Poster abstracts

Poster number 57 submitted by Abul Arif

Two-site Serine Phosphorylation of Glutamyl-prolyl tRNA Synthetase Regulates the Formation of the GAIT Translational Silencing Complex

Abul Arif (Department of Cell Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, 44195), Paul L. Fox (Department of Cell Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, 44195)

Abstract:
Glutamyl-prolyl tRNA synthetase (GluProRS), the bifunctional aminoacyl tRNA synthetase, is an integral component of the tRNA multisynthetase complex (MSC) containing 7 other tRNA synthetases and 3 non-synthetase proteins. In addition to aminoacylation of glutamic acid and proline to cognate tRNAs during protein synthesis, GluProRS also has a regulated, noncanonical activity in translational control. In human monocytic U937 cells, upon activation with gamma-interferon (IFN), GluProRS is phosphorylated and interacts with 3 other proteins to form the Gamma-interferon Activated Inhibitor of Translation (GAIT) complex. The GAIT complex binds to the 29-nucleotide GAIT element in ceruloplasmin (Cp) mRNA and blocks its translation. Here we investigate the mechanism of activation of GluProRS by phosphorylation, and the role of GluProRS phosphorylation in the formation of the GAIT complex. IFN induces rapid phosphorylation of GluProRS at serine residues in the linker domain that joins the two catalytic domains of GluProRS. Immunoaffinity purification, coupled with mass spectrometry, and site-directed mutagenesis has revealed Ser886 and Ser999 as the stimulus-dependent phosphorylation sites. In vitro studies have shown that phosphorylation of the two sites is independent of each other and induced by kinases belonging to two distinct groups. Time course studies suggest that Ser886 phosphorylation precedes phosphorylation of Ser999. Bioinformatic analysis, together with mutation and kinase inhibitor studies has revealed SPXR, a target of proline-dependent Ser/Thr kinases, as the critical kinase recognition motif required for phosphorylation of Ser886. Protein kinase C is a candidate for phosphorylation of Ser999. Phosphorylation induces the release of GluProRS from its usual residence in the MSC and is required for interaction with other protein components to form the active GAIT complex. Thus, two-site serine phosphorylation of GluProRS is required for its translocation from the MSC to the GAIT complex, thereby regulating transcript-selective translational silencing in inflammatory macrophages.

Keywords: GAIT, Phosphorylation, Glutamyl-proly tRNA synthetase