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

Hyun-Duk Jang; Hye Zin Hwang; Hyo-Soo Kim; Soo Young Lee

doi:10.1186/s11658-019-0156-y

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

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

Authors

Hyun-Duk Jang^{1

2

3}, Hye Zin Hwang⁴, Hyo-Soo Kim^{1

2

3

5}, Soo Young Lee⁶

Affiliations

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.

MeSH terms

HEK293 Cells
Humans
Interferon-gamma / pharmacology
Lysine / metabolism*
NF-kappa B / metabolism*
Polyubiquitin / metabolism*
Protein Binding
Proto-Oncogene Proteins c-cbl / chemistry
Proto-Oncogene Proteins c-cbl / metabolism*
RANK Ligand / pharmacology
RING Finger Domains
TNF Receptor-Associated Factor 6 / chemistry
TNF Receptor-Associated Factor 6 / metabolism*
Ubiquitination* / drug effects

Substances

NF-kappa B
RANK Ligand
TNF Receptor-Associated Factor 6
Polyubiquitin
Interferon-gamma
Proto-Oncogene Proteins c-cbl
Lysine