Mitochondrial PKM2 interacts with and phosphorylates Bcl2. Validation from the antibody specificity. cr2016159x6.pdf (29K) GUID:?8C3D002B-E462-43B2-8190-930C2D89F17D Supplementary information, Table S1: Related to Number 1 293T cells were transiently transfected with SFB-PKM2 and then treated with or without H2O2 (0.5 mM, 1 h). cr2016159x7.xlsx (12K) GUID:?59430ADB-A986-45B4-8583-C50AE24D4F07 Supplementary information, Table S2: Related to Figure 2 U87 cells that stably express SFB-PKM2 were treated with or without H2O2 (0.5 mM, 1 h). cr2016159x8.xlsx (13K) GUID:?9F4D533F-7A5F-41AB-AF13-D253E12BBE0C Abstract Pyruvate kinase M2 isoform (PKM2) catalyzes the last step of glycolysis and plays an important role in tumor cell proliferation. Recent studies possess reported that PKM2 also regulates apoptosis. However, the mechanisms underlying such a role of PKM2 remain elusive. Here we display that PKM2 translocates to mitochondria under oxidative stress. In the mitochondria, PKM2 interacts with and phosphorylates Bcl2 at threonine (T) 69. This phosphorylation prevents the binding of Cul3-centered E3 ligase to Bcl2 and subsequent degradation of Bcl2. A chaperone protein, HSP901, is required for this function of PKM2. HSP901’s ATPase activity launches a conformational switch of PKM2 and facilitates connection between PKM2 and Bcl2. Alternative of wild-type Bcl2 with phosphorylation-deficient Bcl2 T69A mutant sensitizes glioma Rabbit polyclonal to AMIGO2 cells to oxidative Panipenem stress-induced apoptosis and impairs mind tumor formation in an orthotopic xenograft model. Notably, a peptide that is composed of the amino acid residues from 389 to 405 of PKM2, through which PKM2 binds to Bcl2, disrupts PKM2-Bcl2 connection, promotes Bcl2 degradation and impairs mind tumor growth. In addition, levels of Bcl2 T69 phosphorylation, conformation-altered PKM2 and Bcl2 protein correlate with one another in specimens of human being glioblastoma individuals. Moreover, levels of Bcl2 T69 phosphorylation Panipenem and conformation-altered PKM2 correlate with both marks and prognosis of glioma malignancy. Our findings uncover a novel mechanism through which mitochondrial PKM2 phosphorylates Bcl2 and inhibits apoptosis directly, highlight the essential part of PKM2 in ROS adaptation of malignancy cells, and implicate HSP90-PKM2-Bcl2 axis like a potential target for therapeutic treatment in glioblastoma. and pre-mRNA results in the generation of pyruvate kinase M1 (PKM1) and M2 (PKM2) from the inclusion of exon 9 and exon 10, respectively8,9. PKM2, but not PKM1, is definitely upregulated in most human being cancers. Substitute of PKM2 with PKM1 in lung malignancy cells inhibits aerobic glycolysis and tumor growth in nude mouse xenograft10. Activation of epidermal growth element receptor (EGFR) in human being glioma cells prospects to increased glucose uptake and lactate production inside a PKM2 expression-dependent manner11. Mediated by extracellular signal-regulated kinase (ERK)-dependent phosphorylation, PKM2 is definitely capable of translocating to the nucleus upon EGRF activation12. In the nucleus, PKM2 binds to c-Src-phosphorylated -catenin and enhances -catenin’s transactivation activity, advertising the manifestation of downstream oncogene cyclin D1 and the progression of cell cycle13. Under hypoxic conditions, prolyl-hydroxylated PKM2 interacts with HIF1a to induce glycolytic gene manifestation, which in turn enhances glucose rate of metabolism in malignancy cells14. These findings demonstrate the crucial functions of PKM2 in tumor cell proliferation. Besides its important roles in promoting cell proliferation, PKM2 is also involved in the rules of apoptosis. It has been demonstrated that depletion of PKM2 manifestation by small interfering RNAs specifically against PKM2 results in decreased viability and improved apoptosis in multiple malignancy cell lines15. Silencing of PKM2 in rat and human being glioma Panipenem spheroids enhances both apoptosis and differentiation16. In non-small cell lung malignancy (NSCLC), PKM2 deficiency enhances ionizing radiation-induced apoptosis and autophagy and (Supplementary info, Number S1A). After hydrogen peroxide (H2O2) or diamide (a thiol-oxidizing compound) treatment, U87 or U251 cells with PKM2 depletion (U87/shPKM2 or U251/shPKM2) experienced much more apoptotic cells than those cells expressing non-targeting shRNA (U87/shNT or U251/shNT), as determined by flow cytometry analysis of Annexin V-positive cells (Number 1A and Supplementary info, Number S1B). Similarly, caspase 3 activity was much more strong in U87/shPKM2 or U251/shPKM2 cells than that in U87/shNT or U251/shNT cells after H2O2 treatment (Number 1B). Cytochrome is definitely released from your mitochondria to the cytosol, where it binds to Apaf1 to activate caspase cascades, during the early stage of mitochondria-dependent apoptosis22. Number 1C showed that more cytochrome was recognized in cytosolic portion in U87/shPKM2 or U251/shPKM2 cells than that in the cells expressing shNT after H2O2 treatment. Immune cells, such as Jurkat T cells, also.
