TRAF6 and the three C-terminal lysine sites on IRF7 are required for its ubiquitination-mediated activation by the tumor necrosis factor receptor family member latent membrane protein 1
TRAF6 and the three C-terminal lysine sites on IRF7 are required for its ubiquitination-mediated activation by the tumor necrosis factor receptor family member latent membrane protein 1
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DOI：
10.1128/mcb.00785-08
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发表时间：
2008-10-01
影响因子：
5.3
通讯作者：
Pagano, Joseph S.
中科院分区：
文献类型：
--
作者：
Ning, Shunbin;Campos, Alex D.;Pagano, Joseph S.
我们最近发现，干扰素调节因子7（IRF 7）被肿瘤坏死因子受体（TNFR）超家族成员EB病毒潜伏膜蛋白1（LMP 1）通过受体相互作用蛋白依赖性K63连接的泛素化（L. E. Huye，S.宁，M. Kelliher和J.S.帕加诺Cell. 27：2910-2918，2007）。在这项研究中，使用小干扰RNA和肿瘤坏死因子相关因子6（TRAF 6）敲除细胞，我们首先表明，TRAF 6及其E3连接酶活性所需的LMP 1刺激的IRF 7泛素化。在潜伏感染并内源性表达高水平LMP 1和IRF 7的Raji细胞中，TRAF 6小发夹RNA构建体的表达降低了内源性泛素化和IRF 7的内源性活性。在TRAF 6（-/-）小鼠胚胎成纤维细胞中，用TRAF 6表达而不是用缺乏E3连接酶活性的TRAF 6（C70 A）重建，恢复了LMP 1刺激K63连接的IRF 7泛素化的能力。此外，我们确定IRF 7作为TRAF 6 E3连接酶的底物，并表明IRF 7在体外和体内的多个位点被TRAF 6泛素化。最重要的是，我们确定了人IRF 7变体A的最后三个C-末端赖氨酸位点（位置444、446和452）对于IRF 7的激活是必不可少的;这些是第一个被鉴定的这样的位点。这些位点突变为丝氨酸的IRF 7的泛素化缺陷突变体不仅完全丧失对LMP 1的应答，而且完全丧失对IRF 7激酶I κ B激酶I κ的应答的反式激活能力。此外，我们发现K63连接的IRF 7的泛素化独立于其C-末端功能磷酸化位点发生。这些数据支持了我们的假设，即调节泛素化的IRF 7是其磷酸化的先决条件。这是第一个暗示泛素化是磷酸化和转录因子激活所必需的证据。
We have recently shown that interferon regulatory factor 7 (IRF7) is activated by Epstein-Barr virus latent membrane protein 1 (LMP1), a member of the tumor necrosis factor receptor (TNFR) superfamily, through receptor-interacting protein-dependent K63-linked ubiquitination (L. E. Huye, S. Ning, M. Kelliher, and J. S. Pagano, Mol. Cell. Biol. 27:2910-2918, 2007). In this study, with the use of small interfering RNA and TNFR-associated factor 6 (TRAF6) knockout cells, we first show that TRAF6 and its E3 ligase activity are required for LMP1-stimulated IRF7 ubiquitination. In Raji cells which are latently infected and express high levels of LMP1 and IRF7 endogenously, expression of a TRAF6 small hairpin RNA construct reduces endogenous ubiquitination and endogenous activity of IRF7. In TRAF6(-/-) mouse embryonic fibroblasts, reconstitution with TRAF6 expression, but not with TRAF6(C70A), which lacks the E3 ligase activity, recovers LMP1's ability to stimulate K63-linked ubiquitination of IRF7. Further, we identify IRF7 as a substrate for TRAF6 E3 ligase and show that IRF7 is ubiquitinated by TRAF6 at multiple sites both in vitro and in vivo. Most important, we determine that the last three C-terminal lysine sites (positions 444, 446, and 452) of human IRF7 variant A are essential for activation of IRF7; these are the first such sites identified. A ubiquitination-deficient mutant of IRF7 with these sites mutated to arginines completely loses transactivational ability in response not only to LMP1 but also to the IRF7 kinase I kappa B kinase epsilon. In addition, we find that K63-linked ubiquitination of IRF7 occurs independently of its C-terminal functional phosphorylation sites. These data support our hypothesis that regulatory ubiquitination of IRF7 is a prerequisite for its phosphorylation. This is the first evidence to imply that ubiquitination is required for phosphorylation and activation of a transcription factor.