Structural and Functional Analysis of Interferon Regulatory Factor 3: Localization of the Transactivation and Autoinhibitory Domains
Structural and Functional Analysis of Interferon Regulatory Factor 3: Localization of the Transactivation and Autoinhibitory Domains
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DOI：
10.1128/mcb.19.4.2465
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发表时间：
1999-04
影响因子：
5.3
通讯作者：
R. Lin;Y. Mamane;J. Hiscott
中科院分区：
文献类型：
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作者：
R. Lin;Y. Mamane;J. Hiscott
摘要干扰素调节因子3(IRF-3)基因编码一个55 kDa的蛋白，在所有组织中都有组成性表达。在未受刺激的细胞中，IRF-3以不活跃的胞浆形式存在；仙台病毒感染后，IRF-3被位于羧基末端的多个丝氨酸和苏氨酸残基的蛋白质磷酸化后修饰。病毒诱导的IRF-3的磷酸化导致磷酸化的IRF-3从胞浆到胞核的移位，与转录辅助激活因子CBP/p300结合，并刺激病毒诱导基因的DNA结合和转录活性。利用酵母和哺乳动物的单杂交分析，我们现在证明了IRF-3的C末端存在一个扩展的、非典型的反式激活结构域，位于氨基酸(AA)134和394之间。我们还表明，位于AA380和427之间的IRF-3的C末端结构域通过与AA98和240之间的N末端区域的分子内结合参与了IRF-3活性的自我抑制。仙台病毒感染后，检测到IRF-3蛋白之间的分子间联系，证明了病毒依赖的IRF-3同源二聚体的形成；在没有病毒感染的情况下，也观察到这种相互作用与IRF-3的成分激活形式。用氨基酸395和407[IRF-3(5D)]之间的ISNSHPLSLTSDQ区的C-末端Ser-Thr磷酸化位点替换，而不是邻近的S385和S386残基，产生了IRF-3的结构性激活的DNA结合形式。相反，用Ala或Asp替换S385和S386会抑制IRF-3(5D)蛋白的DNA结合和反式激活活性。因此，这些研究确定了IRF-3的反式激活结构域，参与IRF-3活性自抑制的两个结构域，以及控制IRF-3二聚体形成、DNA结合活性和与CBP/p300共激活因子结合的调节磷酸化位点。
ABSTRACT The interferon regulatory factor 3 (IRF-3) gene encodes a 55-kDa protein which is expressed constitutively in all tissues. In unstimulated cells, IRF-3 is present in an inactive cytoplasmic form; following Sendai virus infection, IRF-3 is posttranslationally modified by protein phosphorylation at multiple serine and threonine residues located in the carboxy terminus. Virus-induced phosphorylation of IRF-3 leads to cytoplasmic to nuclear translocation of phosphorylated IRF-3, association with the transcriptional coactivator CBP/p300, and stimulation of DNA binding and transcriptional activities of virus-inducible genes. Using yeast and mammalian one-hybrid analysis, we now demonstrate that an extended, atypical transactivation domain is located in the C terminus of IRF-3 between amino acids (aa) 134 and 394. We also show that the C-terminal domain of IRF-3 located between aa 380 and 427 participates in the autoinhibition of IRF-3 activity via an intramolecular association with the N-terminal region between aa 98 and 240. After Sendai virus infection, an intermolecular association between IRF-3 proteins is detected, demonstrating a virus-dependent formation of IRF-3 homodimers; this interaction is also observed in the absence of virus infection with a constitutively activated form of IRF-3. Substitution of the C-terminal Ser-Thr phosphorylation sites with the phosphomimetic Asp in the region ISNSHPLSLTSDQ between amino acids 395 and 407 [IRF-3(5D)], but not the adjacent S385 and S386 residues, generates a constitutively activated DNA binding form of IRF-3. In contrast, substitution of S385 and S386 with either Ala or Asp inhibits both DNA binding and transactivation activities of the IRF-3(5D) protein. These studies thus define the transactivation domain of IRF-3, two domains that participate in the autoinhibition of IRF-3 activity, and the regulatory phosphorylation sites controlling IRF-3 dimer formation, DNA binding activity, and association with the CBP/p300 coactivator.