| Aerobic glycolysis(Warburg effect),a metabolic feature of tumor cells,provides energy and building blocks for rapidly dividing tumor cells.In each phase of the cell cycle,tumor cells have different demands for biosynthesis intermediates and energy from glucose metabolism,however,how cell cycle regulators and glycolytic enzymes orchestrate these requirements are poor characterized.Pyruvate kinase M2(PKM2)is the rate-limiting enzyme of the glycolysis,and its activity regulates the flux and route of glycolysis,which promote the rapid proliferation of tumor cells.The activity of PKM2 can be regulated by various post-translational modifications,such as phosphorylation,acetylation,and oxidation,which can inhibit the activity of PKM2 and promote the synthesis of various biological macromolecules,thereby further promoting tumor cell proliferation.Whether PKM2 participates in crosstalk between metabolic reprogramming and cell cycle regulation is not clear yet.Aurora A is a serine/threonine kinase that plays an important role in centrosome maturation/separation in interphase and spindle formation in mitosis.Studies have shown that the overexpression of Aurora A promotes tumorigenesis and development by promoting cell cycle progression,activating anti-apoptotic signals,inducing genomic instability,and promoting invasion and metastasis.However,it is not clear whether Aurora A in tumor cells promotes tumorigenesis and development by regulating cell metabolism.In this dissertation,I found that Aurora A can bind to several metabolic regulators including PKM2 through co-immunoprecipitation and mass spectrometry experiments.The results of the clinical tumor sample database analysis showed that the highly expressed Aurora A and PKM2 in the tumor were positively correlated,and were negatively correlated with the survival of tumor patients,which suggests that Aurora A and PKM2 may be regulated and involved in tumor progression.Protein-protein interaction and proximity ligation experiments proved that Aurora A can bind to PKM2 and the interaction between them mainly occurs in the interphase of the cell cycle.In vitro kinase experiments proved that Aurora A can phosphorylate PKM2,and mass spectrometry identified the 45th threonine(T45)on PKM2 as the main phosphorylation site of Aurora A.The results of the PKM2 pyruvate kinase activity assay showed that phosphorylation at the T45 site of PKM2 inhibited its enzymatic activity.Cross-linking experiments and protein structure analysis revealed that phosphorylation at the T45 site reduced the stability of the α-helix through the charge repulsion of glutamic acid at position 385 on PKM2,thereby inhibiting the formation of PKM2 tetramers and reducing its activity.Expression of a PKM2 T45A mutant that cannot be phosphorylated by Aurora A significantly inhibited aerobic glycolysis in tumor cells.Meanwhile,the expression of the PKM2 T45A mutant in tumor cells significantly inhibited the accumulation of upstream intermediate metabolites of glycolysis such as G6P,PEP,IMP,serine and NADPH,which can provide the building blocks and energy for proliferating tumor cells.Finally,replacing the endogenous PKM2 with a nonphosphorylated PKM2 T45 A mutant inhibited tumor growth in vitro and in vivo models.In summary,this study found that Aurora A binds to PKM2 and phosphorylates the 45th threonine(T45)on PKM2 in the interphase of the cell cycle.Phosphorylation of PKM2 T45 reduces PKM2 enzymatic activity by inhibiting the composition of PKM2 tetramers.Decreased PKM2 activity promotes aerobic glycolysis and the accumulation of upstream intermediate metabolites in glycolysis and ultimately promotes tumor cell proliferation in vitro and in vivo.These data reveal a new protumor function of Aurora A through modulating a rate-limiting glycolytic enzyme and provide a metabolic advantage to tumor cells.These findings provide a new mechanism for the interconnected regulation bewteen cell cycle and tumor metabolism,which could be applied in preclinical study of target therapy. |
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