Jang HD, Hwang HZ, Kim HS, Lee SY

In its RING domain, tumor necrosis factor receptor-associated factor 6 (TRAF6) has ubiquitin E3 ligase activity that facilitates the formation of lysine 63-linked polyubiquitin chains. This activity is required to activate nuclear factor ?-light-chain-enhancer of activated B cells (NF-?B) and plays an important role in the I?B kinase (IKK) complex.An ...

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in vitro ubiquitination assay was used to establish whether c-Cbl could promote TRAF6 ubiquitination. We assessed direct binding and performed fine mapping between c-Cbl and TRAF6 based on the results of an immunoprecipitation assay with cultured 293?T cells. The luciferase reporter assay was applied to establish if c-Cbl-mediated ubiquitination affected NF-?B activation after stimulus from various TRAF-mediated signals: tumor necrosis factor-? (TNF-?), receptor activator of NF-?B ligand (RANKL), and interleukin-1? (IL-1?). An in vivo ubiquitination assay was performed using endogenous immunoprecipitation of TRAF6 in bone marrow macrophages (BMMs) and osteoclasts.Here, we report on a form of TRAF6 ubiquitination that is mediated by c-Cbl, leading to the formation of lysine 48-linked polyubiquitin chains. The NF-?B activity induced by RANKL and IL-1? treatment is inhibited when c-Cbl is overexpressed, while the NF-?B activity induced by TNF? treatment is not. c-Cbl inhibits NF-?B activity mediated by TRAF6, but not by TRAF2. These findings show that c-Cbl ubiquitin ligase activity is essential for TRAF6 ubiquitination and negative regulation of NF-?B activity. Fine mapping revealed that the proline-rich domain of c-Cbl is critical for interaction with TRAF6. Stimulation with RANKL or interferon-? (IFN-?) caused c-Cbl to bind to polyubiquitinated TRAF6.These findings indicate that the interaction of TRAF6 with c-Cbl causes lysine 48-linked polyubiquitination for both negative feedback regulation and signaling cross-talk between RANKL and IFN-?.

Date: May. 28, 2019

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