TRAF6 autoubiquitination-independent activation of the NFkappaB and MAPK pathways in response to IL-1 and RANKL.
The adapter protein TRAF6 is critical for mediating signal transduction from members of the IL-1R/TLR and TNFR superfamilies. The TRAF6 RING finger domain functions as an ubiquitin E3 ligase capable of generating non-degradative K63-linked ubiquitin chains. It is believed that these chains serve as docking sites for formation of signaling complexes, and that K63-linked autoubiquitination of TRAF6 is essential for formation and activation of a complex involving the kinase TAK1 and its adapters, TAB1 and TAB2. In order to assess independently the E3 ligase and ubiquitin substrate functions of TRAF6, we generated, respectively, RING domain and complete lysine-deficient TRAF6 mutants. We found that while the TRAF6 RING domain is required for activation of TAK1, it is dispensable for interaction between TRAF6 and the TAK1-TAB1-TAB2 complex. Likewise, lysine-deficient TRAF6 was found to interact with the TAK1-TAB1-TAB2 complex, but surprisingly was also found to be fully competent to activate TAK1, as well as NFkappaB and AP-1 reporters. Furthermore, lysine-deficient TRAF6 rescued IL-1-mediated NFkappaB and MAPK activation, as well as IL-6 elaboration in retrovirally-rescued TRAF6-deficient fibroblasts. Lysine-deficient TRAF6 also rescued RANKL-mediated NFkappaB and MAPK activation, and osteoclastogenesis in retrovirally-rescued TRAF6-deficient bone marrow macrophages. While incapable of being ubiquitinated itself, we demonstrate that lysine-deficient TRAF6 remains competent to induce ubiquitination of IKKgamma/NEMO. Further, this NEMO modification contributes to TRAF6-mediated activation of NFkappaB. Collectively, our results suggest that while TRAF6 autoubiquitination may serve as a marker of activation, it is unlikely to underpin RING finger-dependent TRAF6 function.
Walsh
Matthew C
MC
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.
Kim
Gregory K
GK
Maurizio
Paul L
PL
Molnar
Elizabeth E
EE
Choi
Yongwon
Y
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000818:Animals; D027081:DNA Gyrase / Q000737:chemistry; D006801:Humans; D007375:Interleukin-1 / Q000378:metabolism*; D008239:Lysine / Q000737:chemistry; D020935:MAP Kinase Signaling System; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism*; D010010:Osteoclasts / Q000378:metabolism; D017434:Protein Structure, Tertiary; D053245:RANK Ligand / Q000378:metabolism*; D048029:TNF Receptor-Associated Factor 6 / Q000737:chemistry / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D007375:Interleukin-1; D016328:NF-kappa B; D053245:RANK Ligand; C486300:TAB1 protein, human; C410585:TAB2 protein, human; D048029:TNF Receptor-Associated Factor 6; D025801:Ubiquitin; D027081:DNA Gyrase; D008239:Lysine