Fig. 2: Inflammatory-activated AKT phosphorylates EPRS1 at Ser999.

a Immunoblot analysis of EPRS1 Ser999 phosphorylation in U937 cells treated for 4 h with LPS (100 ng/ml) or IFNγ (100 ng/ml). For pharmacological inhibition, cells were either untreated (control) or pretreated with rapamycin (10 nM) 30 min before stimulation with LPS or IFNγ. The ratio of phosphorylated/total EPRS1 is shown as p-EPRS1/EPRS1. b Proteomic analysis identified candidate kinases that bind preferentially to EPRS1. Protein intensity ratios under unstimulated (−)/stimulated (LPS) conditions are shown. c U937 cells, unstimulated (−) or stimulated with LPS (100 ng/ml) for 1 h, were lysed and incubated with vehicle (Veh) or purified Strep-EPRS1. The incubated cells were pulled down with Strep-Tactin beads for 1 h, and precipitates of endogenous kinases were analyzed by immunoblotting with the indicated antibodies. d U937 cells were pretreated with rapamycin (10 nM), roscovitine (10 μM), LY294002 (5 μM), or an AKT1/2 inhibitor (5 μM) for 30 min before LPS (100 ng/ml) stimulation. After 1 h, cells were harvested and the lysates were analyzed by immunoblotting with the indicated antibodies. The calculated ratio of p-EPRS1/EPRS1 is shown. e, f Schematic representation of the AKT kinase assay protocol (e; left). An AKT-specific antibody was used to immunoprecipitate AKT from U937 cell lysates at the indicated times. The AKT-specific antibody bound to protein A/G-agarose beads was incubated with recombinant EPRS1 as a substrate for the in vitro kinase assay. AKT-mediated phosphorylation of Ser999 was determined using the EPRS1 full-length protein (e; right), or a recombinant Linker of wild-type (WT) or phosphorylation-resistant mutant (S886A/999A) EPRS1 (f), followed by immunoblotting with anti-EPRS1 Ser999. Rabbit IgG was used as a negative control for the AKT antibody (e). The purified EPRS1 proteins were subjected to SDS-PAGE and stained with Coomassie Brilliant Blue (CBB). g Effect of AKTs on Ser999 phosphorylation of EPRS1 in AKT1- and/or AKT2-suppressed and control RAW 264.7 cells treated with LPS (100 ng/ml) for 1 h. The fold change in activation of EPRS1 versus total EPRS1 is shown. All data are representative of three independent experiments, each with similar results. Source data are provided as a Source Data file.