γ-6-Phosphogluconolactone, a Byproduct of the Oxidative Pentose Phosphate Pathway, Contributes to AMPK Activation through Inhibition of PP2A

Abstract

The oxidative pentose phosphate pathway (oxiPPP) contributes to cell metabolism through not only the production of metabolic intermediates and reductive NADPH but also inhibition of LKB1-AMPK signaling by ribulose-5-phosphate (Ru-5-P), the product of the third oxiPPP enzyme 6-phosphogluconate dehydrogenase (6PGD). However, we found that knockdown of glucose-6-phosphate dehydrogenase (G6PD), the first oxiPPP enzyme, did not affect AMPK activation despite decreased Ru-5-P and subsequent LKB1 activation, due to enhanced activity of PP2A, the upstream phosphatase of AMPK. In contrast, knockdown of 6PGD or 6-phosphogluconolactonase (PGLS), the second oxiPPP enzyme, reduced PP2A activity. Mechanistically, knockdown of G6PD or PGLS decreased or increased 6-phosphogluconolactone level, respectively, which enhanced the inhibitory phosphorylation of PP2A by Src. Furthermore, γ-6-phosphogluconolactone, an oxiPPP byproduct with unknown function generated through intramolecular rearrangement of δ-6-phosphogluconolactone, the only substrate of PGLS, bound to Src and enhanced PP2A recruitment. Together, oxiPPP regulates AMPK homeostasis by balancing the opposing LKB1 and PP2A.

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Figure 1.. G6PD attenuation activates PP2A to neutralize activated LKB1-AMPK signaling.

(A) Western blot showing effects of G6PD knockdown on AMPK and ACC1 phosphorylation in diverse human cancer cell lines from lung cancer, leukemia, colorectal cancer, prostate cancer, breast cancer and head and neck cancer. (B) Western blot showing effects of G6PD or 6PGD knockdown on AMPK and ACC1 phosphorylation in representative H1299, K562, HT29 and WiDr cells. (C) Western blot showing AMPK and ACC1 phosphorylation in H1299 cells with G6PD or 6PGD CRISPR knockout. (D-E) H1299 cells (left panels) and K562 cells (right panels) with G6PD or 6PGD stable knockdown were tested for lipogenesis (D) and intracellular level of Ru-5-P (E). (F) LKB1 precipitated from K562 cells with or without G6PD (left) and 6PGD (right) knockdown was incubated with recombinant AMPK (rAMPK) in an in vitro kinase assay. Western blot showing AMPK phosphorylation by LKB1. (G) Western blot showing the effect of G6PD (left) or 6PGD (right) stable knockdown on endogenous LKB1 and MO25 interaction in K562 cells. (H) Effect of G6PD or 6PGD stable knockdown on PP2A enzyme activity in H1299 (left) and K562 (right) cells. (I) Western blot showing effects of G6PD knockdown (left) or 6PGD knockdown (right) on AMPK, ACC1, and PP2A phosphorylation in H1299 cells and K562 cells. (J) PP2A enzyme activity (upper panels) and phosphorylation levels of both AMPK and PP2A (lower panels) were tested in H1299 cells with G6PD (left) and 6PGD (right) CRISPR knockout. (K-L) Effect of additional PP2A knockdown on AMPK and ACC1 phosphorylation (K), and cell proliferation rate (L) in K562 cells with G6PD knockdown. Data are mean ± SD (D, E, H, J and L). p values were obtained by a two-tailed Student’s t test except for a two-way ANOVA test in L (ns, not significant; *0.01 < p < 0.05; **0.001 < p < 0.01; *** p< 0.001).

Figure 2.. Two groups of cancer cells respond distinctly to G6PD attenuation due to differential SOD2 expression level and consequent ROS alterations.

(A) Cell proliferation rates normalized to the corresponding control cells in Group I (left) and Group II (right) diverse human cancer cells with G6PD stable knockdown. (B) General ROS levels in diverse Group I and Group II human cancer cells with G6PD stable knockdown were measured and compared. (C) SOD2 mRNA expression level in diverse Group I and Group II human cancer cells was detected by quantitative RT-PCR. (D) Western blot showing expression level of enzyme related to ROS production and scavenging in diverse Group I and Group II human cancer cells. (E) Western blot showing expression level of enzyme related to ROS production and scavenging in diverse Group I and Group II human cancer cells with G6PD stable knockdown. (F) Effect of additional SOD2 transient knockdown on cell proliferation rate and ROS level of H1299 cells with G6PD stable knockdown were detected by cell number counting 96 hours after transfection. Western blot showing the knockdown efficiency. (G) Effect of SOD2 overexpression on cell proliferation rate and ROS level of WiDr cells with G6PD stable knockdown were detected by cell number counting 96 hours after transfection. Western blot showing protein expression level. Data are mean ± SD (A, B, C, F and G). p values were obtained by a two-tailed Student’s t test (ns, not significant; *0.01 <p < 0.05; **0.001 < p < 0.01; ***p < 0.001)

Figure 3.. Tumor growth exhibit different pattern upon G6PD attenuation in the two groups of cancer cells.

(A-B) Xenograft tumor growth and tumor weight (left two panels), ROS level of xenograft tumor tissue (middle panel), Western blot showing AMPK, ACC1 and PP2A phosphorylation, and PP2A enzyme activity of tumor lysates (right 2 panels) in nude mice inoculated with Group I H1299 cells (A) and Group II HT29 cells (B) with G6PD CRISPR-knockout. (C-D) Cell proliferation rates of Group I H1299 and K562 cells (C), and Group II HT29 and WiDr cells (D) with G6PD stable knockdown in the presence or absence of 1mM NAC. Data are mean ± SD (A-D). p values were obtained by a two-tailed Student’s t test except for a two-way ANOVA test for tumor growth rates (A-B, left panels) and cell proliferation rates (D) (ns, not significant; *0.01 < p < 0.05; ***p < 0.001).

Figure 4.. PGLS knockdown inhibits PP2A but activates LKB1, leading to consequent AMPK activation with increased ROS.

(A) Schema showing linear oxidative pentose phosphate pathway with three key enzymes. (B) Western blot showing effects of PGLS knockdown on AMPK and ACC1 phosphorylation in Group I H1299 and K562 cells and Group II HT29 and WiDr cells. (C) Corresponding cell proliferation rates in Group I H1299 and K562 cells (left) and Group II HT29 and WiDr cells (right) with PGLS stable knockdown. (D) General ROS levels in Group I H1299 and K562 cells (left) and Group II HT29 and WiDr cells (right) with PGLS stable knockdown. (E) SOD2 mRNA expression level in H1299 and A549 cells with stable knockdown of G6PD, PGLS, or 6PGD was detected by quantitative RT-PCR. (F) SOD2 mRNA expression level in WiDr and HT29 cells with G6PD stable knockdown was detected by quantitative RT-PCR. (G) LKB1 precipitated from K562 cells with or without PGLS knockdown was incubated with rAMPK in an in vitro kinase assay. Western blot showing the phosphorylation of AMPK by LKB1. (H) Western blot showing effect of PGLS knockdown on endogenous LKB1 and MO25 interaction in K562 cells. (I) Effect of PGLS stable knockdown on PP2A activity in H1299 (left) and K562 (right) cells. (J) Western blot showing effects of PGLS stable knockdown on AMPK, ACC1, and PP2A phosphorylation in H1299 (left) and K562 (right) cells. (K) Cell proliferation rates of H1299 PGLS stable knockdown cells treated with or without 3mM NAC and/or 200nM Compound C. (L) ROS level (upper panel) in H1299 PGLS stable knockdown cells treated with or without 3mM NAC and/or 200nM Compound C was detected. Western blot showing AMPK phosphorylation level (lower panel). (M) NAC (10mg/mL drinking water) and/or Compound C (2 mg/kg, intraperitoneal injection at 4-day intervals) were administrated in H1299 xenograft mice with PGLS knockdown. Tumor weight (left), ROS level in tumor cells (middle), and quantification of IHC staining signal (right) were shown. Data are mean ± SD (C, D, E, F, I, K, L, and M). p values were obtained by a two-tailed Student’s t test except for a two-way ANOVA test for cell proliferation rates (K) (ns, not significant; *0.01 < p < 0.05; ***p < 0.001).

Figure 5.. The oxiPPP intermediate 6-phosphogluconolactone (6PGL) enhances inhibitory phosphorylation of PP2A by upstream kinase Src.

(A) Schema of oxidative pentose phosphate pathway (intermediate 6PGL is highlighted) (upper). Intracellular level of 6PGL (lower left) and 6PG (lower right) were measured in H1299 cells with G6PD, PGLS, and 6PGD individually stable knockdown. (B-C) Effect of 6PGL (B) and 6PG (C) treatment on AMPK, ACC1, and PP2A phosphorylation in K562 cells in a cell-free assay. (D-E) Cell lysates prepared from G6PD (D) or 6PGD (E) knockdown K562 cells were treated with increasing concentration of 6PGL, followed by detection of PP2A enzyme activity (upper panels) and western blot showing AMPK and PP2A phosphorylation (lower panels). Final levels (fold) of 6PGL were normalized to that in control cells without treatment. (F) Cell lysates from G6PD and PP2A double knockdown K562 cells were treated with increasing concentrations of 6PGL, followed by western blot analysis of AMPK phosphorylation (upper). Final levels (fold) of 6PGL were normalized to that in control cells without treatment. (G) Western blot showing effect of G6PD knockdown (left) and 6PGD knockdown (right) on endogenous Src and PP2A interaction in K562 cells. (H- I) Src precipitated from K562 cells was incubated with recombinant PP2A (rPP2A) as substrate in the presence of increasing concentrations of 6PGL in an in vitro kinase assay. PP2A phosphorylation (H) and PP2A-Src interaction (I) were detected using western blot. Data are mean ± SD (A and D). p values were obtained by a two-tailed Student’s t test (ns, not significant, *0.01 < p < 0.05; **0.001 < p < 0.01; ***p < 0.001;).

Figure 6.. OxiPPP byproduct γ-6PGL enhances the inhibition of PP2A by Src.

(A) Schema for the first two steps of the oxidative pentose phosphate pathway. G6P is catalyzed by G6PD to produce δ-6PGL, which is spontaneously exchanged for γ-6PGL through intramolecular rearrangement. (B) The intracellular concentrations of δ-6PGL and γ-6PGL in H1299 cells with individual knockdown of G6PD, PGLS, and 6PGD were measured by quantitative ³¹P nuclear magnetic resonance (³¹PNMR) spectroscopy. Data were shown as one representative result of two independent biological experiments. (C-D) Src precipitated from K562 cells was incubated with purified rPP2A (C) or with recombinant FAK (rFAK) (D) as substrate in the presence of increasing concentrations of γ-6PGL (left) and δ-6PGL (right) in an in vitro kinase assay. Phosphorylation of PP2A and FAK were detected by western blot. (E) Src precipitated from K562 cells was incubated with recombinant myelin basic protein (MBP) as substrate in an in vitro kinase assay in the presence of increasing concentrations of γ-6PGL (upper) and δ-6PGL (lower). Samples were applied to western blot to detect MBP phosphorylation. (F-G) Src precipitated from K562 cells was incubated with purified rPP2A (F) or recombinant FAK (rFAK) (G) as substrate in the presence of increasing concentrations of γ-6PGL and δ-6PGL in an in vitro kinase assay. The interactions between Src and PP2A (F) or Src and FAK (G) were evaluated by western blot.

Figure 7.. γ-6PGL binds to Src to recruit substrate PP2A.

(A) Thermal melt-shift assay was performed to examine purified recombinant protein Src (rSrc) (left two panels) or PP2A (right two panels) and ligand (γ-6PGL or δ-6PGL) interaction. Arrows indicate melting temperature at 0 μM and 40 μM. (B) Thermal melt-shift assay was performed to examine purified recombinant truncated Src domain and ligand γ-6PGL interaction. The full length Src contains amino acids 1–536, the ΔC domain contains amino acids 1–247, the catalytic domain contains amino acids 250–536, and the ΔN domain contains amino acids 88–536. Arrows indicate melting temperature at 0μM and 40μM. (C) Purified rSrc was pre-treated with increasing concentrations of γ-6PGL (left) or δ-6PGL (right), followed by incubation with purified rPP2A in vitro. Src and PP2A interaction was then detected by western blot. (D) Purified rPP2A was pre-treated with increasing concentrations of γ-6PGL (left) or δ-6PGL (right), followed by incubation with purified rSrc protein in vitro. Src and PP2A interaction was then detected by western blot. (E) Purified rSrc was pre-treated with increasing concentrations of γ-6PGL (left) or δ-6PGL (right), followed by incubation with purified recombinant PP2A in vitro. The phosphorylation of PP2A was detected by western blot. (F) Purified recombinant truncated Src domains, catalytic domain (left), ΔN domain (middle), and AC domain (right), were pre-treated with increasing concentrations of γ-6PGL, followed by incubation with rPP2A in vitro. The truncated Src domains and rPP2A interaction were then detected by western blot. (G) Proposed working model. OxiPPP regulates AMPK homeostasis by balancing the opposing LKB1 and PP2A. The byproduct γ-6PGL converted from δ-6PGL promotes Src-dependent inhibition of PP2A by binding to Src and subsequently enhancing PP2A recruitment, which contributes to AMPK activation.