Fig. 5

ATM phosphorylation of PKM2 on T328 regulates PKM2’s activation of CtIP and HR repair. a shRNA-resistant, flag-tagged WT, T328A or T328E PKM2 were expressed in U87 shPKM2 cells. Cells were irradiated, and cell lysates were collected and immunoprecipitated with antibodies against CtIP or Flag. Immunoprecipitates and input protein lysates were resolved by SDS-PAGE, and then probed with the indicated antibodies. exo/endo, exogenously or endogenously expressed PKM2. b shRNA-resistant WT or T328A PKM2 was expressed in U87 shPKM2 cells along with flag-tagged WT or T126A CtIP as indicated. Cells were treated with 0 or 6 Gy, and cell lysates were collected and immunoprecipitated with anti-flag antibody. Immunoprecipitates and input lysates were resolved by SDS-PAGE, then probed with the indicated antibodies. exo/endo, exogenously or endogenously expressed PKM2. c HT1904 cells containing the I-SceI DSB system were used to analyze DNA end-resection adjacent to I-SceI-induced DSBs. Endogenous CtIP and PKM2 were knocked down and replaced with WT, T126A (A), or T126E (E) CtIP and WT or T328A (A) PKM2 as indicated. DSBs were induced by adenoviral delivery of I-SceI. Genomic DNA was isolated, and ssDNA adjacent to I-SceI-induced DSBs was quantitated by quantitative real-time PCR. Data are presented as mean ± SE (n = 3). d Endogenous CtIP and PKM2 were knockdown by siRNA or shRNA in U87 DR-GFP cells and replaced by WT, T126A (A) or T126E CtIP (E) and WT or T328A (A) PKM2. DSBs were induced by adenoviral delivery of I-SceI, and the ability of cells to resolve DSBs by the HR pathway was assessed by the percentage of GFP-positive cells 48 h later. Data are presented as mean ± SE (n = 3). *P < 0.05. ns, not significant