the impairment of the kinase activity of both IRAK and IRAK4 efficiently abolished the IL-1 pathway, demonstrating that the kinase activity of IRAK and IRAK4 is redundant for IL-1-mediated signaling. Moreover, consistent with the fact that IRAK4 is a necessary component of the IL-1 pathway, we found that IRAK4 was required for the efficient recruitment of IRAK to the IL-1 receptor complex.
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pellino 1 is required for interleukin 1 il 1 mediated signaling through its interaction with the il 1 receptor associated kinase 4 irak4 irak tumor necrosis factor receptor associated factor 6 traf6 complex
Journal of Biological Chemistry, 2003Co-Authors: Zhengfan Jiang, Jan H Johnson, Timothy A Bird, Xiaoxia LiAbstract:Abstract The signaling pathway downstream of the mammalian interleukin-1 receptor (IL-1R)/Toll-like receptor (TLR) is evolutionally conserved with that mediated by theDrosophila Toll protein. Toll initiates its signal through the adapter molecule Tube and the serine-threonine kinase Pelle. Pelle is highly homologous to members of the IL-1R-associated kinase (IRAK) family in mammals. Recently, a novel Pelle-interacting protein called Pellino was identified in Drosophila. We now report a mammalian counterpart of Pellino, termed Pellino 1, which is required for NFκB activation and IL-8 gene expression in response to IL-1, probably through its signal-dependent interaction with IRAK4, IRAK, and the tumor necrosis factor receptor-associated factor 6 (TRAF6). The Pellino 1-IRAK-IRAK4-TRAF6 signaling complex is likely to be intermediate, located between the IL-1 receptor complex and the TAK1 complex in the IL-1 pathway.
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interleukin 1 il 1 receptor associated kinase dependent il 1 induced signaling complexes phosphorylate tak1 and tab2 at the plasma membrane and activate tak1 in the cytosol
Molecular and Cellular Biology, 2002Co-Authors: Zhengfan Jiang, Jun Ninomiyatsuji, Kunihiro Matsumoto, Youcun Qian, Xiaoxia LiAbstract:Interleukin-1 (IL-1), a major inflammatory cytokine, exerts its biological effects by activating the transcription of various responsive genes (7). The transcription factors activated by IL-1 include NF-κB, AP1, and ATF (2, 19, 20). The IL-1 receptor complex is composed of the type 1 receptor (IL-1R) and the receptor accessory protein (IL-1RAcp) (8-10). Upon IL-1 stimulation, the cytosolic proteins MyD88 (1, 15, 28) and Tollip (3) are recruited to this receptor complex, where they function as adaptors, recruiting IL-1 receptor-associated kinase (IRAK) in turn. IRAK, a serine-threonine kinase, is phosphorylated at the receptor complex and then interacts with TRAF6 (4, 5, 12-14). Phosphorylated IRAK is eventually ubiquitinated and degraded (31). IRAK4 has recently been shown to be an essential component for the IL-1 signaling pathway and proposed to function as an IRAK kinase (11, 24). IRAK and TRAF6 interact with TAK1, a member of the MAP kinase kinase kinase (MAPKKK) family, and two proteins that bind to it, TAB1 and TAB2 (18, 25). The kinase activity of TAK1 is thus activated upon IL-1 stimulation. While genetic studies show that IRAK is required for the activation of TAK1 (26), in vitro biochemical analyses reveal that TRAF6-mediated ubiquitination may also play an important role in TAK1 activation (27). The activation of TAK1 eventually leads to the activation of IκB kinase (IKK) by an unknown mechanism. Activated IKK phosphorylates the inhibitory IκB proteins, which are then degraded, releasing NF-κB to activate transcription in the nucleus (17, 22, 29, 35). Activated TAK1 has also been implicated in the IL-1-induced activation of MKK6 and JNK (18), leading to the phosphorylation and activation of ATF and AP1, thereby also activating transcription. We have previously taken a genetic approach to study IL-1-dependent signaling pathways; through random mutagenesis, we generated IL-1-unresponsive cell lines lacking specific components of the pathways. Mutant cell line I1A, which lacks both IRAK protein and mRNA (12, 13), has been used effectively to study structure-function relationships of IRAK in IL-1-dependent signaling (12, 13). Neither NF-κB nor JNK is activated in IL-1-treated I1A cells, but these responses are restored in I1A-IRAK cells, indicating that IRAK is required for both. However, the kinase activity of IRAK is not required for IL-1-dependent signaling (12, 13), since kinase-dead IRAK mutants were still able to restore IL-1 responsiveness in mutant I1A cells. On the other hand, IL-1-induced phosphorylation of IRAK probably plays a critical role in its interaction with TRAF6, TAK1, TAB1, and TAB2 and in the activation of TAK1 and IKK. The IRAK phosphorylated in response to IL-1 is membrane-bound, whereas TRAF6 and TAB2 are localized on the membrane ever before stimulation. Previously, Qian et al. (21) reported that IRAK is required for the IL-1-induced translocation of TRAF6 and TAB2 from the membrane to the cytosol, probably through their signal-dependent interaction on the membrane, and proposed that the translocation of both TAB2 and TRAF6 are required to form a TRAF6-TAK1-TAB1-TAB2 complex in the cytosol, leading to the activation of NF-κB and JNK. Although much progress has been made in understanding IL-1-mediated signaling, many questions still remain. For example, we do not know exactly how TAK1 is activated, nor do we understand the precise role of IRAK in TAK1 activation. It is also unknown how activated TAK1 leads to the activation of IKK and JNK. We now attempt to elucidate the details of the molecular mechanism of IL-1-dependent signal transduction by investigating the sequential formation and activation of signaling complexes upon IL-1 stimulation. We find that TRAF6 is recruited to the IL-1 receptor complex (complex I) through IRAK upon IL-1 stimulation. IRAK-TRAF6 then leaves the receptor to form complex II with TAK1, TAB1, and TAB2, which are preassociated on the membrane before stimulation. Importantly, formation of this complex II leads to the phosphorylation of TAK1 and TAB2 but not the activation of TAK1 on the membrane. TRAF6, TAB2, TAK1, and TAB1 (complex III) subsequently dissociate from IRAK and translocate from the membrane to the cytosol, where the TAK1 is activated.
Jeanlaurent Casanova - One of the best experts on this subject based on the ideXlab platform.
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mechanism of dysfunction of human variants of the irak4 kinase and a role for its kinase activity in interleukin 1 receptor signaling
Journal of Biological Chemistry, 2018Co-Authors: Fawziya Karim, Leah Cushing, Jeanlaurent Casanova, Anne Puel, Ezechielle Kiessu, Lih Ling Lin, Pegah Ghandil, Cyrille HoarauAbstract:Interleukin-1 receptor (IL1R)-associated kinase 4 (IRAK4) is a central regulator of innate immune signaling, controlling IL1R and Toll-like receptor (TLR)-mediated responses and containing both scaffolding and kinase activities. Humans deficient in IRAK4 activity have autosomal recessive primary immune deficiency (PID). Here, we characterized the molecular mechanism of dysfunction of two IRAK4 PID variants, G298D and the compound variant R12C (R12C/R391H/T458I). Using these variants and the kinase-inactive D329A variant to delineate the contributions of IRAK4's scaffolding and kinase activities to IL1R signaling, we found that the G298D variant is kinase-inactive and expressed at extremely low levels, acting functionally as a null mutation. The R12C compound variant possessed WT kinase activity, but could not interact with myeloid differentiation primary response 88 (MyD88) and IRAK1, causing impairment of IL-1–induced signaling and cytokine production. Quantitation of IL-1 signaling in IRAK4-deficient cells complemented with either WT or the R12C or D329A variant indicated that the loss of MyD88 interaction had a greater impact on IL-1–induced signaling and cytokine expression than the loss of IRAK4 kinase activity. Importantly, kinase-inactive IRAK4 exhibited a greater association with MyD88 and a weaker association with IRAK1 in IRAK4-deficient cells expressing kinase-inactive IRAK4 and in primary cells treated with a selective IRAK4 inhibitor. Loss of IRAK4 kinase activity only partially inhibited IL-1–induced cytokine and NF-κB signaling. Therefore, the IRAK4–MyD88 scaffolding function is essential for IL-1 signaling, but IRAK4 kinase activity can control IL-1 signal strength by modulating the association of IRAK4, MyD88, and IRAK1.
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interleukin 1 toll like receptor induced autophosphorylation activates interleukin 1 receptor associated kinase 4 and controls cytokine induction in a cell type specific manner
Journal of Biological Chemistry, 2014Co-Authors: Leah Cushing, Wayne Stochaj, Marshall M Siegel, Robert M Czerwinski, Ken Dower, Quentin G Wright, Margaret Hirschfield, Jeanlaurent Casanova, Capucine Picard, Anne PuelAbstract:IRAK4 is a central kinase in innate immunity, but the role of its kinase activity is controversial. The mechanism of activation for IRAK4 is currently unknown, and little is known about the role of IRAK4 kinase in cytokine production, particularly in different human cell types. We show IRAK4 autophosphorylation occurs by an intermolecular reaction and that autophosphorylation is required for full catalytic activity of the kinase. Phosphorylation of any two of the residues Thr-342, Thr-345, and Ser-346 is required for full activity, and the death domain regulates the activation of IRAK4. Using antibodies against activated IRAK4, we demonstrate that IRAK4 becomes phosphorylated in human cells following stimulation by IL-1R and Toll-like receptor agonists, which can be blocked pharmacologically by a dual inhibitor of IRAK4 and IRAK1. Interestingly, in dermal fibroblasts, although complete inhibition of IRAK4 kinase activity does not inhibit IL-1-induced IL-6 production, NF-κB, or MAPK activation, there is complete ablation of these processes in IRAK4-deficient cells. In contrast, the inhibition of IRAK kinase activity in primary human monocytes reduces R848-induced IL-6 production with minimal effect on NF-κB or MAPK activation. Taken together, these studies define the mechanism of IRAK4 activation and highlight the differential role of IRAK4 kinase activity in different human cell types as well as the distinct roles IRAK4 scaffolding and kinase functions play.
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the kinase activity of il 1 receptor associated kinase 4 is required for interleukin 1 receptor toll like receptor induced tak1 dependent nfκb activation
Journal of Biological Chemistry, 2008Co-Authors: Jerzy Fraczek, Jeanlaurent Casanova, Tae Whan Kim, Jianhong Yao, Hui Xiao, Qian Wen, Juliusz PryjmaAbstract:Two parallel interleukin-1 (IL-1)-mediated signaling pathways have been uncovered for IL-1R-TLR-mediated NFkappaB activation: TAK1-dependent and MEKK3-dependent pathways, respectively. The TAK1-dependent pathway leads to IKKalpha/beta phosphorylation and IKKbeta activation, resulting in classic NFkappaB activation through IkappaBalpha phosphorylation and degradation. The TAK1-independent MEKK3-dependent pathway involves IKKgamma phosphorylation and IKKalpha activation, resulting in NFkappaB activation through dissociation of phosphorylated IkappaBalpha from NFkappaB without IkappaBalpha degradation. IL-1 receptor-associated kinase 4 (IRAK4) belongs to the IRAK family of proteins and plays a critical role in IL-1R/TLR-mediated signaling. IRAK4 kinase-inactive mutant failed to mediate the IL-1R-TLR-induced TAK1-dependent NFkappaB activation pathway, but mediated IL-1-induced TAK1-independent NFkappaB activation and retained the ability to activate substantial gene expression, indicating a structural role of IRAK4 in mediating this alternative NFkappaB activation pathway. Deletion analysis of IRAK4 indicates the essential structural role of the IRAK4 death domain in receptor proximal signaling for mediating IL-1R-TLR-induced NFkappaB activation.
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irak4 kinase activity is redundant for interleukin 1 il 1 receptor associated kinase phosphorylation and il 1 responsiveness
Journal of Biological Chemistry, 2004Co-Authors: Zhengfan Jiang, Youcun Qian, Jinzhong Qin, Jeanlaurent CasanovaAbstract:Interleukin-1 (IL-1) stimulation leads to the recruitment of interleukin-1 receptor-associated kinase (IRAK) to the IL-1 receptor, where IRAK is phosphorylated, ubiquitinated, and eventually degraded. Kinase-inactive mutant IRAK is still phosphorylated in response to IL-1 stimulation when it is transfected into IRAK-deficient cells, suggesting that there must be an IRAK kinase in the pathway. The fact that IRAK4, another IRAK family member necessary for the IL-1 pathway, is able to phosphorylate IRAK in vitro suggests that IRAK4 might be the IRAK kinase. However, we now found that the IRAK4 kinase-inactive mutant had the same ability as the wild-type IRAK4 in restoring IL-1-mediated signaling in human IRAK4-deficient cells, including NFκB-dependent reporter gene expression, the activation of NFκB and JNK, and endogenous IL-8 gene expression. These results strongly indicate that the kinase activity of human IRAK4 is not necessary for IL-1 signaling. Furthermore, we showed that the kinase activity of IRAK4 was not necessary for IL-1-induced IRAK phosphorylation, suggesting that IRAK phosphorylation can probably be achieved either by autophosphorylation or by trans-phosphorylation through IRAK4. In support of this, only the impairment of the kinase activity of both IRAK and IRAK4 efficiently abolished the IL-1 pathway, demonstrating that the kinase activity of IRAK and IRAK4 is redundant for IL-1-mediated signaling. Moreover, consistent with the fact that IRAK4 is a necessary component of the IL-1 pathway, we found that IRAK4 was required for the efficient recruitment of IRAK to the IL-1 receptor complex.
