title: Roles of the TRAF6 and Pellino E3 ligases in MyD88 and RANKL signaling.
abstract: 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.
authors.affiliation: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
authors.forename: Sam
authors.initials: S
authors.lastname: Strickson
authors.affiliation: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
authors.forename: Christoph H
authors.initials: CH
authors.lastname: Emmerich
authors.affiliation: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
authors.forename: Eddy T H
authors.initials: ETH
authors.lastname: Goh
authors.affiliation: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
authors.forename: Jiazhen
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
authors.forename: Ian R
authors.identifier: 0000-0002-7501-6504
authors.initials: IR
authors.lastname: Kelsall
authors.affiliation: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
authors.forename: Thomas
authors.initials: T
authors.lastname: Macartney
authors.affiliation: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
authors.forename: C James
authors.initials: CJ
authors.lastname: Hastie
authors.affiliation: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
authors.forename: Axel
authors.initials: A
authors.lastname: Knebel
authors.affiliation: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
authors.forename: Mark
authors.initials: M
authors.lastname: Peggie
authors.affiliation: School of Veterinary Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Bearsden Road, Glasgow G61 1QH, United Kingdom.
authors.forename: Francesco
authors.initials: F
authors.lastname: Marchesi
authors.affiliation: Division of Cell Signalling and Immunology, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
authors.forename: J Simon C
authors.initials: JSC
authors.lastname: Arthur
authors.affiliation: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom; p.cohen@dundee.ac.uk.
authors.forename: Philip
authors.initials: P
authors.lastname: Cohen
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D020930:MAP Kinase Kinase Kinases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D020125:Mutation, Missense; D053594:Myeloid Differentiation Factor 88 / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D025821:Polyubiquitin / Q000235:genetics / Q000378:metabolism; D053245:RANK Ligand / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D048029:TNF Receptor-Associated Factor 6 / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D000099151:MAP Kinase Kinase Kinase 7
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D020930:MAP Kinase Kinase Kinases; D053594:Myeloid Differentiation Factor 88; D009687:Nuclear Proteins; D025821:Polyubiquitin; D053245:RANK Ligand; D048029:TNF Receptor-Associated Factor 6; D044767:Ubiquitin-Protein Ligases; D000099151:MAP Kinase Kinase Kinase 7; C507779:MYD88 protein, human; C507780:Myd88 protein, mouse; C485961:Peli2 protein, mouse; C505688:TNFSF11 protein, human; C000604754:Tifab protein, human; C485959:PELI1 protein, human; C418046:Peli1 protein, mouse
keywords: IL-1; Pellino; TAK1; TRAF6; Ubiquitin