Acetylation accumulates PFKFB3 in cytoplasm to promote glycolysis and protects cells from cisplatin-induced apoptosis

Enhanced glycolysis in cancer cells has been linked to cell protection from DNA damaging signals, although the mechanism is largely unknown. The 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) catalyzes the generation of fructose-2,6-bisphosphate, a potent allosteric stimulator of glycolysis. Intriguingly, among the four members of PFKFB family, PFKFB3 is uniquely localized in the nucleus, although the reason remains unclear. Here we show that chemotherapeutic agent cisplatin promotes glycolysis, which is suppressed by PFKFB3 deletion. Mechanistically, cisplatin induces PFKFB3 acetylation at lysine 472 (K472), which impairs activity of the nuclear localization signal (NLS) and accumulates PFKFB3 in the cytoplasm. Cytoplasmic accumulation of PFKFB3 facilitates its phosphorylation by AMPK, leading to PFKFB3 activation and enhanced glycolysis. Inhibition of PFKFB3 sensitizes tumor to cisplatin treatment in a xenograft model. Our findings reveal a mechanism for cells to stimulate glycolysis to protect from DNA damage and potentially suggest a therapeutic strategy to sensitize tumor cells to genotoxic agents by targeting PFKFB3.

Inhibition of PFKFB3 cooperates with cisplatin to promote cancer cell apoptosis (a) Cisplatin treatment enhanced PFK activity. Endogenous PFK activity were measured in WT or PFKFB3 KO HeLa cells treated with or without cisplatin (10 µM) for 24 h. Data are presented as mean ± s.d. of three biological replicates, and statistical analyses were performed by using two-way ANOVA with Bonferroni's post-test. ** denotes p < 0.01, n.s., not significant.
(b) PFK15 treatment reduces intracellular ATP level. HeLa cells were exposed to PFK15 at the indicated concentrations for 24 h. Intracellular ATP levels were measured as described in the Methods. Data are presented as mean ± s.d. of three biological replicates.
(c) Verification of HeLa cell lines with PFKFB3 deletion. Alignment of genomic sequence of PFKFB3 from PFKFB3 KO cells and WT cells is shown. Both clones are heterozygous. Clone PFKFB3-KO#1: the two alleles contain 5 or 13 nucleotides deletion, respectively. Clone PFKFB3-KO#2: one allele contains one nucleotide deletion, while the other allele contains one nucleotide insertion.  Fig. 3 Acetylation Mimetic Mutant of PFKFB3 disrupts its NLS motif (a) Subcellular localization of GFP fused PFKFB3 wild-type (WT), K472Q or K472R mutants stably expressed in U2OS cells was examined by detecting GFP fluorescence. Scale bars: 20 µm.
(b) Molecular modeling shows acetylation of the first lysine within NLS disrupts its recognition by the importin α subunit. Upper panel, cartoon representation of the crystal structural of mouse importin α1 bound to the classic NLS peptide (PDB ID: 3L3Q) made by the tool from Pymol (www.pymol.org). Lower left, a closer view showing the structure of peptide containing a classic NLS (KKRR) bound to importin α subunit. Lower right, the structure that is mimetic of first lysine acetylation of NLS shows the disruption of its interaction with importin α subunit. The importin α protein is colored in green and the classic NLS peptide is in blue. The red dashed lines represent critical electrostatic interactions between the lysine side chain and the oxygen of importin α subunit.  (a) SIRT1 overexpression decreases K472 acetylation of PFKFB3. Flag-tagged PFKFB3 was coexpressed with either HA-tagged SIRT1 or SIRT2 in HEK293T cells. Cells were treated with low dose of NAM (2.5 mM) for 6 h. Flag-PFKFB3 was immunoprecipitated and the K472 acetylation was determined with α-AcK472 antibody. Relative PFKFB3 K472 acetylation was normalized by Flag protein.
(legend continued on next page) (b) PFKFB3 interacts with SIRT1, but not SIRT2. Flag-tagged PFKFB3 was co-expressed with empty vector or individual HA-tagged SIRTs as indicated in HEK293T cells. SIRTs were immunoprecipitated with HA beads, followed by immunoblotting with Flag antibody to detect PFKFB3.
(c) Verification of HEK293T cell lines with SIRT1 deletion. Alignment of genomic sequence of PFKFB3 from PFKFB3 KO cells and WT cells is shown. Both clones are heterozygous. Clone SIRT1-KO#1: one allele contains 127 nucleotides insertion, while the other allele contains 8 nucleotides deletion. Clone SIRT1-KO#2: the two alleles contain 1 or 209 nucleotides insertion, respectively.
(d) SIRT1 deletion increases K472 acetylation of endogenous PFKFB3. Endogenous PFKFB3 protein was immunoprecipitated from HeLa wild-type and two SIRT1 knockout cell lines. Immunoblotting was performed with the indicated antibodies. Relative PFKFB3 K472 acetylation was normalized by PFKFB3 protein. (legend continued on next page) (b) K472R mutant blocks PCAF and GCN5 induced the acetylation of PFKFB3. Flag-tagged wild-type (WT) or K472R mutant of PFKFB3 was co-expressed with different acetyltransferases indicated and immunoprecipitated by Flag beads. Acetylation of PFKFB3 was examined with the α-AcK antibody.
(c) Ectopic expression of GCN5 increases endogenous PFK activity. Endogenous PFK activity were assayed in HEK293T cells transfected with pCDNA3-Myc empty vector, GCN5 wildtype or acetyltransferase activity-dead mutant of GCN5 (D609A). Data are presented as mean ± s.d. of three biological replicates, and statistical analyses were performed by using one-way ANOVA with Dunnett's post-test. *** denotes p < 0.001 for the indicated comparison; n.s., not significant.
(d) Co-expression of GCN5 disrupts PFKFB3 binding with importin α5. Flag-PFKFB3 and HA-importin α5 were co-transfected with empty vector or different dose of Myc-tag GCN5 construct in HEK293T cells. After 36 h, cells were lysed and immunoprecipitated with Flag beads. Immunoblotting was performed with the indicated antibodies.