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.
Cell Mol Biol Lett. 2019 May 14;24:29. doi: 10.1186/s11658-019-0156-y. eCollection 2019.
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 -light-chain-enhancer of activated B cells (NF-B) 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-B activation after stimulus from various TRAF-mediated signals: tumor necrosis factor- (TNF-), receptor activator of NF-B ligand (RANKL), and interleukin-1 (IL-1). 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-B activity induced by RANKL and IL-1 treatment is inhibited when c-Cbl is overexpressed, while the NF-B activity induced by TNFα treatment is not. c-Cbl inhibits NF-B 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-B activity. Fine mapping revealed that the proline-rich domain of c-Cbl is critical for interaction with TRAF6. Stimulation with RANKL or interferon- (IFN-) 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-.
肿瘤坏死因子受体相关因子 6（TRAF6）在其 RING 结构域中具有泛素 E3 连接酶活性，有助于形成赖氨酸 63 连接的多泛素链。这种活性对于激活核因子-κB 轻链增强子的 B 细胞（NF-κB）是必需的，并在 IκB 激酶（IKK）复合物中发挥重要作用。
采用体外泛素化测定法确定 c-Cbl 是否能促进 TRAF6 的泛素化。我们评估了直接结合，并根据用培养的 293T 细胞进行免疫沉淀测定的结果，对 c-Cbl 和 TRAF6 之间进行精细定位。使用来自各种 TRAF 介导的信号（肿瘤坏死因子-（TNF-），受体激活剂的核因子-κB 配体（RANKL）和白细胞介素-1（IL-1））的刺激，应用荧光素酶报告基因测定法来确定 c-Cbl 介导的泛素化是否影响 NF-κB 的激活。使用骨髓巨噬细胞（BMMs）和破骨细胞中 TRAF6 的内源性免疫沉淀进行体内泛素化测定。
在这里，我们报告了一种由 c-Cbl 介导的 TRAF6 泛素化形式，导致形成赖氨酸 48 连接的多泛素链。当 c-Cbl 过表达时，RANKL 和 IL-1 处理诱导的 NF-κB 活性受到抑制，而 TNFα 处理诱导的 NF-κB 活性不受抑制。c-Cbl 抑制 TRAF6 介导的 NF-κB 活性，但不抑制 TRAF2 介导的 NF-κB 活性。这些发现表明 c-Cbl 泛素连接酶活性对于 TRAF6 泛素化和 NF-κB 活性的负调控是必需的。精细定位显示 c-Cbl 的脯氨酸丰富结构域对于与 TRAF6 的相互作用至关重要。RANKL 或干扰素-（IFN-）的刺激导致 c-Cbl 与多泛素化的 TRAF6 结合。
这些发现表明 TRAF6 与 c-Cbl 的相互作用导致赖氨酸 48 连接的多泛素化，用于 RANKL 和 IFN-之间的负反馈调节和信号交叉对话。
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