The tumor suppressor p53 invokes anti-proliferative processes, of which the best understood include cell cycle arrest, DNA repair, and apoptosis 6,7
. Recent studies suggested that p53 also has a role in modulating metabolism including glycolysis and oxidative phosphorylation 8,9,10
. However, the role of p53 in regulating biosynthesis is less understood. The PPP is a main pathway for glucose catabolism and biosynthesis 5
. In an oxidative phase, the PPP generates NADPH (nicotinamide adenine dinucleotide phosphate), the principal intracellular reductant required for reductive biosynthesis such as the synthesis of lipid, and an essential precursor for biosynthesis of nucleotides. This is followed by a nonoxidative inter-conversion of ribose 5-phosphate to the intermediates in the glycolytic pathways. Despite the vital role of the PPP in biosynthesis and its close link to glycolysis, the regulation of the PPP in tumor cells remains unclear.
To investigate whether p53 modulates the PPP, we compared the oxidative PPP flux in isogenic p53+/+
human colon cancer HCT116 cells 11
. Cells were cultured in medium containing [2-13
C]glucose, and the glucose metabolites were measured by nuclear magnetic resonance (NMR) spectroscopy. As shown in , the absence of p53 resulted in a strong enhancement (~50%) in oxidative PPP flux, suggesting that p53 suppresses the PPP. The absence of p53 concomitantly led to a strong increase in glucose consumption, and this was observed in both HCT116 cells and mouse embryonic fibroblast (MEF) (). Inhibition of G6PD using either small interfering RNA (siRNA) or dehydroepiandrosterone (DHEA) revered the increase in glucose consumption caused by p53 deficiency, while having little effect on glucose consumption in p53+/+
cells (). These results suggest that p53 deficiency increases glucose consumption mainly through an enhanced PPP flux.
p53 deficiency correlates with increases in PPP flux, glucose consumption, and lactate production
The lack of p53 also correlated with elevated lactate production (). However, inhibition of G6PD in these cells increased, rather than decreased, in lactate production regardless of p53 status. Therefore, glucose flux through the PPP may in itself lower lactate production. The suppression of lactate production may be related to the ability of p53 to decrease glycolysis 8
or increase oxidative phosphorylation 9
The PPP plays a major role in the production of cellular NADPH. The lack of p53 led to a strong increase in the NADPH level in HCT116 cells (~ 2 folds, ). Likewise, knocking down of p53 in U2OS cells with small hairpin RNA (shRNA) strongly increased NADPH levels (Supplementary Information, Fig. S1a
). Treatment with G6PD siRNA minimized the difference in NADPH levels between p53 proficient and deficient cells. To verify the cell culture findings in animals, we compared the NADPH levels in various tissues from p53−/−
mice. The tissues from p53−/−
mice – including heart, liver, kidney, and lung – exhibited substantially elevated NADPH levels compared to those in the corresponding tissues from p53+/+
mice (). The exception was found in the spleen. In this tissue, the activity of G6PD was very low (), and the PPP might not contribute substantially to the overall NADPH production. Converse to p53 down-regulation, over-expression of p53 led to a strong decrease in NADPH levels (Supplementary Information, Fig. S1b
p53 regulates NADPH levels, lipid accumulation, and G6PD activity
NADPH is required for the biosynthesis of lipid. To assess the effect of p53 on lipid accumulation, we treated p53+/+
MEF cells with a combination of insulin, rosiglitazone, dexamethasone, and isobutylmethylxanthine, which stimulates lipogenesis 12
. The p53−/−
MEFs showed enhanced lipid levels compared to p53+/+
MEFs as evaluated by Oil Red O staining (). The lack of p53 also resulted in higher levels of lipid In HCT116 cells (Supplementary Information, Fig. S1c
). The difference in lipid accumulation between p53+/+
cells diminished upon treatment with G6PD siRNA or DHEA. We also evaluated the effect of p53 on the formation of fat droplets in the liver. The liver of p53−/−
mice had a larger amount of bigger fat droplets compared to the liver of p53+/+
mice (). Together, these results suggest that p53 inhibits NADPH production and lipid accumulation by lowering the glucose flux through the PPP.
To investigate the mechanism by which p53 regulates the PPP, we assayed the activity of G6PD, a key regulatory point of the PPP. The lack of p53 correlated with a strong elevation in G6PD activity in both MEF and HCT116 cells ( and Supplementary Information, Figs. S1d, e
). Similarly, when p53 was knocked down in U2OS cells with shRNA, G6PD activity nearly doubled (). Furthermore, in mice tissues where G6PD activity could be adequately detected (e.g. liver, lung, and kidney), the lack of p53 was associated with highly elevated G6PD activity (). Conversely, over-expression of wild type p53 in the p53-deficient cell lines (H1299 and p53−/−Mdm2−/−
MEF) caused a noticeable decrease in G6PD activity (Supplementary Information, Fig. S1f, g
). These results show that p53 suppresses G6PD activity.
In each of the cell lines and tissues that were examined, the levels of the G6PD protein remained unchanged when p53 was down regulated or overexpressed ( and Supplementary Information, Fig. S1
). Moreover, p53 did not change the level of G6PD transcript (). To rule out the involvement of other p53 target genes in the inhibition of G6PD, we used an inhibitor of p53 transcriptional activity, pifithrin-α (PFTα)13
. PFTα impeded p53-inuced expression of p21, but did not restore p53-inhibited G6PD activity ( and Supplementary Information, Fig. S2a
). We also used the protein synthesis inhibitor cycloheximide (CHX), alone or together with the DNA damage agent doxorubicin (DOX). Treatment of p53+/+
HCT116 cells with CHX alone resulted in a lower level of p53, which was accompanied by a higher activity of G6PD (). Simultaneous treatment with CHX and DOX led to a stabilization of p53 above the basal level seen in unstressed cells, and a concurrent drop of G6PD activity below its basal level (). As controls, none of these treatments altered G6PD activity in p53−/−
HCT116. In addition, the p53 mutant V122A, which has a transactivation activity comparable to or even higher than wild type p53 dependent on the target gene 14
, failed to inhibit G6PD (Supplementary Information, Fig. S2b
). Moreover, we treated cells with the nuclear export inhibitor leptomycin B to prevent cytoplasmic accumulation of p53 15,16
. Leptomycin B reversed p53-mediated inhibition of G6PD (Supplementary Information, Fig. S2c-e
). Altogether, these results show that inhibition of G6PD by p53 is independent of transcription or translation and is a cytoplasmic, not nuclear, function of p53.
p53 interacts with G6PD and inhibits its activity independently of transcription
We next investigated whether p53 interacts with G6PD. Flag-tagged p53 specifically associated with enhanced green fluorescent protein (eGFP)-G6PD in vivo
(). Likewise, endogenous p53 interacted with endogenous G6PD (). G6PD is a cytoplasmic protein while p53 is present in both the cytoplasm and the nucleus, and consistently, the p53-G6PD interaction occurred in the cytoplasm (Supplementary Information, Fig. S2f-h
). This interaction was enhanced when cells were treated with the proteasome inhibitor MG132 or the DNA damaging agent DOX, both of which stabilized p53 ( and Supplementary Information, Fig. S2h
). The binding between p53 and G6PD is direct as shown by a pull-down assay with purified recombinant proteins (). Analysis of the p53-G6PD binding in real time using surface plasmon resonance (BIAcore) showed that the dissociation constant (KD
) of p53 from G6PD was 173 ± 50 nM (). G6PD is a highly conserved protein, and human p53 interacted with G6PD proteins from both the bacterium L. mesenteroides
and the yeast S. cerevisiae
(Supplementary Information, Figs. S3a–c
p53 inhibits the formation of dimeric G6PD holoenzyme
To delineate the structural determinants for p53’s inhibitory activity towards G6PD, we used a panel of p53 deletion mutants. The G6PD interaction domain was mapped to the C-terminal (CT) region of p53 ( and Supplementary Information, Figs. S3b, c
). However, the CT region alone was not sufficient for G6PD inhibition; the transactivation (TA) and DNA-binding domain (DBD) were also required ( and Supplementary Information, Fig. S1f
). A further deletion analysis showed that within the CT region, the negative regulatory (NR) domain, but not the tetramerization (TET) domain, was required for interaction with and inhibition of G6PD (Supplementary Information, Figs. S4a, b
The involvement of multiple domains of p53 in G6PD inhibition led us to test tumor-associated p53 mutants, the majority of which harbor missense mutations. Unlike wild type p53, three p53 mutants (R175H, R273H, and G279E) showed minimal or no activity in inhibiting G6PD ( and Supplementary Information, Fig. S1g, S2b, S3g
), even though at least two of them (R175H and R273H) retained the ability to bind to G6PD (Supplementary Information, Fig. S4c
). Another tumor-associated, temperature sensitive mutant (A138V) inhibited G6PD activity at the permissive temperature 32 °C, but not at the non-permissive temperature 37 °C. Moreover, the inhibitory effect at 32 °C was abolished with the introduction of R273H (Supplementary Information, Fig. S4d
). To examine the effect of p53 mutants that are expressed at endogenous levels, we used a panel of SW480 cells, in which the endogenous p53 mutant (R273H/P309S) can be inducibly knocked down and, at the same time, exogenous p53 mutants containing either G245S or R248W, alone or in combination with alterations in the activation domain (AD) 1 or 2 17
, were inducibly expressed at levels comparable to the endogenous p53 mutant (Ref. 17
). Replacement of the endogenous mutant p53 with exogenous mutants caused little or no changes in G6PD activity (Supplementary Information, Fig. S4e
). By contrast, a p53 mutant with increased thermodynamic stability (N239Y) 18
, exhibited enhanced ability to inhibit G6PD (). Because tumor-associated mutations impair the native conformation of p53 and the N239Y mutation stabilizes it, the inhibition on G6PD is likely attributed to the native conformation of p53.
How may p53 inhibit G6PD activity? G6PD is in an equilibrium of inactive monomer and active dimer 19,20
. In both HCT116 and MEF cells, the lack of p53 led to a strong increase in G6PD dimer and a corresponding decrease in G6PD monomer (). In a transfection assay, p53 reduced the interaction of two differentially tagged G6PD (Flag-G6PD and eGFP-G6PD) in a dose-dependent manner (). G6PD requires its substrate NADP+
as the cofactor for the formation of holoenzyme, a property that ensures higher G6PD activity and thus more NADPH production when NADPH/NADP+
ratio drops 5
diminished the interaction between G6PD and p53 in a dose-dependent manner (), suggesting that the binding of p53 to G6PD is incompatible with the binding of NADP+
to G6PD. These results suggest that p53 may disrupt the formation of the dimeric G6PD holoenzyme.
The levels of p53 are kept low in unstressed cells due to its rapid degradation in the proteasome. A semi-quantitative western assay showed the levels of cytoplasmic p53 were approximately 3% that of G6PD. The levels of p53 increased to ~10% that of G6PD when p53 was stabilized by either MG132 or doxorubicin (Supplementary Fig. S5a
). Accordingly, the majority of p53 molecules bound to G6PD in unstressed cells, but a small percentage of G6PD bound to p53, as shown by immunodepletion assays (). Only when p53 was stabilized by MG132 and DOX did a significant portion of p53 molecules become separated from G6PD ().
p53 suppresses G6PD through transient interaction and at substoichiometric ratios
The discrepancy between the amounts of p53 that stably bind to G6PD and the strong effect of p53 on overall G6PD activity raises the possibility that p53 is capable of inhibiting G6PD activity through transient interaction and at sub-stoichiometric ratios. To test whether p53 can inhibit G6PD via transient interaction, we incubated lysates of p53−/−
MEF cells with recombinant p53 immobilized on beads and then separated the lysates from the beads. The treated lysates contained virtually all of the G6PD protein and un-detectable amounts of p53 (, and Supplementary Information, Fig. S5b
), but showed much reduced G6PD activity (~50%) and low levels of G6PD dimer (). In the same experiment, un-conjugated control beads and beads conjugated with p53ΔDBD- or p53 R273H protein all failed to inhibit G6PD activity and G6PD dimerization.
To assess whether one p53 molecule can inhibit multiple G6PD molecules, we first mixed extracts of p53+/+ HCT116 cells treated with G6PD siRNA (with low G6PD, but normal p53 level) with extracts of p53−/− HCT116 cells (with normal G6PD level, but no p53). The G6PD activity in the mix was substantially lower than the calculated average of these two extracts (, column 4), suggesting that the small amounts of p53 protein in the former extracts could significantly inhibit the activity of a much larger amount of G6PD in the latter extracts. In a control experiment, when p53+/+ HCT116 extracts were replaced with p53−/− HCT116 extracts, the G6PD activity in the mix was approximately the average of the two extracts that were used, as expected (, column 6).
We next mixed purified recombinant proteins at low molar ratios of p53 versus G6PD. At a ratio of 2.5% (approximately the ratio of these proteins in unstressed cells, Supplementary Fig. S5a
), p53 decreased G6PD activity by 20% (), suggesting that one p53 molecule could inactivate up to eight G6PD molecules. This was likely an underestimate because of the lability of p53 protein. The inhibition efficiency decreased at higher ratios of p53 versus G6PD, but increased at a lower molar ratio (). In contrast, R273H and R175H mutants failed to inhibit G6PD at any ratio examined (). Altogether, these results suggest that wild type p53 may act as a catalyst to inactive G6PD.
To evaluate cytoplasmic and nuclear pools of p53 in the inhibition of G6PD, we purified p53 proteins from these cellular compartments. Cytoplasmic p53 exhibited robust inhibitory activity towards G6PD. However, nuclear p53 protein showed minimal activity (). In the cell lysates, cytoplasmic p53 was mainly in monomeric form, while nuclear p53 was mostly in tetrameric form (Supplementary Information, Fig. S5c
). This difference remained even after these proteins were purified and adjusted to the same concentration (Supplementary Information, Fig. S5d
). Therefore, the cytoplasmic and nuclear p53 proteins appear to be intrinsically different in their oligomerization status and their ability to inhibit G6PD.
The current study identifies an important role for p53 in regulating G6PD. Through this regulation, p53 exerts a powerful surveillance on the metabolic pathways that are critical for both glucose catabolism and biosynthesis. The suppression of G6PD by p53 is evident in unstressed cells and is independent of transcription. Because the majority of cytoplasmic p53 associates with G6PD in unstressed cells (), inhibiting G6PD is likely a main function of cytoplasmic p53 in these cells. In stressed cells where p53 is stabilized, a portion of cytoplasmic p53 becomes free of G6PD and may perform other functions previously attributed to cytoplasmic p53, such as the interaction with Bcl-2 family proteins 21
. Notably, p53 can inhibit G6PD through transient interactions and at levels much lower than that of G6PD, suggesting that p53 may act as a catalyst to induce conformational changes in G6PD. This function is attributed to the entire p53 protein, perhaps with the exception of the tetramerization domain, and appears to be specific to the p53 proteins originated from the cytoplasm, but not the nucleus. p53-mediated inhibition of the PPP likely dominates the effect of TIGAR, a previously identified p53 target gene that stimulates the PPP 22
; this is shown by the enhanced PPP flux and NADPH production in p53 deficient cells. Given the importance of p53 in suppressing the PPP, the prevalent inactivation of p53 in tumor cells likely accelerates glucose consumption, and, at the same time, directs glucose for rapid production of macromolecules via an increase in the PPP flux. Therefore, p53 inactivation not only contributes to the Warburg effect but also links it to enhanced biosynthesis.