The Neddylation Modification Of TRAF6 And The Effects On Different Downstream Signal Pathways

A variety of protein post-translational modifications,such as ubiquitination,are essential during the process of normal life activities.Ubiquitination modification not only participates in the activation of multiple signaling pathways such as NF-?B,MAPKs,but also leads to the degradation of target proteins by proteasome in some cases.At the same time,the ubiquitination-like modification—Neddylation has a large similarities with ubiquitination,and has been reported to play a role in the regulation of multiple life activities.Tumor necrosis factor receptor-associated factor 6(TRAF6)can undergo ubiquitination and thus participates in downstream signaling pathways including NF-?B,MAPKs and IRF that are mediated by IL-1R and Toll-like receptors.The normal function of TRAF6 is necessary for the normal conduct of varieties of life activities.However,whether TRAF6 can occur Neddylation is still unknown.To address this,we overexpressed TRAF6 and NEDD8(the small molecule protein which is essential for Neddylation)in HEK293T cells.The immunoprecipitation assay showed that TRAF6 was modified by NEDD8 in a dose-dependent manner.The addition of Neddylation-specific inhibitor MLN4924 significantly inhibited this phenomenon and confirmed that TRAF6 can be modified by NEDD8.In addition,we found that the Neddylation of TRAF6 promoted the ubiquitination of itself,but its ubiquitination did not affect its Neddylation.Moreover,we constructed the different truncation of TRAF6 and found that the lysine at 124,which has been reported to be required for TRAF6 ubiquitination,also required for TRAF6 Neddylation.However,its ubiquitination activity site cysteine at 70 did not required for its Neddylation.In addition,we found that the Neddylation of TRAF6 could inhibit the activation of IRF signal pathway,but activate NF-?B signal pathway at the same time,and has no significant impact on AP-1 by using SEAP analysis.Moreover,the Neddylation of TRAF6 also played a role in different downstream signal pathways mediated by TLR3 ligand,double-stranded RNA analogue--poly(I:C),by inhibiting IRF and promoting NF-?B signaling pathway.In summary,our study demonstrates that,in addition to ubiquitination,TRAF6 can be modified by NEDD8,one ubiquitination-like modification,and this modification contributes to promoting TRAF6's own ubiquitination.Neddylation of TRAF6 can inhibit the IRF signaling pathway while promote the NF-?B signaling pathway mediated by TRAF6.Our findings show for the first time that TRAF6 could be modified by Neddylation and regulate its different regulatory effects on multiple signal pathways,and provide new insights into TRAF6-mediated signal pathways.