Serine phosphorylation of the linker domain of bifunctional glutamyl‐prolyl tRNA synthetase is critical for transcript‐specific translational silencing

Abstract

Recent studies indicate that some aminoacyl tRNA synthetases (ARSs) have regulatory functions unrelated to aminoacylation. Glutamyl-prolyl tRNA synthetase (EPRS) is a bifunctional ARS that has a regulated, noncanonical activity which blocks the translation of ceruloplasmin (Cp) mRNA. EPRS is a component of the Gamma-interferon Activated Inhibitor of Translation (GAIT) complex along with three other proteins that binds the GAIT element in Cp mRNA and blocks its translation. Here we investigate the mechanism of activation of EPRS by phosphorylation, and the role of EPRS phosphorylation in formation of the GAIT complex. Our studies show that gamma-IFN treatment of U937 monocytic cells causes rapid phosphorylation of EPRS at serine residues in the linker domain that couples the glutamyl and prolyl tRNA synthetase domains. Immunoaffinity purification of EPRS, coupled with mass spectrometry, revealed Ser886 and Ser999/1000 as the sites phosphorylated, possibly by distinct Ser/Thr kinases. Phosphorylation induces release of EPRS from its usual residence in the tRNA multisynthetase complex (MSC). EPRS release from the MSC depends on its phosphorylation, and is reversed upon withdrawal of IFN. Released EPRS, by a phosphorylation-dependent mechanism, interacts with the other 3 protein components to form the active GAIT complex that binds the Cp mRNA GAIT element. Our results show that serine phosphorylation of EPRS is required for its translocation from the MSC to the GAIT complex, thereby regulating transcript-specific translational silencing in inflammatory macrophages. Research support- NIH grant# PO1HL29582