Site-specific Lys-63-linked tumor necrosis factor receptor-associated factor 6 auto-ubiquitination is a critical determinant of IκB kinase activation
Site-specific Lys-63-linked tumor necrosis factor receptor-associated factor 6 auto-ubiquitination is a critical determinant of IκB kinase activation
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DOI：
10.1074/jbc.m609503200
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发表时间：
2007-02-09
影响因子：
4.8
通讯作者：
Darnay, Bryant G.
中科院分区：
文献类型：
--
作者：
Lamothe, Betty;Besse, Arnaud;Darnay, Bryant G.
肿瘤坏死因子（TNF）受体相关因子6（TRAF 6）是白细胞介素-1/Toll样受体和TNF受体超家族近端信号传导的关键介质。对TRAF 6缺陷小鼠的分析揭示了TRAF 6在破骨细胞生成中的基本作用;然而，TRAF 6信号转导在这一生物学过程中的分子机制尚不清楚。最近的生物化学证据表明，TRAF 6具有泛素连接酶活性，其控制IKK和NF-κ B的活化。由于这些研究主要基于无细胞系统，TRAF 6的泛素连接酶活性及其自身泛素化在体内启动NF-κ B途径的作用仍然难以捉摸。在这里，我们表明，TRAF 6的一个完整的RING结构域与E2酶Ubc 13/Uev 1A的结合是必需的赖氨酸-63连接的自身泛素化的TRAF 6和它的能力，激活IKK和NF-κ B。此外，TRAF 6的RING突变体消除了其诱导NF-κ B非依赖性破骨细胞分化的受体激活剂和转录因子NFATc 1的核积累的能力。值得注意的是，我们将TRAF 6的自动遍在化位点映射到单个赖氨酸残基，如果突变，TRAF 6将无法激活转化生长因子β激活激酶1和IKK，并导致自发破骨细胞分化。此外，我们提供了生物化学和体内证据表明TRAF 6作为E3直接泛素化NEMO。用各种TRAF 6突变体重建TRAF 6缺陷细胞，我们清楚地证明了TRAF 6 RING结构域和TRAF 6的位点特异性自身泛素化在响应白细胞介素-1时激活IKK的需要。这些数据建立了一个信号级联反应，其中受调节的位点特异性Lys-63连接的TRAF 6自身泛素化是IKK的关键上游介质。
Tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) is a key mediator in proximal signaling of the interleukin-1/Toll-like receptor and the TNF receptor superfamily. Analysis of TRAF6-deficient mice revealed a fundamental role of TRAF6 in osteoclastogenesis; however, the molecular mechanism underlying TRAF6 signaling in this biological process is not understood. Recent biochemical evidence has indicated that TRAF6 possesses ubiquitin ligase activity that controls the activation of IKK and NF-kappa B. Because these studies are primarily based on cell-free systems, the role of the ubiquitin ligase activity of TRAF6 and its auto-ubiquitination to initiate the NF-kappa B pathway in vivo remain elusive. Here we show that an intact RING domain of TRAF6 in conjunction with the E2 enzyme Ubc13/Uev1A is necessary for Lys-63-linked auto-ubiquitination of TRAF6 and for its ability to activate IKK and NF-kappa B. Furthermore, a RING mutant of TRAF6 abolishes its ability to induce receptor activator of NF-kappa B-independent osteoclast differentiation and nuclear accumulation of the transcription factor NFATc1. Notably, we map the auto-ubiquitination site of TRAF6 to a single Lys residue, which if mutated renders TRAF6 unable to activate transforming growth factor-beta-activated kinase 1 and IKK and to cause spontaneous osteoclast differentiation. Additionally, we provide biochemical and in vivo evidence that TRAF6 serves as an E3 to directly ubiquitinate NEMO. Reconstituting TRAF6-deficent cells with various TRAF6 mutants, we clearly demonstrate the requirement for the TRAF6 RING domain and site-specific auto-ubiquitination of TRAF6 to activate IKK in response to interleukin-1. These data establish a signaling cascade in which regulated site-specific Lys-63-linked TRAF6 auto-ubiquitination is the critical upstream mediator of IKK.