Dynamic acetylation of cytosolic phosphoenolpyruvate carboxykinase toggles enzyme activity between gluconeogenic and anaplerotic reactions

P.L. and J.M. D conceived of the project, designed and performed most experiments, interpreted data and wrote the manuscript. J.B. designed and performed MS experiments and interpreted data. E.A. A analyzed the metabolomics samples. R.H. G and F.C. designed and performed MD simulation experiments and interpreted data. L.E. W and D.S. provided wild-type and SIRT1−/− mice tissue samples. P.L-B. conceived of part of the project and revised the manuscript. J.A. C. conceived of, designed and supervised PCK1 phosphorylation and ubiquitination experiments, interpreted data and revised the manuscript. Here, we investigated how mammalian PCK1 is regulated at the post-translational level in response to varying energy inputs. PCK1 acetylation is regulated by p300 acetyltransferase under high glucose and, conversely, SIRT1 deacetylates PCK1 under low energy conditions. The enzymatic properties of recombinant, site-specifically acetylated enzyme, and of PCK1 purified from cultured cells and from wild-type and SIRT1 knock-out murine tissues reveal a novel mechanism: acetylation under high glucose concentrations triggers PCK1 anaplerotic activity and SIRT1-mediated deacetylation promotes the gluconeogenic activity. Also, we discovered that PCK1 phosphorylation is mediated by the GSK3 β pathway and controls PCK1 stability in cells by increasing PCK1 ubiquitination and degradation. Additionally, our results suggest that phosphorylation affects the efficiency by which SIRT1 deacetylates PCK1. Together, these results reveal a previously unknown intersection of post-translational control of PCK1 involving acetylation, phosphorylation and ubiquitination that regulates central metabolism. our results suggest that PCK1 anaplerotic activity is possible in PC-downregulated HEK293T cells and corrects the metabolic defects of loss-of-Latorre-Muro by SIRT1 when a phosphomimetic S90E+K91AcK variant was utilized, restoring the gluconeogenic ability of PCK1. In conclusion, our results reveal that an interplay between phosphorylation, acetylation and ubiquitination, which is regulated by method involved reversed-phase ion-pairing chromatography on a Vanquish UHPLC coupled with negative mode ESI electrospray ionization and detection on a Q Exactive orbitrap mass spectrometer (Thermo Fisher Scientific). LC separation was performed with a Waters Acquity UPLC BEH C18 column (1.7 μm, 2.1 × 100 mm; Waters Corp.) over a 25’ method with a 14.5’ gradient, ramping methanol from 5% to 95% against a buffer of water with 3% methanol, 10 mM tributyamine, and acetic acid-adjusted pH of 8.3. Injection volume was 10 μL, flow rate 200 μL/min, and column temperature 25 °C. ESI settings were 30/ 10/ 1 sheath/ aux sweep gas rates, 2.50 |kV| spray voltage, S-lense RF level 50, 350˚ C capillary, and 300° C aux gas. MS1 scans were performed over a scan range of 85–1250 m/z at 70,000 resolution, AGC target of 1e6, and 100 ms maximum IT. Data were analyzed using MAVEN/ ElMAVEN (Clasquin and Elucidata and utilizing compound lists with retention times empirically determined in-house or by colleagues. Metabolite expression was captured using the AreaTop metric; exported data were analyzed in Excel (Microsoft).