Identification Of The Upstream Kinase Of GlyRS And Observation Of GlyRS Kinase Activity

The mechanism of milk protein synthesis has been widely regarded as one of the basic scientific questions in the field of lactobiology.At present,the signaling pathways that regulate milk protein production are well known at the level of transcription and translation,but we need to do research on some detailed biochemical regulatory mechanisms.In this study,it was found that Gly RS is phosphorylated at threonine residue at position 544(T544)and serine at position 704(S704).The expression of CSN2 was upregulated by NF?B1 binding to ?-casein(CSN2)promoter,which up-regulates the expression of milk protein synthesis.In addition,our previous laboratory results of the cytoplasmic interactome of Gly RS indicate that several Ser/Thr protein kinases may be the upstream kinases for Gly RS phosphorylation stimulated by the amino acid signals.In this study,we used the method of in vitro kinase activity assay to elucidate whether Gly RS directly activates NF?B1 and identifies MAP3K10 as the upstream kinase of Gly RS.Our findings reveal the molecular mechanism of Gly RS mediating amino acid signaling to the activation of NF?B1 to promote milk protein synthesis.The bovine mammary epithelial cells(BMECs)were cultured and purified by tissue culture method.The purity of these cells were identified by western blotting(WB)and immunofluorescence(IF)analysis of the expressions of keratin 18(CK18)and CSN2.We then explored the upstream kinase of Gly RS.Our previous study found the protein kinase MAP3K10 in the Co-IP of Gly RS by mass spectrometry analysis.We thus detected the interaction between p-MAP3K10 and GlyRS by Co-IP analysis,next exploited in vitro kinase experiments.GST-Gly RS was expressed in prokaryotic cells(E.coli BL21)and purified by GST affinity chromatography;p-MAP3K10 was enriched by Co-IP and subjected to in vitro kinase experiments.The results showed that Gly RS was phosphorylated at T544 and S704.Intriguingly,the molecular weight of p-Gly RS was between 80 kDa-100 kDa,s?ggesting that degradation or cleavage may occur simultaneously during in vitro kinase reactions.Thus,the in vitro kinase assay of p-MAP3K10 activating Gly RS at 6 different time points was further performed,the results showed that truncated p-Gly RS actually appeared during the reaction of p-MAP3K10 to activite GST-Gly RS.These above results indicate that p-MAP3K10 activates GST-Gly RS at its two phosphorylation sites.Previous work in the laboratory has determined that p-Gly RS interacts directly with NF?B1 in vivo.We further exploited experiments to determine whether p-Gly RS directly activates NF?B1.GST-NF?B1 was expressed in prokaryotic cells(E.coli BL21)and purified by GST affinity chromatography.In vitro kinase assay was carried out with enriched p-Gly RS by Co-IP.The in vitro kinase assay of Gly RS to activate NF?B1 was also used as Control.The results showed that Gly RS could not activate NF?B1,whereas p-Gly RS led to NF?B1 phosphorylation.To further determine the direct activation of NF-?B1 by p-Gly RS,an in vitro continuous kinase activation assay for p-MAP3K10-GST-Gly RS-GST-NF?B1 was designed.The results showed that p-NF?B1was continuously activated by p-MAP3K10-GST-Gly RS kinase reaction.To give more direct experimental evidence of p-Gly RS activation on NF?B1,we chose to perform in vitro kinase assay using purified p-Gly RS.The eukaryotic expression vector of pCMV-C-Flag-Gly RS was constructed and transfected into cells.The nucleus was extracted and Flag-Gly RS was purified by Flag Purification Kit and subsequent phosphorylation Kit.The purified product was a single phosphorylated protein,identified as p-Gly RS by western blotting analysis.In vitro kinase experiments were performed using the purified p-Gly RS protein and GST-NF?B1.The anti-phospho-Ser/Thr/Tyr antibody was used to detect phosphorylated protein by western blotting,and the expression of pCMV-C-Flag-Gly RS was detected by using Flag antibody.The results showed that p-NF?B1 appeared at the target molecular weight in the activation group.This experiment further demonstrates that Gly RS has kinase activity.The similarity of Gly RS sequence to the catalytic domain of several typical Ser/Thr kinases was analysized by bioinformatics analysis.It was found that the significant characteristics of Ser/Thr kinase domain were also found in Gly RS,which further raised the possibility of Gly RS to have Ser/Thr protein kinase activity.In summary,we reveal that,p-MAP3K10 phosphorylates Gly RS at T544 and S704 and identify p-MAP3K10 as the upstream kinase of Gly RS.We also show that,p-Gly RS can phosphorylate NF?B1 directly,s?ggesting that Gly RS is possibly a new kinase.Subsequent studies on the molecular structure of Gly RS for its kinase activity will reveal the nonclassical molecular mechanism of Gly RS in depth.