Fig. 5: TMZ-mediated RRM1 T52 phosphorylation promotes DNA damage repair.

a and b WT His-RRM1 or His-RRM1 T52A mutant proteins were mixed with His-RRM2 and incubated with active AMPK in the presence of ATP for 20 min followed by HPLC-MS analysis. (a) Ribonucleotide reductase (RNR) activity was measured according to dCDP production. (b) Kd was calculated. Data represent the mean ± SD from sextuplicate experiments. **P < 0.001. a WT + AMPK vs. T52A + AMPK, P = 2.91e-08; b His-RRM1 WT vs. His-RRM1 WT + AMPK, P = 3.73e-05; His-RRM1 WT + AMPK vs. His-RRM1 T52A + AMPK, P = 0.00032. c Cells with or without HPRT1 depletion were transfected with Flag-RRM1 and treated with or without 0.2 mM of TMZ for 2 h. RNR activity was measured according to dCDP production. Immunoblotting analysis was performed to confirm the AMPK-mediated phosphorylation status of RRM1. Data represent the mean ± SD from sextuplicate experiments. **P < 0.001. U87, Flag-RRM1 + TMZ vs. Flag-RRM1 + TMZ + HPRT1 shRNA, P = 3.18e-08; MES28, Flag-RRM1 + TMZ vs. Flag-RRM1 + TMZ + HPRT1 shRNA, P = 1.56e-11. d Cells with or without HPRT1 depletion were treated with or without 0.2 mM of TMZ, respectively, for 24 h. The rate of apoptotic cells was examined by FACS. Data represent the mean ± SD from sextuplicate experiments. **P < 0.001. U87, TMZ vs. HPRT1 shRNA + TMZ, P = 1.12e-08; MES28, TMZ vs. HPRT1 shRNA + TMZ, P = 1.29e-06. e RRM1-depleted cells with reconstituted expression of Flag-RRM1 WT or Flag-RRM1 T52A were treated with or without 0.2 mM of TMZ, respectively, for 24 h. The rate of apoptotic cells was examined by FACS. Data represent the mean ± SD from sextuplicate experiments. **P < 0.001. U87, Flag-RRM1 WT + TMZ vs. Flag-RRM1 T52A + TMZ, P = 3.68e-05; MES28, Flag-RRM1 WT + TMZ vs. Flag-RRM1 T52A + TMZ, P = 4.7e-09. Statistics: a–e unpaired Student’s t-test for two-group comparison. Source data are provided as a Source Data file.