High-throughput immunoblotting - Ubiquitin-like protein ISG15 modifies key regulators of signal transduction
High-throughput immunoblotting - Ubiquitin-like protein ISG15 modifies key regulators of signal transduction
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DOI：
10.1074/jbc.m208435200
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发表时间：
2003-05-09
影响因子：
4.8
通讯作者：
Zhang, DE
中科院分区：
文献类型：
--
作者：
Malakhov, MP;Kim, KI;Zhang, DE
ISG15是一种泛素样蛋白，可与干扰素或脂多糖处理的细胞中的许多蛋白质结合。小鼠体内蛋白质ISG15修饰的失调会导致预期寿命缩短、脑细胞损伤和对干扰素的超敏反应。虽然ISG15早在二十多年前就被发现，但ISG15修饰的确切生化和生理功能仍不清楚，也没有确定ISG15靶向的蛋白。这项工作的主要目的是从特征良好的蛋白质中确定ISG15靶标，这些蛋白质可以用作生物学研究的模型。我们通过免疫亲和层析从人胸腺中纯化了ISG15蛋白，并用高通量蛋白质印迹技术(PowerBlot(TM))分析了ISG15结合物。我们发现信号转导的三个关键调控因子，磷脂酶CGamma1、JAK1和ERK1被ISG15修饰。除此之外，我们还证明了JAK1的直接底物转录因子STAT1也是ISGyl化的。以全细胞蛋白提取物和磷脂酶CGamma1为例，我们证明了ISG15结合物在蛋白酶体的特定抑制剂处理的细胞中不积累。我们的工作提示了ISG15在多条信号转导通路中的调节作用，并为进一步阐明ISG化的生化功能提供了有吸引力的模型。
ISG15 is a ubiquitin-like protein that conjugates to numerous proteins in cells treated with interferon or lipopolysaccharide. Dysregulation of protein ISG15 modification (ISGylation) in mice leads to decreased life expectancy, brain cell injury, and hypersensitivity to interferon. Although ISG15 was identified more than two decades ago, the exact biochemical and physiological functions of ISG15-modification remain unknown, and the proteins targeted by ISG15 have not been identified. The major purpose of this work was to identify ISG15 targets among well characterized proteins that could be used as models for biological studies. We purified ISGylated proteins from human thymus by immunoaffinity chromatography and analyzed ISG15 conjugates by a high-throughput Western blot screen (PowerBlot(TM)). We found that three key regulators of signal transduction, phospholipase Cgamma1, Jak1, and ERK1 are modified by ISG15. In addition to that, we demonstrate that transcription factor Stat1, an immediate substrate of Jak1 kinase, is also ISGylated. Using whole cell protein extracts and phospholipase Cgamma1 as an example we demonstrate that ISG15 conjugates are not accumulated in cells treated with specific inhibitors of proteasomes. Our work suggests a role for ISG15 in the regulation of multiple signal transduction pathways and offers attractive models to further elucidate the biochemical function of ISGylation.