Activation of the canonical IKK complex by K63/M1-linked hybrid ubiquitin chains

Emmerich, Christoph H.; Ordureau, Alban; Strickson, Sam; Arthur, J. Simon C.; Pedrioli, Patrick G. A.; Komander, David; Cohen, Philip

doi:10.1073/pnas.1314715110

Cited by 382 publications

(470 citation statements)

References 35 publications

(40 reference statements)

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“…IKKβ, which binds to NEMO, was used as the loading control. 30). Importantly, the IL-1β-dependent formation of Ub-IRAK1, Ub-IRAK4, or Ub-MyD88 was still robust in Pellino1/2 double-KO cells and TRAF6 KO IL-1R* cells, but abolished in TRAF6/Pellino1/2 triple-KO IL-1R* cells (Fig.…”

Section: Pellino1 and Pellino2 Generate The K63-ub Chains Required Fomentioning

confidence: 78%

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Roles of the TRAF6 and Pellino E3 ligases in MyD88 and RANKL signaling

Strickson

Emmerich

Goh

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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It is widely accepted that the essential role of TRAF6 in vivo is to generate the Lys63-linked ubiquitin (K63-Ub) chains needed to activate the "master" protein kinase TAK1. Here, we report that TRAF6 E3 ligase activity contributes to but is not essential for the IL-1-dependent formation of K63-Ub chains, TAK1 activation, or IL-8 production in human cells, because Pellino1 and Pellino2 generate the K63-Ub chains required for signaling in cells expressing E3 ligaseinactive TRAF6 mutants. The IL-1-induced formation of K63-Ub chains and ubiquitylation of IRAK1, IRAK4, and MyD88 was abolished in TRAF6/Pellino1/Pellino2 triple-knockout (KO) cells, but not in TRAF6 KO or Pellino1/2 double-KO cells. The reexpression of E3 ligaseinactive TRAF6 mutants partially restored IL-1 signaling in TRAF6 KO cells, but not in TRAF6/Pellino1/Pellino2 triple-KO cells. Pellino1-generated K63-Ub chains activated the TAK1 complex in vitro with similar efficiently to TRAF6-generated K63-Ub chains. The early phase of TLR signaling and the TLR-dependent secretion of IL-10 (controlled by IRAKs 1 and 2) was only reduced modestly in primary macrophages from knockin mice expressing the E3 ligase-inactive TRAF6[L74H] mutant, but the late-phase production of IL-6, IL-12, and TNFα (controlled only by the pseudokinase IRAK2) was abolished. RANKLinduced signaling in macrophages and the differentiation of bone marrow to osteoclasts was similar in TRAF6[L74H] and wild-type cells, explaining why the bone structure and teeth of the TRAF6[L74H] mice was normal, unlike TRAF6 KO mice. We identify two essential roles of TRAF6 that are independent of its E3 ligase activity.T NF receptor-associated factor 6 (TRAF6) is essential for many biological processes (1). These include the myeloid differentiation primary response gene 88 (MyD88) signaling network of the innate immune system, RANK ligand (RANKL)-dependent signaling and osteoclast formation, lymph node organogenesis (2), and the development of hair follicles, sweat glands, and sebaceous glands (3). TRAF6 expression is also needed for CD40 signaling in B cells (4), the maturation and development of dendritic cells (5), and the regulation of T-cell function (6, 7).In innate immunity, nearly all Toll-like receptors (TLRs), as well as the receptors of the interleukin 1 (IL-1) family of cytokines, initiate signaling by recruiting the adaptor protein MyD88. This is followed by the interaction of IL-1-receptor (IL-R)-associated kinase 4 (IRAK4) with MyD88 and then the interaction of other IRAK family members with IRAK4, to form an oligomeric complex, termed the Myddosome (8, 9). IRAK1 and IRAK2 can then interact with TRAF6 (10, 11) and induce TRAF6 dimerization (12), which triggers the activation of its E3 ligase activity (13).TRAF6 catalyzes the formation of Lys63-linked ubiquitin (K63-Ub) chains in vitro in the presence of Ubc13-Uev1a (also called UBE2N-UBE2V1), an E2 conjugating enzyme that directs the formation of this type of ubiquitin linkage (14, 15). Although truncated forms of TRAF6 lacking the really...

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Section: Pellino1 and Pellino2 Generate The K63-ub Chains Required Fomentioning

confidence: 78%

“…Human IL-1β (DU8685) (30) and mouse IL-1α (DU46302) were expressed in E. coli and purified by the PPT of the MRC-PPU. IL-1α was expressed as a GST-fusion protein separated by a PreScission protease cleavage site and purified on glutathione-Sepharose (GE Healthcare).…”

Section: Methodsmentioning

confidence: 99%

Roles of the TRAF6 and Pellino E3 ligases in MyD88 and RANKL signaling

Strickson

Emmerich

Goh

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…These MKKs phosphorylate and activate p38 and JNK, respectively, initiating AP-1-mediated transcription. Second, TAK1 provides a priming phosphorylation to IKKβ, which colocalizes with TAK1 to M1-poly-Ub chains generated by TRAF6 upon TLR activation (2,3). Activation of IKKβ leads to the activation IKKα, degradation of IκBα, and finally, activation of NF-κB-driven transcription.…”

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confidence: 99%

Innate immunity kinase TAK1 phosphorylates Rab1 on a hotspot for posttranslational modifications by host and pathogen

Levin

Hertz

Burlingame

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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TGF-β activated kinase 1 (TAK1) is a critical signaling hub responsible for translating antigen binding signals to immune receptors for the activation of the AP-1 and NF-κB master transcriptional programs. Despite its importance, known substrates of TAK1 are limited to kinases of the MAPK and IKK families and include no direct effectors of biochemical processes. Here, we identify over 200 substrates of TAK1 using a chemical genetic kinase strategy. We validate phosphorylation of the dynamic switch II region of GTPase Rab1, a mediator of endoplasmic reticulum to Golgi vesicular transport, at T75 to be regulated by TAK1 in vivo. TAK1 preferentially phosphorylates the inactive (GDP-bound) state of Rab1. Phosphorylation of Rab1 disrupts interaction with GDP dissociation inhibitor 1 (GDI1), but not guanine exchange factor (GEF) or GTPaseactivating protein (GAP) enzymes, and is exclusive to membranelocalized Rab1, suggesting phosphorylation may stimulate Rab1 membrane association. Furthermore, we found phosphorylation of Rab1 at T75 to be essential for Rab1 function. Previous studies established that the pathogen Legionella pneumophila is capable of hijacking Rab1 function through posttranslational modifications of the switch II region. Here, we present evidence that Rab1 is regulated by the host in a similar fashion, and that the innate immunity kinase TAK1 and Legionella effectors compete to regulate Rab1 by switch II modifications during infection.

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“…Furthermore, MyD88, IRAK1/4, and TAB2/3 are also modified and activated by K63-linked polyubiquitin chains. Such chains were recently described as substrates for M1-polyubiquitination by HOIP, resulting in hybrid chains that may connect the MyD88/ IRAK and TAK1/IKK signaling apparatus (Emmerich et al, 2013). TLR3 signaling is mediated by TIR domain-containing adaptor-inducing IFN-β (TRIF).…”

Section: Tlr Signalingmentioning

confidence: 99%

“…Effector molecule production is initiated by immune sentinels known as pattern recognition receptors (PRRs), which screen the intra-and extracellular environment for molecular motifs uniquely associated with pathogens. PRR engagement transduces pro-immune signals into the nucleus via protein signaling cascades that self-limit to mitigate autoimmunity as the infection clears (Crampton et al, 2012). Protein posttranslational modifications (PTMs) form part of this exquisite system of regulation, with ubiquitin and ubiquitin-like modifications key among them.…”

mentioning

confidence: 99%