Abstract
BackgroundIn 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.MethodsAn 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.ResultsHere, 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.ConclusionsThese 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.
Highlights
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
C-Cbl promotes TRAF6 ubiquitination and inhibits TRAF6-mediated nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB reporter vector (κB)) activation TRAF6 is degraded by Receptor activator of NF-κB ligand (RANKL) stimuli, and degradation of TRAF6 is protected by the proteasome inhibitor MG132 [21–23]. c-Cbl is a known interacting partner of TRAF6 [5, 15, 19] and the famous RING-type E3 ligase in receptor tyrosine kinase signaling, and has recently been reported in other signaling systems [10, 12, 20, 23]
To test whether c-Cbl could promote TRAF6 ubiquitination, TRAF6 was immunoprecipitated from 293 T cells transfected with c-Cbl or controls
Summary
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. NF-κB activation, which affects a wide variety of cellular mechanisms This signal has been implicated in inflammation, immune regulation, bone homeostasis, and development. There are six known mammalian TRAF proteins that show direct or indirect interaction with members of the TNFR superfamily [3] They all share a highly conserved c-terminal domain that is responsible for the interactions between TRAFs or interactions with other proteins. The N-terminal domain of TRAFs, which consists of one or more zinc finger domains, enables the activation of signaling cascades
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