Dynamic Acetylation of Phosphoenolpyruvate Carboxykinase Toggles Enzyme Activity between Gluconeogenic and Anaplerotic Reactions

24 pags, 6 figs. -- Supplemental Information includes five figures, five tables, and four videos and can be found with this article online at https://doi.org/10.1016/j.molcel.2018.07.031.
Cytosolic phosphoenolpyruvate carboxykinase (PCK1) is considered a gluconeogenic enzyme; however, its metabolic functions and regulatory mechanisms beyond gluconeogenesis are poorly understood. Here, we describe that dynamic acetylation of PCK1 interconverts the enzyme between gluconeogenic and anaplerotic activities. Under high glucose, p300-dependent hyperacetylation of PCK1 did not lead to protein degradation but instead increased the ability of PCK1 to perform the anaplerotic reaction, converting phosphoenolpyruvate to oxaloacetate. Lys91 acetylation destabilizes the active site of PCK1 and favors the reverse reaction. At low energy input, we demonstrate that SIRT1 deacetylates PCK1 and fully restores the gluconeogenic ability of PCK1. Additionally, we found that GSK3β-mediated phosphorylation of PCK1 decreases acetylation and increases ubiquitination. Biochemical evidence suggests that serine phosphorylation adjacent to Lys91 stimulates SIRT1-dependent deacetylation of PCK1. This work reveals an unexpected capacity of hyperacetylated PCK1 to promote anaplerotic activity, and the intersection of post-translational control of PCK1 involving acetylation, phosphorylation, and ubiquitination.
Recombinant SIRT1 and SIRT2 were kindly supplied by Beatriz Camacho (UW Madison) and Mark A. Klein (UW Madison), respectively. Jin-Hee Lee (UW Madison) provided the X. laevis Histone H4. Susana Llanos (CNIO, Madrid, Spain) provided the human p300-HA clone. We thank Jing Fan (UW Madison) for discussions on metabolite flux and tracing experiments. We thank S.V. Medaris (UW Madison) for providing graphical support for Rgure 7. P.L.-M. was funded by a predoctoral fellowship from the "la Caixa" Foundation and received financial support from the Universidad de Zaragoza, Fundacion Bancaria lbercaja y Fundacion CAI (CM 1/16) during his stay at the UW Madison. This work has been funded by grants DK100263 and AG028730 (to D.A.S.). We acknowledge grants AGL2015-66177 (to P.L.-B. and J.A.C.) and CTQ2015-67727-R (to F.C.) from the Ministerio de Economia. Industria y Competitividad and UZ 2014-CIE-03 (to P.L-B.) and UZ-2015-B10-01 (to J.A.C.) from the University of Zaragoza. We thank ARAID and MEC (CTQ2013-44367-C2-2-P and BFU2016-75633-P to R.H.-G.). J.M.D acknowledges NIH grant GM065386.