Fig 1: Nox4 deficiency enhances nuclear PP2A activity, this decreases nuclear ?H2AX but increases cytosolic AKT phosphorylation. (A) Interaction between PP2A and ?H2AX as determined by proximity ligation assay. Quantification of colocalization relative to the number of cells stained with DAPI in %. (B) PP2A abundance in cytosolic and nucleus fraction as evaluated by Western blot. (C) Serine-threonine phosphatase activity as measured under basal conditions and after treatment with OA in cytosol and nucleus with the aid of NPP as an artificial substrate. (D and E) ?H2AX abundance as quantified by immunoblotting with and without 1 nM of the PP2A inhibitor OA overnight (D) or H2O2 (24 h, 5 µM (E). (F) Nuclear expression of PP2A after treatment with or without H2O2 as determined by Western blot. All experiments were carried out in isolated fibrosarcoma cells (F). Statistics are represented as numbers below the representative Western blots; *P < 0.05 WT vs. Nox4-/-, #P < 0.05 Nox4-/- CTL vs. Nox4-/- OA/H2O2, n = 5. (G) Interaction between AKT and PP2A as determined by PLA. Quantification of colocalization relative to the number of cells stained with DAPI in % (H). Western blot for phosphorylation of AKT at Ser473 in fibrosarcoma tissue, colon carcinoma tissue, and isolated fibrosarcoma cells of WT and Nox4 -/- mice.
Fig 2: Imidazole Propionate-Induced Basal Akt Activation Is Responsible for Inhibitory AMPK S485 Phosphorylation(A) Effect of 2 h treatment with imidazole propionate (ImP) (at the indicated concentrations) on Akt S473 phosphorylation in amino acid-deprived HEK293 cells (n = 3).(B) Time-dependent effects of ImP (100 µM) on Akt phosphorylation in serum-starved HEK293 cells (n = 3).(C) Effects of mTORC1 inhibition (by 200 nM Rapamycin, Rap) or Akt inhibition (by 200 nM MK2206, MK) on ImP-induced Akt or mTORC1 activation. HEK293 cells preincubated with Rap or MK for 30 min were stimulated with 100 µM ImP for 1 h in the absence of amino acids (n = 4).(D and E) Effects of mTORC1 inhibition by rapamycin (20 nM Rap) or the Akt inhibitor MK (200 nM) on ImP-induced Akt or mTORC1 activation. Serum-starved HEK293 cells were co-incubated with 100 µM ImP and Rap or MK for 6 h (D) (n = 3) and for 8 h (E) (representative of n = 2).(F) mTORC1- and Akt-dependent inhibitory AMPK S485 phosphorylation by ImP. Serum-starved HEK293 cells were co-incubated with ImP and Rap or MK for 6 h (n = 3).Data are mean ± SEM. *p < 0.05, ***p < 0.001. One-way ANOVA with Dunnett’s multiple comparisons test (A–D), one-way ANOVA with Tukey’s multiple comparisons test compared to ImP-treated groups (F).
Fig 3: The interaction of PP2A and AKT is redox sensitive. (A) AKT redox modification as analyzed by BIAM switch assay in fibrosarcoma cells of WT and Nox4-/- (n = 3). (B) Mapping of the H2O2-dependently formed disulfide bridges on the structure of human AKT1 as obtained from 4EJN (25) (SI Appendix, Tables S1 and S2). (C) AKT1 to 3 proteins were immune-captured and copurified interacting proteins were identified by LC/MS. Label-free quantification (LFQ) values were statistically analyzed using permutation test (FDR <0.05, 250 randomizations) (SI Appendix, Table S3). Blue and red dots represent significantly enriched proteins. Black and gray dots were enriched in negative control or background, respectively. Marked protein Ppp2r1a was found to be significantly enriched in AKT1 to 3 pull-down samples. (D) Coimmunoprecipitation of AKT followed by Western blotting for PP2A catalytic subunit after overexpression with GFP, the redox-dead mutants for AKT1 to 3 and phospho-dead AKT1 mutant. (E and F) Proximity ligation in wild-type fibrosarcoma cells after overexpression of AKT1 and AKT3 and AKT1 and AKT3 redox-dead mutants (F). Antibodies used for AKT1 were PP2A and HA and for AKT3 PP2A and GFP. Red indicates dots of proximity, while gray represents DAPI staining of nuclei. Results of cells overexpressed with wild-type AKT were set to 100% (E). *P < 0.05 was considered significant.
Fig 4: PP2A translocation can be rescued by overexpressing AKT. PP2A (catalytic subunit) localization in the cytosol (A) and nucleus (B) was determined by Western blot after nuclear extraction. Cells were overexpressed with the plasmids indicated. *P < 0.05; WT/WT mutated AKT vs. Nox4-/-/Nox4-/- mutated AKT, #P < 0.05 Nox4-/- vs. Nox4-/- AKT OE, n = 5). (C) Comet assay, exemplary photos (C) and statistics (D) of WT and Nox4-/- cells with overexpression of the plasmids indicated *P < 0.05 WT vs. Nox4-/-, #P < 0.05 Nox4 GFP vs. Nox4-/- AKT mutant. n = 5.
Fig 5: Imidazole Propionate-Activated p38? Is a Direct Kinase for Akt, Responsible for Mediating Inhibitory AMPK S485 Phosphorylation(A) In vitro kinase assay (n = 4). p38? and inactive Akt1 were preincubated and the kinase reaction was started by adding ATP in the absence or presence of ImP at the indicated concentrations.(B) Effects of p38? depletion on ImP (100 µM)-induced AMPK and Akt phosphorylation in serum-starved HEK293 cells (n = 3).(C) Effects of the p38? inhibitor pirfenidone (Pirf) on ImP-induced inhibitory action on metformin (Met). Serum-starved HEK293 cells were co-incubated with 0.5 mM Met, 100 µM ImP, and Pirf at the indicated concentrations for 6 h (n = 3).(D and E) Effects of Pirf on ImP-induced inhibition of response to Met. Mice were injected intraperitoneally with vehicle (Veh, 1% DMSO), ImP (100 µg), Pirf (700 µg), or ImP with Pirf followed 1 h later by oral administration of Met (200 mg/kg) or water.(D) Immunoblot (and quantification) of liver lysates taken from chow-fed mice 45 min after Met (n = 4 mice per group).(E) Percent change in fasting blood glucose levels in chow-fed mice 45 min after Met (or water) versus start of experiment: Veh+Water (n = 7), Pirf+Water (n = 6), and Pirf+Met (n = 7) (left); Veh+Met (n = 4), ImP+Met (n = 4), and ImP+Met+Pirf (n = 4) (right).(F) Schematic depiction of imidazole propionate signaling. Interaction between Met and the ImP/p38?/Akt/AMPK axis investigated in this study was shown together with previously reported ImP/alternative p38/p62/mTORC1 axis (Koh et al., 2018).Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. One-way ANOVA followed by Dunnett’s multiple comparisons test (A and B), one-way ANOVA followed by Tukey’s multiple comparisons test (C–E).
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