The Mechanism Of The Differential Sequence Between PIAS3 Isforms In PKM2 Signaling Regulation

Tumor cells have significantly different metabolic patterns than normal cells.Normal cells are metabolized by aerobic oxidation,while tumor cells choose glycolysis to break down glucose and gain energy,which is known as the Warburg effect.As a critical metabolic enzyme in tumor’s Warburg effect,Pyruvate Kinase M2 subtypes(PKM2)exhibit significance in metabolism and growth regulation of tumors.There are two mainly isozymes of Pyruvate Kinase(PKM),PKM1 and PKM2,which show dramatic difference both in tissue localization and gene function.PKM1 is predominantly expressed in normal tissues(such as brain and muscle tissues)that generate enormous energy,while PKM2 is over-expressed in tissues that synthesize large amounts of nucleotides,such as tumor cell.However,upstream regulatory proteins of PKM2 is barely known.PIAS proteins(Protein inhibitor of activated STAT),beyond serve as co-regulatory factor regulating STAT,AR and other signaling pathways,also has the protein SUMO-modified E3 ligase properties,involved in a variety of life activities,such as cell proliferation,senescence and apoptosis.There is only one 68 KD PIAS3 protein(named PIAS3L)in all human tissues.In our previous work,our group found a tumor-specific PIAS3 splicing isoform(64KD,named PIAS3S)that exists in many tumor tissues.In the preliminary work,a differential sequence of PIAS3 Isforms(4KD,named PIAS3-delta)was used as GST fusion proteins to pulldown proteins in prostate cancer cell lysate,and PKM2 was detected by mass spectrometry.In order to systematically analyze the probability of PIAS3 as a PKM2 synergistic protein,this project comprehensively analyzed the the probability and specificity of the interaction between PKM2 and PIAS3 Isforms as well as differential sequence PIAS3-delta,and systematically studied the mechanisms of the regulation of PKM2 by PIAS3 Isforms.Firstly,we detected the functional binding domains of PKM and PIAS3 via GST-Pulldown experiments.293 T cells endogenously express both PKM1 and PKM2,and the results indicated that the GST fusion proteins of PIAS3-delta can specifically bind to PKM2 instead of PKM1.PKM1 and PKM2 are different products of PKM gene precursor m RNA via alternative splicing.PKM1 retains exon 9 while PKM2 retains exon 10.To testify the specificity of the interaction between PKM2 and PIAS3,we constructed PKM-exon 9GST fusion protein(refers to characteristic of PKM1)and PKM-exon 10 GST fusion protein(refers to characteristic of PKM2).Under the condition of overexpressing the PIAS3-delta domain in cells,there was no binding between GST-PKM-exon 9 and PIAS3-delta,while the GST-PKM-exon 10 specifically binds to PIAS3-delta protein.These results indicated that the PIAS3-delta domain specifically binds to PKM2.Furthermore,we examined the interaction between the PIAS3-delta domain and PKM2 in cells,and examined the regulation effect of different PIAS3 Isforms for intracellular localization of PKM2.After PIAS3 delta was over-expressed in 293 T cells,bi-directional co-immunoprecipitation experiments demonstrated that the PIAS3-delta domain specifically interacts with endogenous PKM2.Further,by immunofluorescence experiments,we transfected the FLAG-tagged vector control,PIAS3 S,PIAS3L and PIAS3-delta domains into the cells and found that PIAS3 S promoted the entry of PKM2 into the nucleus,while PIAS3 L and PIAS3 delta inhibited the entry of PKM2 into the nucleus and correlated with PKM2.It is suggested that the PIAS3 L subtype binds to PKM2 via the PIAS33-delta domain and inhibits its entry.There is a special Warburg effect in the tumor cells,and PKM2 is a key enzyme involved in the tumor’s Warburg effect to produce large amounts of lactic acid.We used the lactic acid kit to detect the regulatory function of different subtypes of PIAS3 on lactic acid metabolism of tumor cells.In prostate cancer cells DU145,LNCa P cell over-expression vector control,PIAS3 S,PIAS3L and PIAS3-delta domains,it was found that PIAS3 S can significantly promote tumor cell lactic acid production,PIAS3 L,PIAS3delta can significantly inhibit the generation of tumor cell lactic acid.This effect can be amplified by over-expressed of PKM2 and blocked by sh RNA interference with PKM2.Furthermore,we detected changes in GLUT1 and LDHA expressions related to glucose metabolism and found that PIAS3 L and PIAS3 delta domains can significantly inhibit the expression of GLUT1 and LDHA.The above experimental results demonstrate that PIAS3 can inhibit the Warburg effect of lactic acid metabolism of PKM2 through the PIAS3 delta domain.PKM2 can promote the proliferation of tumor cells.Furthermore,we examined the regulatory functions of different PIAS3 Isforms on PKM2-mediated tumor cell proliferation.In prostate cancer cells DU145,LNCa P cell over-expression vector control,PIAS3 S,PIAS3L and PIAS3-delta domains,PIAS3 S was found to significantly promote the proliferation of tumor cells,PIAS3 L,PIAS3delta significantly inhibited the proliferation of two tumor cells,and PIAS3 L and PIAS3-delta domain can inhibit the expression of the cell proliferative proteins CCND1 and c-Myc;this effect can be magnified by over-expression of PKM2,but it cannot be completely blocked by the sh RNA interference PKM2.It is suggested that PIAS3 mainly inhibits the proliferation of tumor cells through the PIAS3 delta segment,but this effect is independent of the proliferation promoting effect of PKM2.The activity of PKM2 is regulated by two phosphorylation sites of S37 and Y105.The phosphorylation of S37 regulates the entry of PKM2,and the phosphorylation of Y105 regulates the PKM2-mediated Warburg lactate metabolism.We synthesized S37 and Y105 polypeptides and prepared specific PKM2 phosphorylated antibodies.We found that in both breast and prostate cancers,the detection of two PKM2 phosphorylation antibodies was higher than normal cells,suggesting that the two sites of PKM2 phosphorylation levels are closely related to the occurrence and development of tumors.We examined the role of different PIAS3 Isforms in the regulation of PKM2 S37 and Y105 phosphorylation and found that the PIAS3 L and PIAS3 delta domains significantly inhibit phosphorylation at two sites of PKM2,specifically phosphorylation at the Y105 site.PIAS3 S promoted phosphorylation of S37 in PKM2.The results suggest that PIAS3 L inhibits phosphorylation of the Y105 site through the PIAS3 delta domain and inhibits the PKM2-mediated Warburg effect.PIAS protein can be used as the E3 ligase regulatory protein SUMOylation modification process,so we examined the effect of different PIAS3 isoforms on the SUMO modification of PKM2 protein.The co-transfection of different PIAS3 subtypes and GFP-SUMO1,GFP-SUMO3 plasmids in 293 T cells revealed that when PIAS3 L and GFP-SUMO3 were over-expressed,additional modified bands appeared in PKM2,suggesting that PIAS3 L promotes SUMOylation of PKM2.It may be related to its regulation of phosphorylation of PKM2.In summary,we demonstrated that the delta domain of wild-type PIAS3 L has the specific binding effect of PKM2 and inhibits the PKM2-mediated lactic acid metabolism of tumor cells through the delta domain,and this effect is closely related to its inhibition of phosphorylation of Y105 site of PKM2.Additionally,the PIAS3 S subtype of tumor cells loses the delta domain and acts as a promoter of PKM2 phosphorylation and lactic acid metabolism.We also found that PIAS3 L promotes the SUMOylation of PKM2,and the PIAS3 S isoform loses this SUMOylation-modifying regulatory function.This study provides evidence that PIAS3 different Isforms act as cooperative proteins to regulate PKM2 signaling,providing a new target for the pathogenesis and treatment of prostate cancer and other tumors.