Extended Data Figure 1. Identification of S6K1 as EPRS Ser⁹⁹⁹ kinase.

a, Screening of AGC kinase group members for phosphorylation of EPRS Ser⁹⁹⁹ by immunocomplex kinase assay and [γ-³²P]ATP-labeling with S886A linker target in U937 cells. Kinase activity using kinase-specific substrate is shown (bottom; mean ± SEM, n = 3). b, Specificity of S6K1 for Ser⁹⁹⁹ phosphorylation, determined by ³²P incorporation in wild-type (WT), S999A and S886 linker. c, siRNA targeting S6K1 inhibits IFN-γ-stimulated EPRS phosphorylation in U937 cells determined by ³²P-labeling (mean ± SEM, n = 3). d, Active recombinant kinases used for in vitro phosphorylation of linker bearing S886A (Ser⁹⁹⁹ P-acceptor) mutation shows site-specific phosphorylation by S6K1. e, Raptor not rictor is required for Ser⁹⁹⁹ phosphorylation. f, siRNA targeting the S6K1 3’UTR inhibits S6K1 expression and phosphorylation of EPRS Ser⁹⁹⁹, but not Ser⁸⁸⁶ (mean ± SEM, n = 3). g, Phosphorylation of S6K1 Thr³⁸⁹ is required for phosphorylation of EPRS. Cells were co-transfected with siRNA targeting the 3’UTR to knock down endogenous S6K1, and with myc-tagged wild-type or mutant S6K1 cDNA; IFN-γ-stimulated EPRS phosphorylation determined by ³²P-labeling (mean ± SEM, n = 3). h, Cells treated as in (e) but followed by reciprocal co-immunoprecipitation.