Imperial College London

Professor Steve Ley

Faculty of MedicineDepartment of Immunology and Inflammation

Centre Co-Director, Chair in Molecular Immunology
 
 
 
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Commonwealth BuildingHammersmith Campus

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Summary

 

Publications

Publication Type
Year
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77 results found

Nanou A, Bourbouli M, Vetrano S, Schaeper U, Ley S, Kollias Get al., 2021, Endothelial Tpl2 regulates vascular barrier function via JNK-mediated degradation of claudin-5 promoting neuroinflammation or tumor metastasis, CELL REPORTS, Vol: 35, ISSN: 2211-1247

Journal article

Breyer F, Hartlova A, Thurston T, Flynn HR, Chakravarty P, Janzen J, Peltier J, Heunis T, Snijders AP, Trost M, Ley SCet al., 2021, TPL-2 kinase induces phagosome acidification to promote macrophage killing of bacteria, The EMBO Journal, Vol: 40, Pages: 1-19, ISSN: 0261-4189

Tumour progression locus 2 (TPL-2) kinase mediates Toll-like receptor (TLR) activation of ERK1/2 and p38α MAP kinases in myeloid cells to modulate expression of key cytokines in innate immunity. This study identified a novel MAP kinase-independent regulatory function for TPL-2 in phagosome maturation, an essential process for killing of phagocytosed microbes. TPL-2 catalytic activity was demonstrated to induce phagosome acidification and proteolysis in primary mouse and human macrophages following uptake of latex beads. Quantitative proteomics revealed that blocking TPL-2 catalytic activity significantly altered the protein composition of phagosomes, particularly reducing the abundance of V-ATPase proton pump subunits. Furthermore, TPL-2 stimulated the phosphorylation of DMXL1, a regulator of V-ATPases, to induce V-ATPase assembly and phagosome acidification. Consistent with these results, TPL-2 catalytic activity was required for phagosome acidification and the efficient killing of Staphylococcus aureus and Citrobacter rodentium following phagocytic uptake by macrophages. TPL-2 therefore controls innate immune responses of macrophages to bacteria via V-ATPase induction of phagosome maturation.

Journal article

Manils J, Webb L, Howes A, Janzen J, Boeing S, Bowcock AM, Ley SCet al., 2020, CARD14(E138A) signalling in keratinocytes induces TNF-dependent skin and systemic inflammation, ELIFE, Vol: 9, ISSN: 2050-084X

Journal article

Webb LV, Ventura S, Ley SC, 2019, ABIN-2, of the TPL-2 Signaling Complex, Modulates Mammalian Inflammation, TRENDS IN IMMUNOLOGY, Vol: 40, Pages: 799-808, ISSN: 1471-4906

Journal article

Webb L, Barbarulo A, Huysentruyt J, Vanden Berghe T, Takahashi N, Ley S, Vandenabeele P, Seddon Bet al., 2019, Survival of Single Positive Thymocytes Depends upon Developmental Control of RIPK1 Kinase Signaling by the IKK Complex Independent of NF-kappa B, IMMUNITY, Vol: 50, Pages: 348-+, ISSN: 1074-7613

Journal article

Ventura S, Cano F, Kannan Y, Breyer F, Pattison MJ, Wilson MS, Ley SCet al., 2018, A20-binding inhibitor of NF-kappa B (ABIN) 2 negatively regulates allergic airway inflammation, Journal of Experimental Medicine, Vol: 215, Pages: 2737-2747, ISSN: 0022-1007

TPL-2 MAP 3-kinase promotes inflammation in numerous mouse disease models and is an attractive anti-inflammatory drug target. However, TPL-2–deficient (Map3k8−/−) mice develop exacerbated allergic airway inflammation to house dust mite (HDM) compared with wild type controls. Here, we show that Map3k8D270A/D270A mice expressing kinase dead TPL-2 had an unaltered response to HDM, indicating that the severe airway inflammation observed in Map3k8−/− mice is not due to blockade of TPL-2 signaling and rather reflects a TPL-2 adaptor function. Severe allergic inflammation in TPL-2–deficient mice was likely due to reduced levels of ABIN-2 (TNIP2), whose stability depends on TPL-2 expression. Tnip2E256K knock-in mutation, which reduced ABIN-2 binding to A20, augmented the HDM-induced airway inflammation, but did not affect TPL-2 expression or signaling. These results identify ABIN-2 as a novel negative regulator of allergic airway responses and importantly indicate that TPL-2 inhibitors would not have unwanted allergic comorbidities.

Journal article

Kannan Y, Li Y, Coomes SM, Okoye IS, Pelly VS, Sriskantharajah S, Gückel E, Webb L, Czieso S, Nikolov N, MacDonald AS, Ley SC, Wilson MSet al., 2017, Tumor progression locus 2 reduces severe allergic airway inflammation by inhibiting Ccl24 production in dendritic cells, Journal of Allergy and Clinical Immunology, Vol: 139, Pages: 655-666.e7, ISSN: 0091-6749

Journal article

Howes A, O'Sullivan PA, Breyer F, Ghose A, Cao L, Krappmann D, Bowcock AM, Ley SCet al., 2016, Psoriasis mutations disrupt CARD14 autoinhibition promoting BCL10-MALT1-dependent NF-κB activation, Biochemical Journal, Vol: 473, Pages: 1759-1768, ISSN: 1470-8728

Inherited and <em>de novo</em> mutations in the <em>CARD14</em> gene promote the development of psoriasis, an inflammatory disease of the skin. CARD14 is a member of the CARMA protein family that includes the structurally related CARD11 adaptor that mediates NF-κB activation by antigen receptors. We investigated the mechanism by which <em>CARD14 </em>mutation in psoriasis activates NF-κB. In contrast to wild type CARD14, CARD14E138A and CARD14G117S psoriasis mutants interacted constitutively with BCL10 and MALT1, and triggered BCL10 and MALT1 dependent activation of NF-κB in keratinocytes. These alterations disrupted the inhibitory effect of the CARD14 linker region on NF-κB activation by facilitating BCL10 binding. Therefore, psoriasis mutations activated CARD14 by a mechanism analogous to oncogenic CARD11 mutations in non-Hodgkin B cell lymphomas. CARD14E138A also stimulated MALT1 paracaspase activity and activated both ERK1/2 and p38α MAP kinases. Inhibition of MALT1 with mepazine reduced CARD14E138A-induced expression of specific psoriasis-associated transcripts in keratinocytes. Our results establish the mechanism whereby gain-of-function CARD14 variants, which induce psoriatic disease in affected individuals, activate pro-inflammatory signaling.

Journal article

Jacque E, Schweighoffer E, Tybulewicz VL, Ley SCet al., 2015, BAFF activation of the ERK5 MAP kinase pathway regulates B cell survival, Journal of Experimental Medicine, Vol: 212, Pages: 883-892, ISSN: 1540-9538

B cell activating factor (BAFF) stimulation of the BAFF receptor (BAFF-R) is essential for the homeostatic survival of mature B cells. Earlier in vitro experiments with inhibitors that block MEK 1 and 2 suggested that activation of ERK 1 and 2 MAP kinases is required for BAFF-R to promote B cell survival. However, these inhibitors are now known to also inhibit MEK5, which activates the related MAP kinase ERK5. In the present study, we demonstrated that BAFF-induced B cell survival was actually independent of ERK1/2 activation but required ERK5 activation. Consistent with this, we showed that conditional deletion of ERK5 in B cells led to a pronounced global reduction in mature B2 B cell numbers, which correlated with impaired survival of ERK5-deficient B cells after BAFF stimulation. ERK5 was required for optimal BAFF up-regulation of Mcl1 and Bcl2a1, which are prosurvival members of the Bcl-2 family. However, ERK5 deficiency did not alter BAFF activation of the PI3-kinase-Akt or NF-κB signaling pathways, which are also important for BAFF to promote mature B cell survival. Our study reveals a critical role for the MEK5-ERK5 MAP kinase signaling pathway in BAFF-induced mature B cell survival and homeostatic maintenance of B2 cell numbers.

Journal article

Ley SC, Beyaert R, 2014, Priming IKKβ kinase for action., Biochem J, Vol: 463, Pages: e1-e2

IKKβ (IκB kinase β) is a core component of signalling pathways that control the activation of NF-κB (nuclear factor κB) transcription factors, which regulate many physiological processes, including cell survival, immunity and DNA-damage responses. Like many kinases, activation of IKKβ requires phosphorylation of the activation loop of its kinase domain. Different upstream protein kinases, and IKKβ itself, have been reported to directly phosphorylate and activate IKKβ in vitro, but the exact molecular mechanism of IKKβ activation in cells has remained unclear. In a recent article in the Biochemical Journal, Zhang and co-workers showed that IKKβ is activated by two sequential phosphorylations of its activation loop in response to TNF (tumour necrosis factor), IL-1 (interleukin-1) and TLR (Toll-like receptor) ligands. Using a combination of biochemical and genetic approaches, they demonstrate that IKKβ is first phosphorylated by the upstream kinase TAK1 [TGFβ (transforming growth factor β)-activated kinase-1] at Ser177, which then serves as a priming signal for subsequent IKKβ autophosphorylation at Ser181. This study resolves two apparently conflicting earlier models of IKKβ activation into a single unified model, and suggests that the IKKβ activation loop may integrate distinct 'upsteam' signals to activate NF-κB.

Journal article

Jacque E, Schweighoffer E, Visekruna A, Papoutsopoulou S, Janzen J, Zillwood R, Tarlinton DM, Tybulewicz VLJ, Ley SCet al., 2014, IKK-induced NF-kappa B1 p105 proteolysis is critical for B cell antibody responses to T cell-dependent antigen, Journal of Experimental Medicine, Vol: 211, Pages: 2085-2101, ISSN: 0022-1007

The importance of IκB kinase (IKK)–induced proteolysis of NF-κB1 p105 in B cells was investigated using Nfkb1SSAA/SSAA mice, in which this NF-κB signaling pathway is blocked. Nfkb1SSAA mutation had no effect on the development and homeostasis of follicular mature (FM) B cells. However, analysis of mixed bone marrow chimeras revealed that Nfkb1SSAA/SSAA FM B cells were completely unable to mediate T cell–dependent antibody responses. Nfkb1SSAA mutation decreased B cell antigen receptor (BCR) activation of NF-κB in FM B cells, which selectively blocked BCR stimulation of cell survival and antigen-induced differentiation into plasmablasts and germinal center B cells due to reduced expression of Bcl-2 family proteins and IRF4, respectively. In contrast, the antigen-presenting function of FM B cells and their BCR-induced migration to the follicle T cell zone border, as well as their growth and proliferation after BCR stimulation, were not affected. All of the inhibitory effects of Nfkb1SSAA mutation on B cell functions were rescued by normalizing NF-κB activation genetically. Our study identifies critical B cell-intrinsic functions for IKK-induced NF-κB1 p105 proteolysis in the antigen-induced survival and differentiation of FM B cells, which are essential for T-dependent antibody responses.

Journal article

Ley SC, Beyaert R, 2014, Commentary priming IKKβ kinase for action, Biochemical Journal, Vol: 463, Pages: e1-e2, ISSN: 0264-6021

IKKβ (IκB kinase β) is a core component of signalling pathways that control the activation of NF-κB (nuclear factor κB) transcription factors, which regulate many physiological processes, including cell survival, immunity and DNA-damage responses. Like many kinases, activation of IKKβ requires phosphorylation of the activation loop of its kinase domain. Different upstream protein kinases, and IKKβ itself, have been reported to directly phosphorylate and activate IKKβ in vitro, but the exact molecular mechanism of IKKβ activation in cells has remained unclear. In a recent article in the Biochemical Journal, Zhang and co-workers showed that IKKβ is activated by two sequential phosphorylations of its activation loop in response to TNF (tumour necrosis factor), IL-1 (interleukin- 1) and TLR (Toll-like receptor) ligands. Using a combination of biochemical and genetic approaches, they demonstrate that IKKβ is first phosphorylated by the upstream kinase TAK1 [TGFβ (transforming growth factor β)-activated kinase-1] at Ser177, which then serves as a priming signal for subsequent IKKβ autophosphorylation at Ser181. This study resolves two apparently conflicting earlier models of IKKβ activation into a single unified model, and suggests that the IKKβ activation loop may integrate distinct 'upsteam' signals to activate NF-κB.

Journal article

Ben-Addi A, Mambole-Dema A, Brender C, Martin SR, Janzen J, Kjaer S, Smerdon SJ, Ley SCet al., 2014, IκB kinase-induced interaction of TPL-2 kinase with 14-3-3 is essential for Toll-like receptor activation of ERK-1 and -2 MAP kinases, Proceedings of the National Academy of Sciences of USA, Vol: 111, Pages: E2394-E2403, ISSN: 0027-8424

The MEK-1/2 kinase TPL-2 is critical for Toll-like receptor activation of the ERK-1/2 MAP kinase pathway during inflammatory responses, but it can transform cells following C-terminal truncation. IκB kinase (IKK) complex phosphorylation of the TPL-2 C terminus regulates full-length TPL-2 activation of ERK-1/2 by a mechanism that has remained obscure. Here, we show that TPL-2 Ser-400 phosphorylation by IKK and TPL-2 Ser-443 autophosphorylation cooperated to trigger TPL-2 association with 14-3-3. Recruitment of 14-3-3 to the phosphorylated C terminus stimulated TPL-2 MEK-1 kinase activity, which was essential for TPL-2 activation of ERK-1/2. The binding of 14-3-3 to TPL-2 was also indispensible for lipopolysaccharide-induced production of tumor necrosis factor by macrophages, which is regulated by TPL-2 independently of ERK-1/2 activation. Our data identify a key step in the activation of TPL-2 signaling and provide a mechanistic insight into how C-terminal deletion triggers the oncogenic potential of TPL-2 by rendering its kinase activity independent of 14-3-3 binding.

Journal article

Sriskantharajah S, Gueckel E, Tsakiri N, Kierdorf K, Brender C, Ben-Addi A, Veldhoen M, Tsichlis PN, Stockinger B, O'Garra A, Prinz M, Kollias G, Ley SCet al., 2014, Regulation of Experimental Autoimmune Encephalomyelitis by TPL-2 Kinase, JOURNAL OF IMMUNOLOGY, Vol: 192, Pages: 3518-3529, ISSN: 0022-1767

Journal article

Silva A, Cornish G, Ley SC, Seddon Bet al., 2014, NF-κB signaling mediates homeostatic maturation of new T cells., Proc Natl Acad Sci U S A, Vol: 111, Pages: E846-E855

Interleukin (IL)-7 is critical for the maintenance of the peripheral T-cell compartment of the adaptive immune system. IL-7 receptor α (IL-7Rα) expression is subject to developmental regulation and new T cells induce expression as they leave the thymus, which is essential for their long-term survival. It is not understood how this expression is regulated. Here, we identify a role for the Nuclear Factor κ-B (NF-κB) signaling pathway in controlling expression of IL-7Rα in new T cells. Perturbations to NF-κB signaling, either by deletion of Inhibitor of Kappa-B Kinase-2 (IKK2) or by inhibiting Rel dimer activity, prevented normal IL-7Rα expression in new T cells. Defective IL-7Rα expression resulted in impaired survival and homeostatic cell division responses by T cells that could be attributed to their failure to express IL-7Rα normally. Surprisingly, NF-κB signaling was only required transiently in new T cells to allow their normal expression of IL-7Rα, because IKK2 deletion in mature T cells had no effect on IL-7Rα expression or their normal homeostatic responsiveness. Therefore, we identify a developmental function for NF-κB signaling in the homeostatic maturation of new T cells, by regulating IL-7Rα expression.

Journal article

Arthur JSC, Ley SC, 2013, Mitogen-activated protein kinases in innate immunity., Nat Rev Immunol, Vol: 13, Pages: 679-692

Following pathogen infection or tissue damage, the stimulation of pattern recognition receptors on the cell surface and in the cytoplasm of innate immune cells activates members of each of the major mitogen-activated protein kinase (MAPK) subfamilies--the extracellular signal-regulated kinase (ERK), p38 and Jun N-terminal kinase (JNK) subfamilies. In conjunction with the activation of nuclear factor-κB and interferon-regulatory factor transcription factors, MAPK activation induces the expression of multiple genes that together regulate the inflammatory response. In this Review, we discuss our current knowledge about the regulation and the function of MAPKs in innate immunity, as well as the importance of negative feedback loops in limiting MAPK activity to prevent host tissue damage. We also examine how pathogens have evolved complex mechanisms to manipulate MAPK activation to increase their virulence. Finally, we consider the potential of the pharmacological targeting of MAPK pathways to treat autoimmune and inflammatory diseases.

Journal article

McNab FW, Ewbank J, Rajsbaum R, Stavropoulos E, Martirosyan A, Redford PS, Wu X, Graham CM, Saraiva M, Tsichlis P, Chaussabel D, Ley SC, O'Garra Aet al., 2013, TPL-2-ERK1/2 Signaling Promotes Host Resistance against Intracellular Bacterial Infection by Negative Regulation of Type I IFN Production, JOURNAL OF IMMUNOLOGY, Vol: 191, Pages: 1732-1743, ISSN: 0022-1767

Journal article

Gantke T, Boussouf S, Janzen J, Morrice NA, Howell S, Mühlberger E, Ley SCet al., 2013, Ebola virus VP35 induces high-level production of recombinant TPL-2-ABIN-2-NF-κB1 p105 complex in co-transfected HEK-293 cells., Biochem J, Vol: 452, Pages: 359-365

Activation of PKR (double-stranded-RNA-dependent protein kinase) by DNA plasmids decreases translation, and limits the amount of recombinant protein produced by transiently transfected HEK (human embryonic kidney)-293 cells. Co-expression with Ebola virus VP35 (virus protein 35), which blocked plasmid activation of PKR, substantially increased production of recombinant TPL-2 (tumour progression locus 2)-ABIN-2 [A20-binding inhibitor of NF-κB (nuclear factor κB) 2]-NF-κB1 p105 complex. VP35 also increased expression of other co-transfected proteins, suggesting that VP35 could be employed generally to boost recombinant protein production by HEK-293 cells.

Journal article

Roget K, Ben-Addi A, Mambole-Dema A, Gantke T, Yang H-T, Janzen J, Morrice N, Abbott D, Ley SCet al., 2012, IκB kinase 2 regulates TPL-2 activation of extracellular signal-regulated kinases 1 and 2 by direct phosphorylation of TPL-2 serine 400., Mol Cell Biol, Vol: 32, Pages: 4684-4690

Tumor progression locus 2 (TPL-2) functions as a MEK-1/2 kinase, which is essential for Toll-like receptor 4 (TLR4) activation of extracellular signal-regulated kinase 1 and 2 (ERK-1/2) mitogen-activated protein (MAP) kinases in lipopolysaccharide (LPS)-stimulated macrophages and for inducing the production of the proinflammatory cytokines tumor necrosis factor and interleukin-1β. In unstimulated cells, association of TPL-2 with NF-κB1 p105 prevents TPL-2 phosphorylation of MEK-1/2. LPS stimulation of TPL-2 MEK-1/2 kinase activity requires TPL-2 release from p105. This is triggered by IκB kinase 2 (IKK-2) phosphorylation of the p105 PEST region, which promotes p105 ubiquitination and degradation by the proteasome. LPS activation of ERK-1/2 additionally requires transphosphorylation of TPL-2 on serine 400 in its C terminus, which controls TPL-2 signaling to ERK-1/2 independently of p105. However, the identity of the protein kinase responsible for TPL-2 serine 400 phosphorylation remained unknown. In the present study, we show that TPL-2 serine 400 phosphorylation is mediated by IKK2. The IKK complex therefore regulates two of the key regulatory steps required for TPL-2 activation of ERK-1/2, underlining the close linkage of ERK-1/2 MAP kinase activation to upregulation of NF-κB-dependent transcription.

Journal article

Yang H-T, Papoutsopoulou S, Belich M, Brender C, Janzen J, Gantke T, Handley M, Ley SCet al., 2012, Coordinate regulation of TPL-2 and NF-κB signaling in macrophages by NF-κB1 p105., Mol Cell Biol, Vol: 32, Pages: 3438-3451

The role of IκB kinase (IKK)-induced proteolysis of NF-κB1 p105 in innate immune signaling was investigated using macrophages from Nfkb1(SSAA/SSAA) mice, in which the IKK target serines on p105 are mutated to alanines. We found that the IKK/p105 signaling pathway was essential for TPL-2 kinase activation of extracellular signal-regulated kinase (ERK) mitogen-activate protein (MAP) kinase and modulated the activation of NF-κB. The Nfkb1(SSAA) mutation prevented the agonist-induced release of TPL-2 from its inhibitor p105, which blocked activation of ERK by lipopolysaccharide (LPS), tumor necrosis factor (TNF), CpG, tripalmitoyl-Cys-Ser-Lys (Pam(3)CSK), poly(I · C), flagellin, and R848. The Nfkb1(SSAA) mutation also prevented LPS-induced processing of p105 to p50 and reduced p50 levels, in addition to decreasing the nuclear translocation of RelA and cRel. Reduced p50 in Nfkb1(SSAA/SSAA) macrophages significantly decreased LPS induction of the IκBζ-regulated Il6 and Csf2 genes. LPS upregulation of Il12a and Il12b mRNAs was also impaired although specific blockade of TPL-2 signaling increased expression of these genes at late time points. Activation of TPL-2/ERK signaling by IKK-induced p105 proteolysis, therefore, induced a negative feedback loop to downregulate NF-κB-dependent expression of the proinflammatory cytokine interleukin-12 (IL-12). Unexpectedly, TPL-2 promoted soluble TNF production independently of IKK-induced p105 phosphorylation and its ability to activate ERK, which has important implications for the development of anti-inflammatory drugs targeting TPL-2.

Journal article

Risco A, del Fresno C, Mambol A, Alsina-Beauchamp D, MacKenzie KF, Yang H-T, Barber DF, Morcelle C, Arthur JSC, Ley SC, Ardavin C, Cuenda Aet al., 2012, p38γ and p38δ kinases regulate the Toll-like receptor 4 (TLR4)-induced cytokine production by controlling ERK1/2 protein kinase pathway activation., Proc Natl Acad Sci U S A, Vol: 109, Pages: 11200-11205

On the basis mainly of pharmacological experiments, the p38α MAP kinase isoform has been established as an important regulator of immune and inflammatory responses. However, the role of the related p38γ and p38δ kinases has remained unclear. Here, we show that deletion of p38γ and p38δ impaired the innate immune response to lipopolysaccharide (LPS), a Toll-like receptor 4 (TLR4) ligand, by blocking the extracellular signal-regulated kinase 1/2 (ERK1/2) activation in macrophages and dendritic cells. p38γ and p38δ were necessary to maintain steady-state levels of tumor progression locus 2 (TPL2), the MKK kinase that mediates ERK1/2 activation after TLR4 stimulation. TNFα, IL-1β, and IL-10 production were reduced in LPS-stimulated macrophages from p38γ/δ-null mice, whereas IL-12 and IFNβ production increased, in accordance with the known effects of TPL2/ERK1/2 signaling on the induction of these cytokines. Furthermore, p38γ/δ-deficient mice were less sensitive than controls to LPS-induced septic shock, showing lower TNFα and IL-1β levels after challenge. Together, our results establish p38γ and p38δ as key components in innate immune responses.

Journal article

Bi Y, Zeng N, Chanudet E, Huang Y, Hamoudi RA, Liu H, Dong G, Watkins AJ, Ley SC, Zou L, Chen R, Zhu X, Du M-Qet al., 2012, A20 inactivation in ocular adnexal MALT lymphoma, HAEMATOLOGICA-THE HEMATOLOGY JOURNAL, Vol: 97, Pages: 926-930, ISSN: 0390-6078

Journal article

Gantke T, Sriskantharajah S, Sadowski M, Ley SCet al., 2012, IκB kinase regulation of the TPL-2/ERK MAPK pathway., Immunol Rev, Vol: 246, Pages: 168-182

Nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) activation play central roles in the induction of gene expression in innate immune cells following pathogen recognition. TPL-2 (tumor progression locus 2) is the MAP 3-kinase component of an ERK-1/2 (extracellular signal-regulated kinase 1/2) MAPK pathway activated by Toll-like receptor and tumor necrosis factor receptor family stimulation. In this review, we discuss results obtained from our laboratory and others that show that TPL-2 signaling function is directly controlled by the inhibitor of NF-κB (IκB) kinase (IKK) complex. Significantly, this means that IKK controls both NF-κB and ERK activation. TPL-2 is stoichiometrically complexed with the NF-κB inhibitory protein, NF-κB1 p105, and the ubiquitin-binding protein ABIN-2, both of which are required to maintain TPL-2 protein stability. Binding to p105 also prevents TPL-2 from phosphorylating MEK (MAPK/ERK kinase), its downstream target. Agonist stimulation releases TPL-2 from p105-inhibition by IKK-mediated phosphorylation of p105, which triggers degradation of p105 by the proteasome. This facilitates TPL-2 phosphorylation of MEK, in addition to liberating p105-associated Rel subunits to translocate into the nucleus. We also examine evidence that TPL-2 is critical for the induction of inflammation and may play a role in development and/or progression of certain types of cancer. Finally, we consider the potential of TPL-2 as an anti-inflammatory drug target for treatment of certain types of inflammatory disease and cancer.

Journal article

Cornish GH, Tung SL, Marshall D, Ley S, Seddon BPet al., 2012, Tissue specific deletion of inhibitor of kappa B kinase 2 with OX40-Cre reveals the unanticipated expression from the OX40 locus in skin epidermis., PLoS One, Vol: 7

NF-κB signalling plays an essential role in T cell activation and generation of regulatory and memory populations in vivo. In the present study, we aimed to investigate the role of NF-κB signalling in post-activation T cells using tissue specific ablation of inhibitor of kappa-B kinase 2 expression, an important component of the inhibitor of kappa-B kinase complex in canonical NF-κB signalling. The OX40 antigen is expressed on activated T cells. Therefore, we used previously described mouse strain expressing Cre recombinase from the endogenous OX40 locus. Ablation of IKK2 expression using OX40(Cre) mice resulted in the development of an inflammatory response in the skin epidermis causing wide spread skin lesions. The inflammatory response was characterised by extensive leukocytic infiltrate in skin tissue, hyperplasia of draining lymph nodes and widespread activation in the T cell compartment. Surprisingly, disease development did not depend on T cells but was rather associated with an unanticipated expression of Cre in skin epidermis, and activation of the T cell compartment did not require Ikbk2 deletion in T cells. Employment of Cre reporter strains revealed extensive Cre activity in skin epidermis. Therefore, development of skin lesions was rather more likely explained by deletion of Ikbk2 in skin keratinocytes in OX40(Cre) mice.

Journal article

Gao L, Coope H, Grant S, Ma A, Ley SC, Harhaj EWet al., 2011, ABIN1 protein cooperates with TAX1BP1 and A20 proteins to inhibit antiviral signaling., J Biol Chem, Vol: 286, Pages: 36592-36602

Upon virus infection, the innate immune response provides the first line of protection and rapidly induces type I interferons (IFNα/β), which mediate potent antiviral effects. To maintain homeostasis and prevent autoimmunity, IFN production is tightly regulated; however, the mechanisms of negative regulation are poorly understood. Herein, we demonstrate that the A20 binding inhibitor of NF-κB 1 (ABIN1) is a novel negative regulator of antiviral signaling. Overexpression of ABIN1 inhibited IFN-β promoter activation in response to virus infection or poly(I:C) transfection, whereas siRNA-mediated knockdown of ABIN1 enhanced IFN-β production upon virus infection. ABIN1 interacted with the A20 regulatory molecule TAX1BP1 and was essential for the recruitment of TAX1BP1 and A20 to the noncanonical IκB kinases TBK1 and IKKi in response to poly(I:C) transfection. ABIN1 and TAX1BP1 together disrupted the interactions between the E3 ubiquitin ligase TRAF3 and TBK1/IKKi to attenuate lysine 63-linked polyubiquitination of TBK1/IKKi. Finally, an intact ubiquitin binding domain of ABIN1 was essential for ABIN1 to interact with TBK1/IKKi and inhibit IFN-β production upon poly(I:C) transfection or virus infection. Together, these results suggest that ABIN1 requires its ubiquitin binding domain and cooperates with TAX1BP1 and A20 to restrict antiviral signaling.

Journal article

Yang HT, Wang Y, Zhao X, Demissie E, Papoutsopoulou S, Mambole A, O'Garra A, Tomczak MF, Erdman SE, Fox JG, Ley SC, Horwitz BHet al., 2011, NF-κB1 inhibits TLR-induced IFN-β production in macrophages through TPL-2-dependent ERK activation., Journal of Immunology, Vol: 4, Pages: 1989-1996

Although NF-κB1 p50/p105 has critical roles in immunity, the mechanism by which NF-κB1 regulates inflammatory responses is unclear. In this study, we analyzed the gene expression profile of LPS-stimulated Nfkb1(-/-) macrophages that lack both p50 and p105. Deficiency of p50/p105 selectively increased the expression of IFN-responsive genes, which correlated with increased IFN-β expression and STAT1 phosphorylation. IFN Ab-blocking experiments indicated that increased STAT1 phosphorylation and expression of IFN-responsive genes observed in the absence of p50/p105 depended upon autocrine IFN-β production. Markedly higher serum levels of IFN-β were observed in Nfkb1(-/-) mice than in wild-type mice following LPS injection, demonstrating that Nfkb1 inhibits IFN-β production under physiological conditions. TPL-2, a mitogen-activated protein kinase kinase kinase stabilized by association with the C-terminal ankyrin repeat domain of p105, negatively regulates LPS-induced IFN-β production by macrophages via activation of ERK MAPK. Retroviral expression of TPL-2 in Nfkb1(-/-) macrophages, which are deficient in endogenous TPL-2, reduced LPS-induced IFN-β secretion. Expression of the C-terminal ankyrin repeat domain of p105 in Nfkb1(-/-) macrophages, which rescued LPS activation of ERK, also inhibited IFN-β expression. These data indicate that p50/p105 negatively regulates LPS-induced IFN signaling in macrophages by stabilizing TPL-2, thereby facilitating activation of ERK.

Journal article

Gantke T, Sriskantharajah S, Ley SC, 2011, Regulation and function of TPL-2, an IκB kinase-regulated MAP kinase kinase kinase., Cell Res, Vol: 21, Pages: 131-145

The IκB kinase (IKK) complex plays a well-documented role in innate and adaptive immunity. This function has been widely attributed to its role as the central activator of the NF-κB family of transcription factors. However, another important consequence of IKK activation is the regulation of TPL-2, a MEK kinase that is required for activation of ERK-1/2 MAP kinases in myeloid cells following Toll-like receptor and TNF receptor stimulation. In unstimulated cells, TPL-2 is stoichiometrically complexed with the NF-κB inhibitory protein NF-κB1 p105, which blocks TPL-2 access to its substrate MEK, and the ubiquitin-binding protein ABIN-2 (A20-binding inhibitor of NF-κB 2), both of which are required to maintain TPL-2 protein stability. Following agonist stimulation, the IKK complex phosphorylates p105, triggering its K48-linked ubiquitination and degradation by the proteasome. This releases TPL-2 from p105-mediated inhibition, facilitating activation of MEK, in addition to modulating NF-κB activation by liberating associated Rel subunits for translocation into the nucleus. IKK-induced proteolysis of p105, therefore, can directly regulate both NF-κB and ERK MAP kinase activation via NF-κB1 p105. TPL-2 is critical for production of the proinflammatory cytokine TNF during inflammatory responses. Consequently, there has been considerable interest in the pharmaceutical industry to develop selective TPL-2 inhibitors as drugs for the treatment of TNF-dependent inflammatory diseases, such as rheumatoid arthritis and inflammatory bowel disease. This review summarizes our current understanding of the regulation of TPL-2 signaling function, and also the complex positive and negative roles of TPL-2 in immune and inflammatory responses.

Journal article

Verstrepen L, Verhelst K, van Loo G, Carpentier I, Ley SC, Beyaert Ret al., 2010, Expression, biological activities and mechanisms of action of A20 (TNFAIP3)., Biochem Pharmacol, Vol: 80, Pages: 2009-2020

A20 (also known as TNFAIP3) is a cytoplasmic protein that plays a key role in the negative regulation of inflammation and immunity. Polymorphisms in the A20 gene locus have been identified as risk alleles for multiple human autoimmune diseases, and A20 has also been proposed to function as a tumor suppressor in several human B-cell lymphomas. A20 expression is strongly induced by multiple stimuli, including the proinflammatory cytokines TNF and IL-1, and microbial products that trigger pathogen recognition receptors, such as Toll-like receptors. A20 functions in a negative feedback loop, which mediates its inhibitory functions by downregulating key proinflammatory signaling pathways, including those controlling NF-κB- and IRF3-dependent gene expression. Activation of these transcription factors is controlled by both K48- and K63- polyubiquitination of upstream signaling proteins, respectively triggering proteasome-mediated degradation or interaction with other signaling proteins. A20 turns off NF-κB and IRF3 activation by modulating both types of ubiquitination. Induction of K48-polyubiquitination by A20 involves its C-terminal zinc-finger ubiquitin-binding domain, which may promote interaction with E3 ligases, such as Itch and RNF11 that are involved in mediating A20 inhibitory functions. A20 is thought to promote de-ubiquitination of K63-polyubiquitin chains either directly, due to its N-terminal deubiquitinase domain, or by disrupting the interaction between E3 and E2 enzymes that catalyze K63-polyubiquitination. A20 is subject to different mechanisms of regulation, including phosphorylation, proteolytic processing, and association with ubiquitin binding proteins. Here we review the expression and biological activities of A20, as well as the underlying molecular mechanisms.

Journal article

Mieulet V, Yan L, Choisy C, Sully K, Procter J, Kouroumalis A, Krywawych S, Pende M, Ley SC, Moinard C, Lamb RFet al., 2010, TPL-2-mediated activation of MAPK downstream of TLR4 signaling is coupled to arginine availability., Sci Signal, Vol: 3

The innate immune response is influenced by the nutrient status of the host. Mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinase 1 (ERK1) and ERK2, are activated after the stimulation of macrophages with bacterial lipopolysaccharide (LPS) and are necessary for the optimal production of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha). We uncovered a role for the extracellular nutrient arginine in the activation of ERK1/2 in LPS-stimulated macrophages. Arginine facilitated the activation of MAPKs by preventing the dephosphorylation and inactivation of the MAPK kinase kinase tumor-promoting locus 2 (TPL-2). Starvation of mice decreased the concentration of arginine in the plasma and impaired the activation of ERK1/2 by LPS. Supplementation of starved mice with arginine promoted the subsequent activation of ERK1/2 and the production of TNF-alpha in response to LPS. Thus, arginine is critical for two aspects of the innate immune response in macrophages: It is the precursor used in the generation of the antimicrobial mediator nitric oxide, and it facilitates MAPK activation and consequently cytokine production.

Journal article

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