Negative role of RIG-I serine 8 phosphorylation in the regulation of interferon-beta production.
Negative role of RIG-I serine 8 phosphorylation in the regulation of interferon-beta production.
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DOI：
10.1074/jbc.m109.089912
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发表时间：
2010-06-25
期刊：
影响因子：
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通讯作者：
García-Sastre A
中科院分区：
文献类型：
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作者：
Nistal-Villán E;Gack MU;Martínez-Delgado G;Maharaj NP;Inn KS;Yang H;Wang R;Aggarwal AK;Jung JU;García-Sastre A
RIG-I（视黄酸诱导基因I）和TRIM 25（三重基序蛋白25）已成为诱导干扰素（IFN）介导的先天免疫应答以限制病毒复制的关键调节因子。在识别病毒RNA后，TRIM 25 E3连接酶结合RIG-I的第一个半胱天冬酶募集结构域（CARD），随后诱导RIG-I的第二个CARD的赖氨酸172泛素化，这对于与下游MAVS/IPS-1/CARDIF/VISA的相互作用以及由此产生IFN-β mRNA是必需的。虽然泛素化已成为参与RIG-I激活的主要因素，但其他翻译后修饰（如磷酸化）对RIG-I活性调节的潜在贡献尚未得到解决。在这里，我们报告了在RIG-I的第一个CARD处的丝氨酸8磷酸化的鉴定，作为RIG-I介导的IFN-β产生的负调节机制。用磷酸特异性抗体进行的免疫印迹分析显示，在用IFN-β或病毒感染刺激细胞后，RIG-I丝氨酸8磷酸化稳态水平降低。用磷酸模拟天冬氨酸或谷氨酸取代CARD RIG-I功能结构域中的丝氨酸8导致TRIM 25结合、RIG-I泛素化、MAVS结合和下游信号传导降低。最后，序列比较显示，只有灵长类动物携带丝氨酸8，而其他动物携带天冬酰胺，表明丝氨酸8磷酸化可能代表灵长类特异性调节RIG-I激活。总的来说，这些数据表明RIG-I丝氨酸8的磷酸化通过抑制TRIM 25相互作用作为RIG-I激活的负开关，进一步强调了RIG-I和TRIM 25连接在I型IFN信号转导中的重要性。
RIG-I (retinoic acid-inducible gene I) and TRIM25 (tripartite motif protein 25) have emerged as key regulatory factors to induce interferon (IFN)-mediated innate immune responses to limit viral replication. Upon recognition of viral RNA, TRIM25 E3 ligase binds the first caspase recruitment domain (CARD) of RIG-I and subsequently induces lysine 172 ubiquitination of the second CARD of RIG-I, which is essential for the interaction with downstream MAVS/IPS-1/CARDIF/VISA and, thereby, IFN-β mRNA production. Although ubiquitination has emerged as a major factor involved in RIG-I activation, the potential contribution of other post-translational modifications, such as phosphorylation, to the regulation of RIG-I activity has not been addressed. Here, we report the identification of serine 8 phosphorylation at the first CARD of RIG-I as a negative regulatory mechanism of RIG-I-mediated IFN-β production. Immunoblot analysis with a phosphospecific antibody showed that RIG-I serine 8 phosphorylation steady-state levels were decreased upon stimulation of cells with IFN-β or virus infection. Substitution of serine 8 in the CARD RIG-I functional domain with phosphomimetic aspartate or glutamate results in decreased TRIM25 binding, RIG-I ubiquitination, MAVS binding, and downstream signaling. Finally, sequence comparison reveals that only primate species carry serine 8, whereas other animal species carry an asparagine, indicating that serine 8 phosphorylation may represent a primate-specific regulation of RIG-I activation. Collectively, these data suggest that the phosphorylation of RIG-I serine 8 operates as a negative switch of RIG-I activation by suppressing TRIM25 interaction, further underscoring the importance of RIG-I and TRIM25 connection in type I IFN signal transduction.