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

Hyun Duk Jang, Hye Zin Hwang, Hyo Soo Kim, Soo Young Lee

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18 Scopus citations


Background: 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. Methods: 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. Results: 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. Conclusions: 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.

Original languageEnglish
Article number29
JournalCellular and Molecular Biology Letters
Issue number1
StatePublished - 14 May 2019

Bibliographical note

Funding Information:
This study was supported by the Korean Health Technology R&D Project (HI-14 C-1277) through the Korean Health Industry Development Institute (KHIDI), and funded by the Ministry of Health & Welfare (MHW) and by the National Research Foundation (NRF) of Korea (grant nos 2012R1A5A1048236 and NRF-2017R1D1A1A02017804).

Publisher Copyright:
© 2019, The Author(s).


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


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