Regulatory serine residues mediate phosphorylation-dependent and phosphorylation-independent activation of interferon regulatory factor 7
Regulatory serine residues mediate phosphorylation-dependent and phosphorylation-independent activation of interferon regulatory factor 7
复制标题
DOI：
10.1074/jbc.m411389200
复制
发表时间：
2005-05-06
影响因子：
4.8
通讯作者：
Marié, IJ
中科院分区：
文献类型：
--
作者：
Caillaud, A;Hovanessian, AG;Marié, IJ
干扰素调节因子7（Interferon Regulatory Factor，IRF）是一种重要的转录因子，是建立抗病毒耐药性的关键因子.响应感染，IRF 7通过非典型I κ B激酶、I κ B激酶-β和TANK结合激酶1的作用被磷酸化激活。激活导致核保留、DNA结合和反式激活能力的去抑制。位于IRF 7的羧基末端调节结构域中的丝氨酸残基簇是病毒活化激酶的推定靶标。然而，磷酸化的确切位点尚未确定。在这里，我们报告了一个全面的结构活性检查潜在的IRF 7磷酸化位点通过分析突变蛋白，其中特定的丝氨酸残基被改变为丙氨酸或天冬氨酸。通过双向凝胶电泳分析这些突变体的磷酸化模式，并通过报告基因分析监测其转录活性。将必需磷酸化事件定位于氨基酸437-438和氨基酸429-431或441处的冗余位点组。从感染的细胞中回收的IRF 7在这些位点被不均匀磷酸化，并且更大的磷酸化与增加的反式激活相关。有趣的是，在相关蛋白IRF 3中保守的独特丝氨酸簇对于IRF 7活化和远端磷酸化也是必不可少的。然而，这个基序的重要作用似乎并没有通过磷酸化来实现。相反，这些丝氨酸残基和相邻的亮氨酸需要在远端位点磷酸化，并可能决定功能所需的构象元素。
Interferon regulatory factor (IRF)7 is a key transcription factor required for establishment of antiviral resistance. In response to infection, IRF7 is activated by phosphorylation through the action of the non-canonical I kappa B kinases, I kappa B kinase-epsilon and TANK-binding kinase 1. Activation leads to nuclear retention, DNA binding, and derepression of transactivation ability. Clusters of serine residues located in the carboxyl-terminal regulatory domain of IRF7 are putative targets of virus-activated kinases. However, the exact sites of phosphorylation have not yet been established. Here, we report a comprehensive structure-activity examination of potential IRF7 phosphorylation sites through analysis of mutant proteins in which specific serine residues were altered to alanine or aspartate. Phosphorylation patterns of these mutants were analyzed by two-dimensional gel electrophoresis, and their transcriptional activity was monitored by reporter assays. Essential phosphorylation events were mapped to amino acids 437-438 and a redundant set of sites at either amino acids 429-431 or 441. IRF7 recovered from infected cells was heterogeneously phosphorylated at these sites, and greater phosphorylation correlated with increased transactivation. Interestingly, a distinct serine cluster conserved in the related protein IRF3 was also essential for IRF7 activation and distal phosphorylation. However, the essential role of this motif did not appear to be fulfilled by phosphorylation. Rather, these serine residues and an adjacent leucine were required for phosphorylation at distal sites and may determine a conformational element required for function.