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Cell Mol Biol Lett. 2019 May 14;24:29. doi: 10.1186/s11658-019-0156-y. eCollection 2019.

C-Cbl negatively regulates TRAF6-mediated NF-κB activation by promoting K48-linked polyubiquitination of TRAF6.

Jang HD1,2,3, Hwang HZ4, Kim HS1,2,3,5, Lee SY6.

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1National Leading Laboratory for Stem Cell Research, Seoul National University College of Medicine, Seoul, South Korea.
2Korea Research-Driven Hospital, Biomedical Research Institute, Seoul National University Hospital, Seoul, South Korea.
3Strategic Center of Cell & Bio Therapy, Seoul National University Hospital, Seoul, South Korea.
4Department of Biotechnology, The Catholic University of Korea, Bucheon, South Korea.
5Cardiovascular Center & Department of Internal Medicine, Seoul National University Hospital, Seoul, South Korea.
6Department of Life Science and the Research Center for Cellular Homeostasis, Ewha Woman's University, Seoul, South Korea.



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 κ-light-chain-enhancer of activated B cells (NF-κB) and plays an important role in the IκB kinase (IKK) complex.


An 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-κB activation after stimulus from various TRAF-mediated signals: tumor necrosis factor-α (TNF-α), receptor activator of NF-κB ligand (RANKL), and interleukin-1β (IL-1β). 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-κB activity induced by RANKL and IL-1β treatment is inhibited when c-Cbl is overexpressed, while the NF-κB activity induced by TNFα treatment is not. c-Cbl inhibits NF-κB 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-κB activity. Fine mapping revealed that the proline-rich domain of c-Cbl is critical for interaction with TRAF6. Stimulation with RANKL or interferon-γ (IFN-γ) 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-γ.


C-Cbl; E3 ligase; Tumor necrosis factor receptor-associated factor 6; Ubiquitin

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