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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.

Author information

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

Abstract

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.

KEYWORDS:

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

PMID:
31123462
PMCID:
PMC6518801
DOI:
10.1186/s11658-019-0156-y
[Indexed for MEDLINE]
Free PMC Article

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