Characterization Of Impact From O-GlcNAc Modification At T405/S406 On Non-metabolic Functions Of PKM2

Metabolic reprogramming is one of the features of tumors,which intake of large amounts of glucose for glycolysis to produce lactate in the case of sufficient O₂,this phenomenon is known as Warburg effect.It is believed that the Warburg effect allows cancer cells to meet high biosynthetic demand in both ATP and biomass production.The key metabolic enzymes can regulate the metabolic process.Pyruvate kinase is the final rate-limiting enzymes in glycolysis.Pyruvate kinase M2?PKM2?,one of the isoform of PK family,is highly expressed in tumors of many different types,and it can promote Warburg effect and tumor growth through its activity and nuclear non-metabolizing enzyme function.PKM2 function can be regulated by a variety of protein post-translational modifications such as acetylation,phosphorylation.Recent studies have reported that PKM2 can also undergo O-GlcNAcylation.Our previous work in the laboratory showed that O-GlcNAcylation could promote the disassociation of PKM2 into the nucleus and enhance its nuclear non-metabolic enzyme function.It is identified by mass spectrometry that T405 and S406 are PKM2O-GlcNAcylation sites,but the double T405/S406 mutation do not completely eliminate the PKM2 O-GlcNAcylation,indicating that there are other O-GlcNAcylation sites in PKM2.Therefore,we speculate that T405 and S406 may be the two most abundant sites for the modification of PKM2 sites.However,whether T405/S406 O-GlcNAcylation can modulate the function of PKM2 is unclear.Therefore,the effect of the T405/S406 O-GlcNAcylation on the function of PKM2 will be studied.In this study,firstly we constructed eukaryotic expression plasmid OGT to increase the level of O-GlcNAcylation of PKM2.Our results indicated ransient transfection of OGT plasmid in MCF-7 cells expressing PKM2 wild-type(PKM2^WT)and PKM2 T405/S406double mutants(PKM2^T405A/S406A),could significantly up-regulate the O-GlcNAcylation of PKM2^WT.However,there is no significant effect on PKM2^T405A/S406A.Subsequently,Through the use of glutaraldehyde cross-linking experiments and Western Blot,it was found that O-GlcNAcylation at these two sites can promote the disassociation of PKM2 tetramers.By the pyruvate kinase activity detection kit,it was found that T405/S406 O-GlcNAcylation reduced PKM2 activity.Next,by extracting nuclear proteins and cytoplasmic proteins and detecting PKM2 distribution,we found that T405/S406 O-GlcNAcylation facilitated nuclear translocation of PKM2.Using Western Blot,we found that T405/S406 O-GlcNAcylation can enhance histone H3 T11 phosphorylation and H3 K9 acetylation levels,thereby promoting the expression of MYC gene,indicating T405/S406 O-GlcNAcylation cound enhance PKM2non-metabolizing enzyme function.Finally,by using the glucose assay kit and the lactate assay kit,we found that T405/S406 O-GlcNAcylation increased cell glucose consumption and lactate production,indicating that these two sites O-GlcNAcylation can promote the Warburg effect.In summary,this study finds that T405/S406 O-GlcNAcylation promotes the disassociation of PKM2 tetramer and enhances nuclear translocation and non-metabolic enzyme function,in order to lay the foundation for further revealing the effect of O-GlcNAcylation on the Warburg effect by regulating the structure and function of PKM2.