Functional And Mechanistic Characterization Of PKM2 O-GlcNAcylation In Tumor Cell Proliferation

The Warburg effect,characterized by increased glucose uptake and lactate production,is a well-known universal across cancer-and other proliferating-cells.PKM2,a splice isoform of the pyruvate kinase?PK?specifically expressed in these cells,serves as a major regulator of this metabolic reprogramming with an adjustable activity subjected to numerous allosteric effectors and posttranslational modifications.Besides acetylation and phosphorylation,O-GlcNAcylation,which occurs at serine or threonine residues of proteins,has recently been found on PKM2.However,the consequences of O-GlcNAc modification on PKM2 still remains unknown.Here we show that PKM2 O-GlcNAcylation is upregulated in various types of human tumor cells and patient tumor tissues.O-GlcNAcylation specifically targets Thr⁴⁰⁵ and Ser⁴⁰⁶ on PKM2,residues of the region encoded by the alternatively spliced exon 10 in cancer cells.The presence of O-GlcNAc modification at Thr⁴⁰⁵ and Ser⁴⁰⁶destabilizes PKM2 tetramers and leads to a shift of the oligomeric equilibrium towards dimers and monomers by blocking the key interaction along“C-C”interface.On one hand,the disassociation of active tetrameric PKM2 causes reduced PK activity,which directly rewires metabolic fluxes towards anabolic pathways to promote lipid and DNA synthesis for rapid cell proliferation.On the other hand,O-GlcNAcylation mediated destabilization of PKM2 tetramers facilitates the exposure of NLS as well as phosphorylation at Ser³⁷ for the subsequent PKM2 nuclear localization.Once PKM2was translocated into the nucleus,it stimulated c-Myc-dependent expression of two key glycolysis components,namely Glut1 and LDHA,to enhance glucose consumption and lactate production Eventually,our in vivo experiments showed that blocking PKM2 O-GlcNAcylation attenuated tumor growth.Collectively,these findings argue that O-GlcNAcylation at Thr⁴⁰⁵ and Ser⁴⁰⁶ on PKM2 promotes the Warburg effect and contributes to the rapid proliferation of tumor cells.In this thesis,we demonstrate that O-GlcNAcylation is a new regulatory mechanism for PKM2 in cancer cells and serves as a bridge linking the adjustment of PKM2 functions to metabolic reprogramming.Alteration of the PKM2 oligomeric state triggered by O-GlcNAcylation allows simultaneous control of the metabolic and nuclear?non-metabolic?roles of this key enzyme in proliferating cells.By controlling PKM2 structure and function,O-GlcNAcylation couples metabolic state to dynamically changing nutritional environment.Notably,the O-GlcNAcylated residues of PKM2,Thr⁴⁰⁵ and Ser⁴⁰⁶,are encoded by the alternatively spliced exon 10.Thus,O-GlcNAcylation appears to be a unique regulatory mode for PKM2 function and serves as a bridge translating genomic gene expression switch from PKM1 to PKM2 towards metabolic reprogramming.