Arif Abul, Jia Jie, Halawani Dalia, Fox Paul L
Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA.
Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA.
Methods. 2017 Jan 15;113:72-82. doi: 10.1016/j.ymeth.2016.10.004. Epub 2016 Oct 8.
Phosphorylation of many aminoacyl tRNA synthetases (AARSs) has been recognized for decades, but the contribution of post-translational modification to their primary role in tRNA charging and decryption of genetic code remains unclear. In contrast, phosphorylation is essential for performance of diverse noncanonical functions of AARSs unrelated to protein synthesis. Phosphorylation of glutamyl-prolyl tRNA synthetase (EPRS) has been investigated extensively in our laboratory for more than a decade, and has served as an archetype for studies of other AARSs. EPRS is a constituent of the IFN-γ-activated inhibitor of translation (GAIT) complex that directs transcript-selective translational control in myeloid cells. Stimulus-dependent phosphorylation of EPRS is essential for its release from the parental multi-aminoacyl tRNA synthetase complex (MSC), for binding to other GAIT complex proteins, and for regulating the binding to target mRNAs. Importantly, phosphorylation is the common driving force for the context- and stimulus-dependent release, and non-canonical activity, of other AARSs residing in the MSC, for example, lysyl tRNA synthetase (KARS). Here, we describe the concepts and experimental methodologies we have used to investigate the influence of phosphorylation on the structure and function of EPRS. We suggest that application of these approaches will help to identify new functional phosphorylation event(s) in other AARSs and elucidate their possible roles in noncanonical activities.
数十年来，人们已经认识到许多氨酰tRNA合成酶（AARS）会发生磷酸化，但翻译后修饰对其在tRNA充电和遗传密码解密中的主要作用的贡献仍不清楚。相比之下，磷酸化对于AARS与蛋白质合成无关的多种非经典功能的发挥至关重要。在我们实验室中，谷氨酰胺-脯氨酸tRNA合成酶（EPRS）的磷酸化已经被广泛研究了十多年，并已成为研究其他AARS的范例。EPRS是干扰素γ激活的翻译抑制剂（GAIT）复合物的组成部分，该复合物在髓样细胞中指导转录本选择性的翻译控制。EPRS的刺激依赖性磷酸化对于其从亲本多氨酰tRNA合成酶复合物（MSC）中释放、与其他GAIT复合物蛋白结合以及调节与靶mRNA的结合至关重要。重要的是，磷酸化是位于MSC中的其他AARS（例如赖氨酰tRNA合成酶（KARS））的背景和刺激依赖性释放以及非经典活性的共同驱动力。在这里，我们描述了我们用于研究磷酸化对EPRS结构和功能影响的概念和实验方法。我们认为，应用这些方法将有助于识别其他AARS中新的功能性磷酸化事件，并阐明它们在非经典活性中的可能作用。
