p53 regulates biosynthesis through direct inactivation of glucose-6-phosphate dehydrogenase

Journal name:
Nature Cell Biology
Volume:
13,
Pages:
310–316
Year published:
DOI:
doi:10.1038/ncb2172
Received
Accepted
Published online

Cancer cells consume large quantities of glucose and primarily use glycolysis for ATP production, even in the presence of adequate oxygen1, 2. This metabolic signature (aerobic glycolysis or the Warburg effect) enables cancer cells to direct glucose to biosynthesis, supporting their rapid growth and proliferation3, 4. However, both causes of the Warburg effect and its connection to biosynthesis are not well understood. Here we show that the tumour suppressor p53, the most frequently mutated gene in human tumours, inhibits the pentose phosphate pathway5 (PPP). Through the PPP, p53 suppresses glucose consumption, NADPH production and biosynthesis. The p53 protein binds to glucose-6-phosphate dehydrogenase (G6PD), the first and rate-limiting enzyme of the PPP, and prevents the formation of the active dimer. Tumour-associated p53 mutants lack the G6PD-inhibitory activity. Therefore, enhanced PPP glucose flux due to p53 inactivation may increase glucose consumption and direct glucose towards biosynthesis in tumour cells.

At a glance

Figures

  1. p53 deficiency correlates with increases in PPP flux, glucose consumption and lactate production.
    Figure 1: p53 deficiency correlates with increases in PPP flux, glucose consumption and lactate production.

    (ap53+/+ and p53−/− HCT116 cells were cultured in medium containing [2-13C]glucose. Oxidative PPP flux (top) was measured based on the rate of glucose consumption and the ratio of 13C incorporated into carbon 2 (indicating glycolysis) and carbon 3 (indicating PPP) of lactate by NMR spectroscopy. Data are means ± s.d. (n=3). Protein expression is shown at the bottom, with molecular weight standards indicated on the left. (bp53+/+ and p53−/− HCT116 cells were treated with G6PD siRNA and control siRNA (−). Top: glucose consumption. Data are means ± s.d. (n=3). Bottom: the expression of p53, G6PD and actin (a loading control). (cp53+/+ and p53−/− MEF cells were treated with 1mM DHEA or vehicle (−) for 24h. Top: glucose consumption. Data are means ± s.d. (n=3). Bottom: protein expression (d,e). Lactate levels in cells from b (d) and c (e). Data are means ± s.d. (n=3 for each panel).

  2. p53 regulates NADPH levels, lipid accumulation and G6PD activity.
    Figure 2: p53 regulates NADPH levels, lipid accumulation and G6PD activity.

    (a) NADPH levels (means ± s.d., n=3) in p53+/+ and p53−/− HCT116 cells treated with G6PD siRNA or control siRNA. Protein expression is shown below. (b) NADPH levels (means ± s.d., n=3) in tissues from p53+/+ and p53−/− mice maintained on a normal diet. Protein expression is shown below. (cp53+/+ and p53−/− MEF cells treated with or without DHEA were cultured in the presence of insulin, rosiglitazone, isobutylmethylxanthine and dexamethasone. Lipid contents were analysed by Oil Red O staining. Left: Oil Red O-stained dishes. Right: staining was quantified by absorbance at 500nm. Data are means ± s.d. (n=3). (d) Histological sections of liver tissue from p53+/+ and p53−/− mice were stained with haematoxylin and eosin. Arrows indicate fat droplets. (e) G6PD activity (means ± s.d., n=3) in p53+/+ and p53−/− MEF cells treated with or without DHEA. (f) U2OS cells stably expressing p53 shRNA or control shRNA (−) were transfected with G6PD siRNA or control siRNA (−). G6PD activity (top) and protein expression (bottom) were analysed. Data are means ± s.d. (n=3). (g) G6PD activity in tissues from p53−/− and p53+/+ mice maintained on a normal diet. G6PD activity is the mean ± s.d. of three p53+/+ or four p53−/− mice.

  3. p53 interacts with G6PD and inhibits its activity independently of transcription.
    Figure 3: p53 interacts with G6PD and inhibits its activity independently of transcription.

    (ap53+/+ and p53−/− HCT116 cells, and U2OS cells transfected with p53 shRNA and control shRNA, were analysed by PCR with reverse transcription (top) and western blot (bottom). (b,c) p53+/+ and p53−/− HCT116 cells were treated with or without 20μM of PFTα for 24h (b), or treated with or without doxorubicin (2μM) for 1h, and then with or without cycloheximide (20μM) for 2h (c). Cells were analysed for G6PD activity (top) and protein expression (bottom). Data are means ± s.d. (n=3). (d) H1299 cells were transfected with eGFP–G6PD alone or together with increasing amounts of Flag–p53. Cell lysates were immunoprecipitated with an anti-Flag antibody and an isotype-matching control antibody (IgG). Immunoprecipitated proteins (IP) and 5% input were analysed by western blot. (e) p53+/+ HCT116 cells were treated with MG132 (20μM), doxorubicin (2μM) or vehicle (dimethylsulphoxide). Cell lysates were incubated with anti-G6PD antibody or a control antibody (IgG). Immunoprecipitates and input were analysed by western blot. (f) Left, schematic representation of p53 and its deletion mutants. WT, wild-type; TA, transactivation domain; DBD, DNA-binding domain; CT, C-terminal region; TET, tetramerization domain; NR, negative regulation domain. The amino acids at the domain boundaries are indicated. Right, purified GST and GST fusions of wild-type and mutant p53 proteins were incubated separately with recombinant Flag–G6PD protein conjugated to beads. Beads-bound and input proteins were analysed by western blot using anti-Flag (top) and Coomassie blue staining (bottom). (g) The dissociation constant (Kd) for p53 from immobilized G6PD was determined by surface plasmon resonance (BIAcore). A real-time graph of response units against time is shown. The on rate was 7.53±1.59×103M−1s−1, and the off rate was 1.30±0.03×10−3s−1.

  4. p53 inhibits the formation of dimeric G6PD holoenzyme.
    Figure 4: p53 inhibits the formation of dimeric G6PD holoenzyme.

    (a,b) Activity of the G6PD protein after being incubated with increasing amounts of purified wild-type or mutant p53 proteins (top). Proteins were analysed by silver staining (middle) and anti-Flag western blot (bottom). The p53 proteins were tagged with either three copies (a) or one copy (b) of the Flag epitope. Data are means ± s.d. (n=3). (c,d) H1299 cells (c) and p53−/−Mdm2−/− MEF cells (d) were transfected separately with wild-type and mutant p53 proteins as indicated. G6PD activity (top) and protein expression (bottom) were analysed. The K386R mutation blocks p53 SUMOylation. Data are means ± s.d. (n=3). (e) Extracts of p53+/+ and p53−/− HCT116 and MEF cells were treated with and without 5mM disuccinimidyl suberate (DSS) and analysed by western blot with antibodies against G6PD and p53, and as controls, tubulin and actin. The positions of various forms of G6PD and p53 are indicated. (f) H1299 cells were transfected with Flag–G6PD, eGFP–G6PD and different amounts of haemagglutinin (HA)–p53. Cell lysates were incubated with anti-Flag antibody. Input and immunoprecipitates were analysed by western blot. (g) Lysates from p53−/−Mdm2−/− MEF cells expressing eGFP or eGFP–G6PD were incubated with Flag–p53 immobilized on M2 beads or control beads in the presence of increasing amounts of NADP+ (0, 0.1 and 1mM). Input and beads-bound (pulldown) proteins were analysed by western blot. HC, IgG heavy chain; asterisk, non-specific band.

  5. p53 suppresses G6PD through transient interaction and at substoichiometric ratios.
    Figure 5: p53 suppresses G6PD through transient interaction and at substoichiometric ratios.

    (a,bp53+/+ HCT116 cells were treated with dimethylsulphoxide, MG132 and doxorubicin. The cytosolic fraction was immunoprecipitated separately with a control antibody and anti-p53 (a) or G6PD (b) antibody, plus protein A/G beads. The lysates before (−) and after immunoprecipitation were analysed by western blot. (c,d) Lysates from p53−/− MEF cells were first incubated with Flag-tagged p53 proteins immobilized on beads and control beads, and then were separated from the beads. (c) Levels of proteins (top and middle) and G6PD dimerization (bottom) in the input lysates (Input) and supernatant after being incubated with the beads. (d) G6PD activity in the input lysates and supernatants. Data in d are means ± s.d. (n=3). (ep53+/+ and p53−/− HCT116 cells were either treated separately with G6PD siRNA and control siRNA, or untreated. Filled columns, actual G6PD activity in the 1:1 mixtures of the indicated lysates (lanes 4 and 6) and in individual lysates (the other lanes). Dashed columns, expected G6PD activity in mixtures based on the averages of the individual lysates. Protein expression is shown at the bottom. The samples were derived from the same conditions. Data are means ± s.d. (n=3). (f,g) Left, activity of G6PD after being incubated with total wild-type and mutant p53 proteins (f) or cytosolic, nuclear and total wild-type p53 (g). p53 proteins were used at molar ratios of 1, 2.5, 5 and 10% that of G6PD. Data are means ± s.d. (n=3). Right, Coomassie blue staining of purified proteins.

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Author information

  1. These authors contributed equally to this work

    • Peng Jiang &
    • Wenjing Du

Affiliations

  1. Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, 230027, China

    • Peng Jiang,
    • Wenjing Du,
    • Xingwu Wang &
    • Mian Wu
  2. Department of Cancer Biology and Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19096, USA

    • Peng Jiang,
    • Wenjing Du,
    • Anthony Mancuso &
    • Xiaolu Yang
  3. Model Animal Research Center, State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, China

    • Xiang Gao

Contributions

P.J., W.D., M.W. and X.Y. designed the experiments and interpreted results. P.J. and W.D. carried out all the experiments, except those mentioned below. X.W. carried out the experiments on G6PD activity in yeast, the surface plasmon resonance, and lipid droplets in mouse liver. A.M. and P.J. analysed the oxidative PPP flux. X.G. supplied the p53 wild-type and knockout mice. X.Y. wrote the manuscript with the help of P.J. and W.D.

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The authors declare no competing financial interests.

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