The Research Of Glycyl-tRNA Synthetase Regulates The NFκB1 Signaling Pathway In Bovine Mammary Epithelial Cells

The early stage of the laboratory experiment found that glycyl-t RNA synthetase(GlyRS) in bovine mammary epithelial cells(BMECs) could reply the methionine signal, the site of threonine 544(T544) and serine 704(S704) were phosphorylated, and then phosphorylated GlyR S entered into the nucleus, and positively regulated the expression of transcription level. However, the specific adjustment mechanism is unclear. Clarified the mechanism can help to reveal GlyRS regulation the molecular mechanisms of milk protein synthesi s.0.6mmol/L methionine(Met) was added to BMECs culture to establish amino acids-stimulating BMECs in vitro model. Western blotting and IF were used to detect the expression and localization of NFκB1 and p-NFκB1 after adding Met. NFκB1 had two molecular forms in cytoplasm, which molecular weights were about 141 k Da and 105 k Da, and in nucleus only 105 k Da form existed. The p-NFκB1(50kDa) mainly existed in the nucleus. After Met stimulation, the protein level of NFκB1 was slightly upregulated, the protein level and nuclear localization of p-NFκB1 were both obviously increased. These results suggest that amino acids promote the cytoplasm NFκB1 dissociation with IκBα, and enter into the nucleus and phosphorylated, then to regulate milk synthesis transcriptional expression of related genes.Then research the different amino acids treatment on the expression of p-GlyRS. The results showed that the p-GlyRS, GlyRS and CSN2 expression significantly increased after adding methionine, leucine and lysine. In subcellular localization found, GlyRS existed only in the cytoplasm, and the p-GlyRS(T544) and p-GlyRS(S704) were 80 k Da form in the cytoplasm, and 70 k Da form in the nucleus. The form of p-GlyRS 70 k Da in the nucleus significantly increased after adding amino acids, showed that amino acids induce GlyRS phosphorylation in the cytoplasm, then the p-GlyRS rapidly translocated to the nucleus, and cleaved into a shortened form of phosphorylation.The interaction between NFκB1 and GlyRS were identified by co-immunoprecipitation(Co-IP) in the BMECs. The result showed that NFκB1 can not bind with GlyRS, whereas NFκB1 bind with p-GlyRS(T544) and p-GlyRS(S704) in the nucleus.In order to study whether p-GlyRS affected the expression of NFκB1 gene by GCN2/e IF2 pathway. In this study, overexpression and gene silencing of GlyRS were completed, Western blotting to detect the expression of NFκB1, e IF2α and GCN2, and the level of phosphorylation. The results showed that amino acids promote GlyRS phosphorylation and then rapidly tra nslocated to the nucleus, inhibition of GCN2 phosphorylation, regulation transcription activation of NFκB1, so as to promote milk protein synthesis.The study used cycloheximide inhibition of total protein synthesis, observed in methionine stimuli, the expression and phosphorylation of Gly RS, NFκB1, mTOR and Stat5. The results showed that cycloheximide reduced the expression level of the proteins, the proteins level increased after adding methionine. The results showed that the GlyRS sensing amino acids sig nal was not dependent on the protein translation process. The study continued to verify whether GlyRS phosphorylation was dependent on the m TOR pathway. m TOR activity was inhibited by rapamycin, the expression and phosphorylation level of NFκB1, m TOR and GlyRS were detected by Western blotting. The results showed that the expression of GlyRS, p-GlyRS, NFκB1 and p-NFκB1 proteins were slightly increased after adding methionine. The phosphorylation of GlyRS and NFκB1 were not inhibited by rapamycin, indicating that the phosphorylation of GlyRS was not depend on the m TOR pathway, that is, m TOR was not the upstream kinase of GlyRS.In summary, under the promoting effect of amino acids, GlyRS in 544 threonine and 704 serine occurred phosphorylation, entered the nucleus and cleaved into a shortened form of phosphorylation, inhibition of GCN2 phosphorylation, then bound with NFκB1, phosphorylated NFκB1 and bound enhancer sites of downstream target genes to activate target genes transcription and expression.