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

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 114; no. 17; pp. E3481 - E3489
• Main Authors: Strickson, Sam, Emmerich, Christoph H., Goh, Eddy T. H., Zhang, Jiazhen, Kelsall, Ian R., Macartney, Thomas, Hastie, C. James, Knebel, Axel, Peggie, Mark, Marchesi, Francesco, Arthur, J. Simon C., Cohen, Philip
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 25.04.2017
• Series: PNAS Plus
• Subjects: Amino Acid Substitution; Animals; Biocompatibility; Biological Sciences; Biomedical materials; Bone marrow; Cells; Differentiation; Gene Knockdown Techniques; HEK293 Cells; Humans; In vitro methods and tests; Interleukin 1; Interleukin 10; Interleukin 12; Interleukin 6; Interleukin 8; Intracellular Signaling Peptides and Proteins; IRAK protein; Kinases; Macrophages; MAP Kinase Kinase Kinases - genetics; MAP Kinase Kinase Kinases - metabolism; Mice; Mice, Knockout; Mutants; Mutation; Mutation, Missense; MyD88 protein; Myeloid Differentiation Factor 88 - genetics; Myeloid Differentiation Factor 88 - metabolism; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Osteoclastogenesis; Osteoclasts; PNAS Plus; Polyubiquitin - genetics; Polyubiquitin - metabolism; Protein kinase; RANK Ligand - genetics; RANK Ligand - metabolism; Rodents; Signal Transduction; TAK1 protein; Teeth; TNF Receptor-Associated Factor 6 - genetics; TNF Receptor-Associated Factor 6 - metabolism; TRAF6 protein; TRANCE protein; Tumor necrosis factor-α; Ubiquitin; Ubiquitin-protein ligase; Ubiquitin-Protein Ligases - genetics; Ubiquitin-Protein Ligases - metabolism; Ubiquitin; TAK1; IL-1; Pellino; TRAF6
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1702367114

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-knockout (KO) cells, but not in TRAF6 KO or Pellino1/2 double-KO cells. The reexpression of E3 ligase-inactive 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. 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.