The Modulation Of P53 By Pyruvate Kinase M2 And Its Role In DNA Damage Response

Pyruvate kinase M2(PKM2)is a rate-limiting enzyme of glycolysis process,which catalytic the last step of glycolysis,by transferring one phosphate of PEP to adenosine diphosphate glucose pyrophospheralase(ADP),producing a pyruvate and one adenosine triphosphate(ATP).In recent years,PKM2 has been found to modulate the tumor cells to perform the Warburg effect.However,the exact functional mechanism has not been clarified clearly.By using the immunoprecipitation-coupled LC-MS/MS,we found that PKM2 could interact directly with P53 during DNA damage response.Based on this,we further studied the mechanism of the interaction and the functions during DNA damage response both in vivo and in vitro.Finally,we have some findings as below:1)By using the immunoprecipitation-coupled LC-MS/MS in MCF7 cells,we found that the nuclear PKM2 directly interacts with P53 during DNA damage response.2)By using co-IP,GST-pulldown and immunofluorescence,we confirmed that PKM2 and P53 co-localized in nucleus and has direct interaction.Further using GST-Pulldown assay,we identified the functional domains of P53 that interacts with PKM2 protein is the C terminal of its DNA binding domain.3)The Ch IP(Chromatin Immunoprecipitation),luciferase gene reporter and co-IP assays showed that PKM2 inhibited P53 from binding with the P53 Response Element to down-regulate the expression of the cyclin-dependent kinase inhibitor protein P21,leading to a nonstop of phase G1 of the cell cycle during DNA damage response,thus ensuring tumor cells continue to proliferate.This indicated that PKM2 could promote cell growth during DNA damage response.4)In a mouse xenograft model,we found that under the treatment of etoposide,the growth of the tumor of the PKM2 knockout group significantly slowed down.5)P53 mutations appear in more than 50% of various types of tumors.In these parts of tumors,point mutations of P53 is the most common ones,and the hotspots focused on the DNA binding domain.These mutations mainly cause the loss of the function of regulating the transcripsion of some target genes.Based on the previous reports,we selected a hotspot P53 mutation type R248 Q,which has the highest mutant frequency among the mutations and the acute promyelocytic leukemia cell line NB4,which expresses the P53-R248 Q to do further investigation.By using the immunoprecipitations and immunofluorescence,we found that PKM2 could also directly interact with P53-R248 Q.Furthermore,by knocking down P53-R248 Q in NB4 cells,we found that the existence of P53-R248 Q could increase the percentage of the tetramer of PKM2 and influence the pyruvate kinase activity of PKM2.In summary,our work found that PKM2 acted as a co-repressor of P53,by interacting with P53 to down-regulate the transcription of the target gene P21,thus influencing the growth of tumors during DNA damage response.PKM2 can still interact with the P53-R248 Q in NB4 cells leading to the variation of the structure of PKM2 and the different activity of pyruvate kinase.This study illustrated that PKM2 can interact with P53 as a co-repressor in nucleus to influence the proliferation of tumor cells for the first time,which revealed the new mechanism of PKM2 to promote the tumor initiation and development.In the meanwhile,this work provided a new theoretical basis for PKM2 targeted therapy.