Fig. 5: AKT1 phosphorylation of ME2fl induces a metabolic switch to glycolytic activity from mitochondrial respiration.
From: AKT1 phosphorylation of cytoplasmic ME2 induces a metabolic switch to glycolysis for tumorigenesis
a Total lysates or anti-Flag immunoprecipitants from transfected HEK293T cells expressing 3’HA-tagged ME2flS9D (ME2flS9D-3’HA) together with Flag-tagged glycolytic enzymes or Flag vector control as indicated were analyzed by immunoblotting. Representative results are shown from three independent experiments. b Cytoplasmic fractions of H1299 cells were immunoprecipitated with an anti-ME2fl antibody and then analyzed by western blotting with the anti-PFK, anti-GAPDH, anti-PKM, and anti-LDH antibodies, respectively. Representative results are shown from three independent experiments. c HCT116 cells were stably infected with control lenti-virus (pCDH-Flag Vec) or lenti-viruses expressing wild-type ME2fl (pCDH-ME2fl-3’Flag), ME2flS9A (pCDH-ME2flS9A-3’Flag) or ME2flS9D (pCDH-ME2flS9D-3’Flag). The activity of the indicated glycolytic enzymes was measured (left) and protein expression was analyzed by western blotting (right). Data are means ± SD, two-tailed Student’s t test. d, e HCT116 cells stably expressing wild-type ME2fl (pCDH-ME2fl-3’Flag), ME2flS9A (pCDH-ME2flS9A-3’Flag), ME2flS9D (pCDH-ME2flS9D-3’Flag), or vector control (pCDH-Flag Vec) were used for ECAR (d) or OCR (e) analysis. Cells were supplied with 25 mM glucose, 1 μM oligomycin and 100 mM 2-DG at the indicated times for ECAR analysis, and 1 μM oligomycin, 1 μM FCCP, and 2.5 μM antimycin/rotenone at the indicated times for OCR analysis by using a Seahorse XFe96 analyzer system. n = 6 biologically independent experimental repeats for each group in (d) and n = 4 biologically independent experimental repeats for each group in (e). Data are means ± SD, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; NS, no significance; two-tailed Student’s t test. Exact P values are shown in Source data. f–h The abundance of NAD+ and NADH, and the NAD+/NADH ratio (f), and NADPH levels (g) in HCT116 cells stably expressing ME2fl (pCDH-ME2fl-3’Flag), ME2flS9A (pCDH-ME2flS9A-3’Flag), ME2flS9D (pCDH-ME2flS9D-3’Flag), or vector control (pCDH-Flag Vec) were measured by LC-MS (n = 3 biologically independent wells from distinct biological sources). ROS level (h) was determined by 2′,7′-di-chlorofluorescein (DCF) staining and flow cytometry analysis. Data are means ± SD; two-tailed Student’s t test.
