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
Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
School of Veterinary Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Bearsden Road, Glasgow G61 1QH, United Kingdom.
Proc Natl Acad Sci U S A. 2017 Apr 25;114(17):E3481-E3489. doi: 10.1073/pnas.1702367114. Epub 2017 Apr 12.
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.
人们普遍认为，TRAF6在体内的重要作用是生成激活“主”蛋白激酶TAK1所需的赖氨酸63连接的泛素（K63-Ub）链。在此，我们报告TRAF6 E3连接酶活性对人细胞中IL-1依赖性K63-Ub链的形成、TAK1激活或IL-8产生有促进作用，但并非必不可少，因为Pellino1和Pellino2会在表达E3连接酶失活的TRAF6突变体的细胞中生成信号传导所需的K63-Ub链。在TRAF6/Pellino1/Pellino2三敲除（KO）细胞中，IL-1诱导的K63-Ub链形成以及IRAK1、IRAK4和MyD88的泛素化被消除，但在TRAF6 KO或Pellino1/2双敲除细胞中未被消除。E3连接酶失活的TRAF6突变体的重新表达部分恢复了TRAF6 KO细胞中的IL-1信号传导，但在TRAF6/Pellino1/Pellino2三敲除细胞中未恢复。Pellino1生成的K63-Ub链在体外激活TAK1复合物的效率与TRAF6生成的K63-Ub链相似。在表达E3连接酶失活的TRAF6[L74H]突变体的敲入小鼠的原代巨噬细胞中，TLR信号传导的早期阶段和TLR依赖性IL-10分泌（由IRAKs 1和2控制）仅适度降低，但IL-6、IL-12和TNFα的晚期产生（仅由假激酶IRAK2控制）被消除。TRAF6[L74H]细胞和野生型细胞中巨噬细胞中RANKL诱导的信号传导以及骨髓向破骨细胞的分化相似，这解释了为什么TRAF6[L74H]小鼠的骨骼结构和牙齿正常，这与TRAF6 KO小鼠不同。我们确定了TRAF6的两个与其E3连接酶活性无关的重要作用。
