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

Sam Strickson, Christoph H. Emmerich, Eddy T. H. Goh, Jiazhen Zhang, Ian R. Kelsall, Thomas Macartney, C. James Hastie, Axel Knebel, Mark Peggie, Francesco Marchesi, J. Simon C. Arthur, Philip Cohen (Lead / Corresponding author)

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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 ligase-inactive 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 knock-out (KO) cells, but not in TRAF6 KO or Pellino1/2 double KO cells. The re-expression 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 knock-in 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. RANKL-induced 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.
Original languageEnglish
Pages (from-to)E3481-E3489
Number of pages9
JournalProceedings of the National Academy of Sciences
Issue number7
Early online date12 Apr 2017
Publication statusPublished - 25 Apr 2017


  • TRAF6
  • TAK1
  • ubiquitin
  • IL-1
  • TLR
  • MyD88


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