title: Experimental approaches for investigation of aminoacyl tRNA synthetase phosphorylation.
abstract: 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.
authors.affiliation: Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA.
authors.forename: Abul
authors.initials: A
authors.lastname: Arif
authors.affiliation: Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA.
authors.forename: Jie
authors.initials: J
authors.lastname: Jia
authors.affiliation: Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA.
authors.forename: Dalia
authors.initials: D
authors.lastname: Halawani
authors.affiliation: Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA. Electronic address: foxp@ccf.org.
authors.forename: Paul L
authors.initials: PL
authors.lastname: Fox
mesh_terms: D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D000906:Antibodies / Q000737:chemistry; D001681:Biological Assay; D045744:Cell Line, Tumor; D051360:Cyclin-Dependent Kinase 5 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics / Q000378:metabolism; D008264:Macrophages / Q000166:cytology / Q000378:metabolism; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D009000:Monocytes / Q000166:cytology / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D061251:Primary Cell Culture; D011392:Proline / Q000378:metabolism*; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D012361:RNA, Transfer, Pro / Q000235:genetics / Q000378:metabolism*
chemical_list: D000906:Antibodies; D010761:Phosphorus Radioisotopes; D034741:RNA, Small Interfering; D012361:RNA, Transfer, Pro; D007371:Interferon-gamma; D011392:Proline; D051360:Cyclin-Dependent Kinase 5; C495905:CDK5 protein, human; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D000604:Amino Acyl-tRNA Synthetases; C100134:prolyl T RNA synthetase
keywords: Aminoacyl tRNA synthetase; Cdk5; EPRS; Glutamyl-prolyl tRNA; Kinase; Multisynthetase complex; Phosphorylation; Synthetase