The Signaling Pathway Via Which Amino Acids Activate Glycyl TRNA Synthetase In Bovine Mammary Epithelial Cells

Aminoyl-tRNA synthetases(aaRSs)are key enzymes in the process of protein synthesis and have classical aminoacylation.In recent years,it has been found that many members of the aminoacyl-tRNA synthetase play important roles in the regulation of gene expression,the promotion of angiogenesis,and the occurrence of inflammatory responses.These study reveal that the noncanonical function of aminoacyl-tRNA synthetases besides aminoacylation is a new molecular mechanism in the field of biology.Previous studies have shown that glycyl tRNA synthetase(GlyRS),as a member of the family of aaRSs,may play an regulatory role in the process of protein expression.GlyRS in bovine mammary epithelial cells(BMECs)may be associated with the stimulation on protein synthesis by hormones like prolactin and estrogen,nutrients(like amino acids),and other environmental stimuli,but the specific mechanism is unclear.Our preliminary results have revealed that GlyRS can accept amino acid signal,and is phosphorylated,then enters into the nucleus.Previous experiments also revealed the protein interactome of GlyRS in the cytoplasm.Based on the bioinformatics analysis of the GlyRS cytoplasmic interactome,we speculate that amino acids might activate MAP3K10 through GPR87 and its downstream CDC42/Rac1/Rho A signaling pathway,to activate the signal transduction pathway of GlyRS.The study would provide an experimental basis to reveal the mechanism of GlyRS signaling for milk synthesis under amino acid stimulation.In this study,immunoprecipitation(Co-IP)was used to identify the interaction between MAP3K10 and GlyRS in the cytoplasm.Western blotting analysis detected that,when BMECs were stimulated by amino acids(Met)and MAP3K10 was simultaneously inhibited by siRNA,we found the amino acid stimulation of GlyRS phosphorylation was significantly reduced by the inhibition of MAP3K10.Using siRNA to knock down CDC42 or Rac1,we found the expression of MAP3K10 was not significantly changed but the level of p-MAP3K10 was significantly decreased.The level of p-MAP3K10 was further decreased after BMECs were transfected with CDC42 siRNA together with Rac1 siRNA,suggesting that CDC42/Rac1 are upstream of MAP3K10,furthermore CDC42 and Rac1 are functional redundancy.The expression of GPR87 was increased after individually adding Met,Leu and Lys.When expression of GPR87 was inhibited by siRNA,amino acids-induced GlyRS phosphorylation were significantly inhibited.We next constructed the eukaryotic expression vector GPR87,and overexpressed GPR87 in BMECs,and we found that the levels of CDC42 or Rac1 GTP binding forms weresignificantly increased,furthermore p-MAP3K10 was increased significantly.Using siRNA to inhibit Rac1 or CDC42 and using vector to overexpress GPR87 simultanously,we found the expression of p-MAP3K10 enhanced by GPR87 overexpression was significantly reduced by Rac1 or CDC42 knock down.We further overexpressed MAP3K10 and simultaneously inhibited GlyRS by siRNA,and found that GlyRS mediated the activation of MAP3K10 on NFκB1 phosphorylation.In summary,the above studies reveal that,amino acids signal to GPR87,then GPR87 activates the downstream CDC42/Rac1 GTP kinase activities,leading to the phosphorylation of MAP3K10.p-MAP3K10 further activates of GlyRS,which mediates the activation of p-MAP3K10 on NFκB1phosphorylation that promotes gene expression in milk synthesis related signaling pathways.