Glycyl-tRNA synthetase (GlyRS) has non-canonical roles beyond aminoacylation, but the molecular mechanism is largely unknown. We have previously found that GlyRS is phosphorylated in the cytoplasm of bovine mammary epithelial cells (bMECs) in response to amino acid stimulation, and the phosphorylated GlyRS enters nucleus to stimulate gene expression for milk synthesis. In this study, we aim to uncover the upstream kinase of GlyRS and reveal the signaling pathways that methionine (Met) stimulates GlyRS phosphorylation. We show that mitogen-activated protein kinase 10 (MAP3K10) interacts with GlyRS in bMECs by Co-IP, mass spectrometry, and Western blotting analysis. We further identify that MAP3K10 is an upstream kinase of GlyRS by in vitro kinase assay and MAP3K10 stimulates NF kappa B1 phosphorylation via activating GlyRS. We also uncover that Met stimulates GlyRS phosphorylation via the GPR87-CDC42/Rac1- MAP3K10 signaling pathway. Our findings help to understand the molecular mechanism of GlyRS in cellular signaling transduction. (C) 2020 Elsevier Inc. All rights reserved.