Study On Effect And Mechanisms Of MCF-7 Cells Proliferation Induced By O-GlcNAcylation Of PKM2 T405/S406

Pyruvate kinase(PK)is the last rate-limiting enzyme in the glycolytic pathway,which catalyzes the conversion of phosphoenolpyruvate(PEP)to pyruvate.Pyruvate kinase M2(PKM2),one of the isoform of PK family,is highly expressed during embryogenesis and in tumors of many different types.There is abundant evidence that PKM2 plays a key role in the metabolic reprogramming of tumor cells.It is known that the activity of PKM2 can be modified by a variety of post-translational modifications,such as phosphorylation,acetylation and so on.Previous studies of our laboratory found that O-GlcNAcylation could promote tumor cell proliferation,while the O-GlcNAcylation of PKM2 may play an keyt role.Mass spectrometry detected that T405/S406 was the O-GlcNAcylation sites of PKM2.Subsequent experiments showed that the mutation of PKM2 T405/S406 could not eliminate O-GlcNAcylation completely,indicating that PKM2 has other O-GlcNAcylation sites.The analysis of the crystal structure of PKM2 revealed that T405/S406 were located at the contact surface of PKM2 tetramer subunits,and its O-GlcNAcylation could destroy the main force between PKM2 tetramer subunits and regulate PKM2 activity.To investigate whether O-GlcNAcylation of PKM2 T405/S406 plays an important role in promoting tumor cell proliferation and its possible mechanism,we first constructed PKM2knockdown cell line and then established PKM2~WTT and PKM2~T405A/S406A405A/S406A rescue cell lines.The results showed that PKM2 O-GlcNAcylation level in PKM2~T405A/S406A405A/S406A cell line was significantly down-regulated,as well as the proliferation rate,this proved that O-GlcNAcylation of PKM2 T405/S406 plays an important role in tumor cells proliferation.Subsequent experiments showed that O-GlcNAcylation of PKM2 T405/S406 could promote the phosphorylation of Stat3 Y705,enabling it to be activated and thus promoting the high expression of its target gene MEK5.Since MEK5 can further activate the signaling pathway associated with cell proliferation,our experimental results provide a possible mechanism for the effect of PKM2 T405/S406 O-GlcNAcylation on promoting tumor cell proliferation.This study not only helps to deepen the understanding of PKM2 in the mechanism of metabolic reprogramming of tumor cells,but also provides a valuable basis for further exploring the relationship between metabolic reprogramming and cell proliferation of tumor cells.