Fig. 6
Cisplatin induces K472 acetylation and S461 phosphorylation of PFKFB3. a DNA damage signals induced PFKFB3 K472 acetylation. Flag-tagged PFKFB3 was expressed in HEK293T cells, which were then treated with etoposide (10 μM), adriamycin (1 μM), UV irradiation (10 J/m2) and cisplatin (50 or 100 μM) for 24 h. Flag-PFKFB3 was immunoprecipitated with Flag beads and immunoblotting was performed with the antibodies indicated. Relative PFKFB3 K472 acetylation and phosphorylation were normalized by Flag protein. b The amino acid sequence near K472 of PFKFB3 displays high similarity with the sequence near K320 of TP53. c Cisplatin treatment induces PFKFB3 cytoplasmic accumulation. HEK293T cells were treated with or without cisplatin (50 μM) for 24 h before harvest. Cells were then suspended in PBS and treated with a gradient concentration of digitonin. Supernatant and precipitate were collected for immunoblotting with indicated antibodies. S, supernatant; P, precipitate. d Cisplatin induces K472 acetylation and S461 phosphorylation of endogenous PFKFB3. Endogenous PFKFB3 protein was purified from HEK293T cells after cisplatin treatment as indicated for 24 h. e Cisplatin or etoposide treatment enhances K472 acetylation in SIRT1 knockout cells. Endogenous PFKFB3 protein were purified from WT or SIRT1 knockout HEK293T cells treated with EX527 (10 μM), cisplatin (50 μM) or etoposide (10 μM) for 24 h. f Combined knockdown of PCAF and GCN5 abolishes cisplatin- or etoposide-induced PFKFB3 K472 acetylation. HEK293T cells were transfected with siRNAs targeting PCAF and GCN5. After 60 h, cells were treated with cisplatin (50 μM) or etoposide (10 μM) for 24 h. g, h Cisplatin treatment induces pan-acetylation of PCAF and GCN5. Flag-tagged PCAF or GCN5 was expressed in HEK293T cells, which were treated with cisplatin for the duration indicated at a concentration of 50 μM. Relative pan-acetylation level of PCAF or GCN5 was normalized by Flag protein. i Cisplatin increases acetyltransferase activity of PCAF and GCN5. Flag-tag PCAF or GCN5 was purified from HEK293T cells treated with or without cisplatin (50 μM) for 24 h, then incubated with recombinant His-PFKFB3 in acetylation assay buffer. Purified proteins visualized by Coomassie blue staining are shown (lower panel). Data are representative of at least two independent experiments
