Oxidative stress activates SIRT2 to deacetylate and stimulate phosphoglycerate mutase

Abstract

Glycolytic enzyme phosphoglycerate mutase (PGAM) plays an important role in coordinating energy production with generation of reducing power and the biosynthesis of nucleotide precursors and amino acids. Inhibition of PGAM by small RNAi or small molecule attenuates cell proliferation and tumor growth. PGAM activity is commonly upregulated in tumor cells, but how PGAM activity is regulated in vivo remains poorly understood. Here we report that PGAM is acetylated at lysine 100 (K100), an active site residue that is invariably conserved from bacteria, to yeast, plant, and mammals. K100 acetylation is detected in fly, mouse, and human cells and in multiple tissues and decreases PGAM2 activity. The cytosolic protein deacetylase sirtuin 2 (SIRT2) deacetylates and activates PGAM2. Increased levels of reactive oxygen species stimulate PGAM2 deacetylation and activity by promoting its interaction with SIRT2. Substitution of endogenous PGAM2 with an acetylation mimetic mutant K100Q reduces cellular NADPH production and inhibits cell proliferation and tumor growth. These results reveal a mechanism of PGAM2 regulation and NADPH homeostasis in response to oxidative stress that impacts cell proliferation and tumor growth.

Figure 1

PGAM2 is acetylated at K100. A, sequence alignment of PGAM surrounding K100 from various species, including human (Homo sapiens, NCBI reference number: NP_002620.1), mouse (Mus musculus, NP_0075907), chicken (Gallus gallus, NP_001026727), frog (Xenopus laevis, NP_001084996), zebrafish (Danio rerio, NP_942099.1), fruitfly (Drosophila melanogaster, AAF56866.2), thale cress (Arabidopsis thaliana, O04499), budding yeast (Saccharomyces cerevisiae, P00950), fission yeast (Schizosaccharomyces pombe, P36623), and bacterial (E. coli, WP_001333397.1). Bold, lysine 100. B, molecular modeling of acetylation of K100 in PGAM. The 3-PG binding site of PGAM (from Burkholderia pseudomallei, which shares 60% sequence identity with human PGAM) is rendered in the slate blue cartoon. The phosphorylated H11 is shown in slate blue (carbon), blue (nitrogen), red (oxygen), and orange (phosphorus). 3-PG is shown in sticks with carbon atoms colored white, oxygen atoms colored red, and phosphorus atoms colored orange. The green dashed lines represent critical electrostatic interactions between the lysine side chain and the oxygen of the phosphate of 3-PG. The acetyl group is colored in yellow (carbons). C, PGAM2 is acetylated. Flag-PGAM2 was transfected into HEK293T cells followed by treatment with TSA and NAM for indicated time. PGAM2 acetylation and protein levels were analyzed by Western blotting with indicated antibody. D, K100 is the primary acetylation site of PGAM2. The indicated plasmids were transfected into HEK293T cells and proteins were immunoprecipitated, followed by Western blot for acetylation analyses. E, the plasmids were transfected into HEK293T cells; acetylation levels of immunoprecipitated Flag-PGAM2 and K100R mutant were probed with the site-specific K100 acetylation antibody (α-K100Ac). F, endogenous PGAM2 is acetylated at K100. The HEK293T cells lysate were prepared after TSA and NAM treatment. Endogenous PGAM2 protein and K100 acetylation levels were determined by Western blotting with indicated antibodies. G, K100 acetylation is evolutionarily conserved. The A549, MEF, and S2 cells lysate were prepared after TSA and NAM treatment, respectively. Endogenous PGAM2 protein and K100 acetylation levels were determined by Western blotting with indicated antibodies. H, K100 acetylation is broadly distributed in different tissues. The indicated tissues were isolated from C57BL6 mouse and the lysate was prepared after homogenized. Endogenous PGAM2 protein and K100 acetylation levels were determined by Western blotting with indicated antibodies. IP, immunoprecipitation.

Figure 2

Acetylation at K100 reduces PGAM2 activity. A, inhibition of deacetylases decreases PGAM2 enzyme activity. Flag-tagged PGAM2 was expressed in HEK293T cells, which were treated with or without TSA and NAM, followed by immunoprecipitation, and enzyme activity was measured and normalized against protein levels. The protein levels and acetylation levels were determined by Western blotting. Mean values ± SD of relative enzyme activity of triplicate experiments are presented. B, PGAM2 is activated by in vitro deacetylation. Flag-PGAM2 was expressed in HEK293T cells, purified, and incubated with recombinant CobB. Samples were analyzed for acetylation levels and PGAM2 enzyme activity. Relative enzyme activities of triplicate experiments ± SD are presented. C, K100 mutation decreases PKM2 enzyme activity. Flag-tagged WT and mutant PGAM2 proteins were expressed in HEK293T cells and purified by immunoprecipitation. The enzyme activity was measured and normalized against protein level. Error bars represent ± SD for triplicate experiments. D, inhibition of deacetylases with TSA and NAM treatment decreases the activity of WT, but not K100-mutant PGAM2. Flag-tagged WT and mutant PGAM2 proteins were expressed in HEK293T cells, followed by treatment with TSA and NAM, and then purified by immunoprecipitation. The PGAM2 enzyme activity was measured and normalized against protein level. The mean value of triplicates and ± SD are presented. E, WT PGAM2, but not K100-mutant PGAM2, is activated by in vitro deacetylation. Flag-tagged WT and mutant PGAM2 proteins were expressed in HEK293T cells, then purified and incubated with recombinant CobB, followed by enzyme activity assay. Relative enzyme activities of triplicate experiments ± SD are presented. F, K100-acetylated PGAM2 has lower enzyme activity. Recombinant WT and K100-acetylated PGAM2 protein were purified in E. coli. The enzyme activity was measured and normalized against protein level. Mean values of relative enzyme activity of triplicate experiments with ± SD are presented.

Figure 3

SIRT2 deacetylates PGAM2 at K100. A, NAM, but not TSA, increases PGAM2 K100 acetylation. HEK293T cells were treated with either NAM or TSA, endogenous PGAM2 protein and K100 acetylation levels were determined by Western blotting with PGAM2 antibody or anti-acetyl-K100 antibody, respectively. B, SIRT2 interacts with PGAM2. HEK293T cells were transfected with indicated plasmids, and interactions between PGAM2 and SIRT1 or SIRT2 were examined by immunoprecipitation and Western blot analysis. C, WT SIRT2 overexpression decreases PGAM2 K100 acetylation. K100 acetylation levels of PGAM2 in HEK293T cells expressing indicated plasmids were detected by Western blotting. D, salermide, the SIRT2 inhibitor, increases the K100 acetylation of PGAM2. HEK293T cells were cultured at different concentrations of salermide. The K100 acetylation levels were probed by anti-acetyl-K100 antibody. E, knocking down SIRT2 increases endogenous K100 acetylation of PGAM2. HEK293T cells were infected with retrovirus targeting SIRT2, and the levels of PGAM2 protein and K100 acetylation were determined by Western blotting. SIRT2 knockdown efficiency was determined by quantitative PCR. Error bars represent ± SD for triplicate experiments. F, the K100 acetylation level of PGAM2 is increased in sirt2 knockout MEFs and decreased in hSIRT2 putting-back MEFs. The indicated cell lysates were prepared, and levels of PGAM2 protein and K100 acetylation were detected by Western blotting using indicated antibodies.

Figure 4

SIRT2 activates PGAM2. A, SIRT2 increases PGAM2 activity. HEK293T cells were transfected with indicated plasmids, Flag-PGAM2 was immunoprecipitated, and PGAM2 activity was assayed. Mean values of relative enzyme activity of triplicate experiments with ± SD are presented. B, SIRT2 knockdown decreases PGAM2 activity. Scramble and SIRT2 knocking-down stable HEK293T cells were transfected with Flag-PGAM2, and PGAM2 protein was immunoprecipitated and activity was assayed. Error bars represent ± SD for triplicate experiments. C, inhibition of SIRT2 decreases PGAM2 activity. HEK293T cells were transfected with Flag-PGAM2, followed by treatment with salermide. Flag-PGAM2 was immunoprecipitated, and PGAM2 activity was measured. Relative enzyme activities of triplicate experiments ± SD are presented. D, SIRT2 overexpression does not activate K100 mutants of PGAM2. WT and mutant PGAM2 were co-expressed in HEK293T cells with SIRT2, respectively, and purified by Flag beads, followed by enzyme assay and Western blotting. The mean value of triplicates and ± SD are presented. E, SIRT2 deacetylates and activates PGAM2 in vitro. Flag-PGAM2, HA-SIRT1, and HA-SIRT2 were purified from HEK293T cells, respectively, and the in vitro deacetylation assay was performed as described. Samples were determined by Western blotting with indicated antibodies.

Figure 5

Oxidative stress decreases K100 acetylation levels and induces PGAM2 activity. A, H₂O₂ treatment decreases endogenous K100 acetylation level. HEK293T and A549 cells were treated with H₂O₂ for different lengths of time as indicated. The levels of K100 acetylation were determined by Western blotting with anti-acetyl-PGAM2 (K100) antibody. B, H₂O₂-induced oxidative stress increases PGAM2 activity. HEK293T cells were transfected with Flag-PGAM2 and then treated with H₂O₂ for indicated time, followed by immunoprecipitation, and enzyme activity was measured and normalized against protein levels. Mean values of relative enzyme activity of triplicate experiments with ± SD are presented. C, H₂O₂ increases WT PGAM2 activity, but not K100 mutants. HEK293T cells were transfected with indicated plasmids, followed by H₂O₂ treatment for 2 hours, immunoprecipitated, and then PGAM2 activity was assayed. Relative enzyme activities of triplicate experiments ± SD are presented. D, oxidative stress increases the endogenous PGAM activity via reducing the K100 acetylation level. HEK293T cells or WT MEF cells were treated with H₂O₂ for 2 hours or Menadione (Mena) for 30 minutes, respectively, and then endogenous PGAM activity was assayed. The K100 acetylation levels of PGAM2 were determined by Western blot analysis. Relative enzyme activities of triplicate experiments ± SD are presented. E, oxidative stress increases the interaction between SIRT2 and PGAM2. HEK293T cells were transfected with indicated plasmids, and the SIRT2–PGAM2 association was examined by immunoprecipitation (IP)–Western blot analysis. F, Sirt2 mediates K100 deacetylation in response to oxidative stress. Sirt2^−/− MEFs and Sirt2^−/− MEFs stably expressing human SIRT2 were treated with H₂O₂ for indicated time. The change of K100 acetylation levels of PGAM2 was determined by Western blot analysis.

Figure 6

Acetylation mimetic K100Q mutant reduces NADPH amount and impairs the ability of cells protection from oxidative damage. A, identification of PGAM2 knocking-down and putting-back cell lines. Whole-cell lysates were prepared from PGAM2 knocking-down and putting-back stable cells, PGAM2 knockdown efficiency and re-expression were determined by Western blotting with indicated antibodies. B, A549 cells stably expressing acetylation mimetic K100Q mutant reduces NADPH production. Stable cells identified above were prepared, and NADPH was measured using an NADPH kit. Error bars represent ± SD for triplicate experiments. C, A549 cells expressing acetylation mimetic K100Q mutant accumulates ROS. The same stable cells identified above were prepared, and DCF staining was performed to measure ROS levels. Error bars represent ± SD for triplicate experiments. D, acetylation mimetic K100Q mutant impairs the ability of cells protection from oxidative damage. Stable cells were exposed to different concentrations of H₂O₂ for 24 hours, and the viability of cells was measured by trypan blue exclusion. Error bars represent ± SD for triplicate experiments. E, sirt2 KO MEFs are more sensitive to oxidative stress. sirt2 KO MEFs and human SIRT2 putting-back (PB) MEFs were exposed to different concentrations of H₂O₂ as indicated for 24 hours, and the viability of cells was measured applying trypan blue exclusion. Error bars represent ± SD for triplicate experiments. F, overexpression of WT PGAM2 in sirt2 KO MEFs can partially rescue the viability of sirt2 KO MEFs in response to oxidative stress. Indicated cells were infected with virus expressing PGAM2 wild-type or K100Q mutant for 30 hours, dispersed to 6-well plates, and then exposed to different concentrations of H₂O₂ for 24 hours. The viability of cells was measured by trypan blue exclusion. Error bars represent ± SD for triplicate experiments.

Figure 7

Acetylation mimetic K100Q mutant inhibits cell proliferation and tumor growth. A, acetylation mimic mutant K100Q suppresses cell proliferation. A total of 5 × 10⁴ indicated stable cells were seeded in each well. Cell numbers were counted every 24 hours. Error bars represent cell numbers ± SD for triplicate experiments. B, the expression of PGAM2 and PGAM2^K100Q in stable cell lines and xenograft tumors. Whole-cell extracts were prepared from either original A549 stable cell pools or xenograft tumors, followed analysis by Western blotting. C, acetylation mimic mutant K100Q inhibits xenograft tumor growth in vivo. Nude mice were injected with A549 PGAM2 cells or PGAM2^K100Q cells. The xenograft tumors were dissected and measured after 7 weeks and shown. D and E, quantification of average volume and weight of xenograft tumors are shown in D and E, respectively. Error bars represent ± SD for 7 tumors.