Virus-dependent phosphorylation of the IRF-3 transcription factor regulates nuclear translocation, transactivation potential, and proteasome-mediated degradation
Virus-dependent phosphorylation of the IRF-3 transcription factor regulates nuclear translocation, transactivation potential, and proteasome-mediated degradation
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DOI：
10.1128/mcb.18.5.2986
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发表时间：
1998-05-01
影响因子：
5.3
通讯作者：
Hiscott, J
中科院分区：
文献类型：
--
作者：
Lin, RT;Heylbroeck, C;Hiscott, J
干扰素调节因子（IRF）由一个不断增长的相关转录蛋白家族组成，该家族首先被确定为α - β干扰素（ifn - α / β）基因启动子的调节因子，以及一些干扰素刺激基因的干扰素刺激反应元件（ISRE）。IRF-3最初被确定为IRF家族的成员，基于与其他IRF家族成员的同源性以及与ISG15启动子的ISRE结合，IRF-3在多种组织中组成性表达，IRF-3 mRNA的相对水平在病毒感染或ifn处理的细胞中没有变化。在本研究中，我们证明在仙台病毒感染后，IRF-3在位于IRF-3碳末端的多个丝氨酸和苏氨酸残基上被蛋白磷酸化，从而进行翻译后修饰。IRF-3缺失和点突变的结合将诱导磷酸化位点定位在395和407氨基酸之间的i (S)区，位于bar N (S)区，bar HPL (S)区，bar L (T)区，bar L (S)区，bar dq区，bar L (S)区；残基Ser-396和Ser-398的点突变消除了病毒诱导的IRF-3蛋白磷酸化，尽管残基Ser-402、ser -404和Ser-405也是目标。磷酸化导致IRF-3的细胞质到细胞核的易位、DNA结合和转录激活增加。用拟磷Asp取代Ser-Thr位点产生了一种组成活性形式的IRF-3，它作为含有PRDI-PRDIII或ISRE调控元件的启动子的非常强的激活剂。磷酸化似乎也代表了病毒介导降解的信号，因为病毒诱导的IRF-3的翻转被IRF-3 Ser-Thr簇的突变或蛋白酶体抑制剂所阻止。有趣的是，病毒感染导致IRF-3与CREB结合蛋白（CBP）共激活因子的关联，通过与抗CBP抗体的共免疫沉淀检测到，这是一种由两种蛋白的c端结构域介导的相互作用。IRF-3中Ser-396和Ser-398残基的突变使其与CBP的结合失效。这些结果是根据一个模型来讨论的，在这个模型中，病毒诱导的IRF-3的c端磷酸化改变了蛋白质的构象，从而允许核易位，与转录伙伴的关联，以及IFN和IFN应答基因的初级激活。
The interferon regulatory factors (IRF) consist of a growing family of related transcription proteins first identified as regulators of the alpha beta interferon (IFN-alpha/beta) gene promoters, as well as the interferon-stimulated response element (ISRE) of some IFN-stimulated genes. IRF-3 was originally identified as a member of the IRF family based on homology with other IRF family members and on binding to the ISRE of the ISG15 promoter, IRF-3 is expressed constitutively in a variety of tissues, and the relative levels of IRF-3 mRNA do not change in virus-infected or IFN-treated cells. In the present study, we demonstrate that following Sendai virus infection, IRF-3 is posttranslationally modified by protein phosphorylation at multiple serine and threonine residues, which are located in the carbon terminus of IRF-3. A combination of IRF-3 deletion and point mutations localized the inducible phosphorylation sites to the region -I (S) under bar N (S) under bar HPL (S) under bar L (T) under bar (S) under bar DQ-between amino acids 395 and 407; point mutation of residues Ser-396 and Ser-398 eliminated virus-induced phosphorylation of IRF-3 protein, although residues Ser-402, Thr-404, and Ser-405 were also targets. Phosphorylation results in the cytoplasm-to-nucleus translocation of IRF-3, DNA binding, and increased transcriptional activation, Substitution of the Ser-Thr sites with the phosphomimetic Asp generated a constitutively active form of IRF-3 that functioned as a very strong activator of promoters containing PRDI-PRDIII or ISRE regulatory elements. Phosphorylation also appears to represent a signal for virus-mediated degradation, since the virus-induced turnover of IRF-3 was prevented by mutation of the IRF-3 Ser-Thr cluster or by proteasome inhibitors, Interestingly, virus infection resulted in the association of IRF-3 with the CREB binding protein (CBP) coactivator, as detected by coimmunoprecipitation with anti-CBP antibody, an interaction mediated by the C-terminal domains of both proteins. Mutation of residues Ser-396 and Ser-398 in IRF-3 abrogated its binding to CBP. These results are discussed in terms of a model in which virus-inducible, C-terminal phosphorylation of IRF-3 alters protein conformation to permit nuclear translocation, association with transcriptional partners, and primary activation of IFN- and IFN-responsive genes.