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.