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. 2009 Feb 20;284(8):5148-57.
doi: 10.1074/jbc.M808890200. Epub 2008 Dec 22.

A PGC-1alpha-O-GlcNAc transferase complex regulates FoxO transcription factor activity in response to glucose

Michael P Housley  1 , Namrata D Udeshi, Joseph T Rodgers, Jeffrey Shabanowitz, Pere Puigserver, Donald F Hunt, Gerald W Hart
Affiliations

A PGC-1alpha-O-GlcNAc transferase complex regulates FoxO transcription factor activity in response to glucose

Michael P Housley et al. J Biol Chem. .

Abstract

Metabolic and stress response gene regulation is crucial for the survival of an organism to a changing environment. Three key molecules that sense nutrients and broadly affect gene expression are the FoxO transcription factors, the transcriptional co-activator PGC-1alpha, and the dynamic post-translational modification, O-linked beta-N-acetylglucosamine (O-GlcNAc). Here we identify novel post-translational modifications of PGC-1alpha, including O-GlcNAc, and describe a novel mechanism for how PGC-1alpha co-activates transcription by FoxOs. In liver, in cultured cells, and in vitro with recombinant proteins, PGC-1alpha binds to O-GlcNAc transferase and targets the enzyme to FoxOs, resulting in their increased GlcNAcylation and increased transcriptional activity. Furthermore, glucose-enhanced activation of FoxO1 occurs via this PGC-1alpha-O-GlcNAc transferase-mediated GlcNAcylation. Therefore, one mechanism by which PGC-1alpha can serve as a co-activator of transcription is by targeting the O-GlcNAc transferase to increase GlcNAcylation of specific transcription factors important to nutrient/stress sensing and energy metabolism.

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Figures

FIGURE 1.
FIGURE 1.
O-GlcNAc site mapping on Pgc-1α via liquid chromatography-MS/MS. A, a high resolution MS1 spectrum (mass range m/z 473–537) recorded using a LTQ Orbitrap mass spectrometer operated in the data-dependent mode. The [M+4H]+4 ion at m/z 478.7236 corresponds to the 12C isotope of the LysC-generated, carbamidomethylated PGC-1α peptide, RARYSECSGTQGSHSTK. This mass agrees to <1 ppm with the calculated monoisotopic mass. Signals at m/z 529.4929 correspond to the [M+4H]+412C isotope of RARYSECSGTQGSHSTK harboring one GlcNAc moiety; this mass agrees with the calculated mass to <1 ppm. Signals for the GlcNAcylated species are >700-fold lower in abundance relative to the unmodified species. B, a CAD MS/MS spectrum recorded on [M+4H]+4 ions (m/z 529.4) of GlcNAcylated peptide RARYSECSGTQGSHSTK. The CAD spectrum contains signature GlcNAc oxonium ions at m/z 204.1 that are generated from loss of the GlcNAc moiety upon collision-activated dissociation (56). Charge-reduced ions, [(M + 4H)+4-(GlcNAc +H)+1]+3 at m/z 638.1, are also observed in this spectrum (56). C, an ETD MS/MS spectrum recorded on [M+4H]+4 ions (m/z 529.8) of GlcNAcylated peptide RARYSECSGTQGSHSTK. An ETD-enabled LTQ mass spectrometer was operated to record a MS/MS spectrum on m/z 529.8 followed by four data-dependent scans after every MS1 scan to generate the ETD spectrum, which is presented as a subtracted spectrum. Predicted product ions of types c′- and z′·- are listed above and below the peptide sequence, respectively. Singly and doubly charged ions are listed as monoisotopic and average masses, respectively. Observed product ions are underlined and are sufficient to define the O-GlcNAc residue at Ser-333. ETD product ions are labeled in the ETD spectrum. A triangle (▾) is positioned above m/z peaks that are within the precursor isolation window. Signals corresponding to charge-reduced species and species resulting from neutral losses are bracketed.
FIGURE 2.
FIGURE 2.
PGC-1α is GlcNAcylated. A, FLAG-PGC-1α was immunoprecipitated (IP) from Fao cells subjected to SDS-PAGE and blotted (IB) using anti-O-GlcNAc antibodies (CTD110.6) or a terminal GlcNAc-specific lectin (sWGA). Specificity was confirmed by GlcNAc competition. BSA, bovine serum albumin. B, FLAG-PGC-1α was GalNAz-labeled with a GlcNAc specific β-1-4-galactosyltransferase and reacted with biotin-alkyne, then subjected to SDS-PAGE and blotted using streptavidin-horseradish peroxidase (streptavidin-HRP). Immunoblots are representative of three experiments.
FIGURE 3.
FIGURE 3.
PGC-1α interacts with OGT. A, Fao cells were infected with Ad-FLAG-PGC-1α and treated with insulin for 1 h prior to harvesting and OGT co-immunoprecipitation (IP). Immunoprecipitates were subjected to SDS-PAGE and immunoblotted (IB) for the presence of PGC-1α. Membranes were then stripped and blotted for OGT. Immunoblots are representative of three experiments. B, Fao cells were infected with Ad-FLAG-PGC-1α and treated with insulin for 1 h prior to harvesting and FLAG co-immunoprecipitation. Immunoprecipitates were subjected to SDS-PAGE and immunoblotted for the presence of OGT. Membranes were then stripped and blotted for PGC-1α. C, gel filtration chromatography (SMART system, Superdex 200 column, phosphate-buffered saline, 1% Nonidet P-40 buffer) of lysates from rat liver incubated on ice for 30 min with either normal IgG or anti-OGT antibodies (AL28). Fractions were subjected to SDS-PAGE and blotted using anti-PGC-1α, anti-OGT (DM-17), and anti CPB. Data are representative of two experiments.
FIGURE 4.
FIGURE 4.
PGC-1α enhances high glucose activation and GlcNAcylation of FoxO1. A, overexpression of PGC-1α in Fao hepatoma cells increases O-GlcNAc levels on co-expressed FLAG-FoxO1. The bar graph represents densitometry of relative anti-O-GlcNAc immunoreactivity from three experiments. Co-IP, co-immunoprecipitations; IB, immunoblot. B, inputs from immunoprecipitations were subjected to SDS-PAGE and blotted for O-GlcNAc, PGC-1α, OGT, and tubulin. Total O-GlcNAc levels are unchanged by overexpression of PGC-1α.
FIGURE 5.
FIGURE 5.
PGC-1α enhances the in vitro GlcNAcylation of FoxO1. A, bacterial lysates expressing either HIS-PGC-1α (amino acids (aa) 200–665) or empty formula image HIS vector. B, an autoradiograph (Autorad) of in vitro OGT labeled FoxO1 in the presence of HIS-PGC-1α or control. Data are representative of three experiments. C, scintillation counting of in vitro OGT labeled FoxO1 in the presence of HIS-PGC-1α or control (*, p < 0.05 by Student's t test). The bar graph represents the mean of three experiments. The error bars depict stand errors. D, a GST-pull down assay of GST-FoxO1 in the presence of HIS-PGC-1α or control.
FIGURE 6.
FIGURE 6.
PGC-1α enhances the in vitro GlcNAcylation of FoxO3. An autoradiograph (Autorad) of in vitro OGT labeled FoxO3 wild type (wt), a truncated FoxO3 (amino acids (aa) 1–525), and a mutant lacking the protein kinase B (PKB/AKT) phosphorylation sites (amino acids 1–525 triple mutant (TM)) in the presence of lysates expressing either HIS-PGC-1α (amino acids 200–665) or empty formula image HIS vector is shown.
FIGURE 7.
FIGURE 7.
PGC-1α enhances high glucose activation and GlcNAcylation of FoxO1. A, PGC-1α co-transfection increases FoxO1-dependent luciferase expression (plotted as relative luciferase activity normalized to β-galactosidase; error bars indicate standard errors from three experiments; *, p < 0.05 by Student's t test). Expression was confirmed by immunoblotting (IB) for HA. B, short hairpin RNA knockdown of PGC-1α decreases FoxO-dependent luciferase expression. Knockdown was confirmed by real-time-PCR analysis of steady-state mRNA levels.
FIGURE 8.
FIGURE 8.
Model depicting the regulation of FoxO by the PGC-1α-OGT complex in response to glucose. In the diabetic liver, GlcNAcylation of FoxO (12) and CRTC2 (TORC2) (13) mediates inappropriate gluconeogenesis in response to glucose by activating these key transcription factors. In a second step of activation of gluconeogenesis, elevated expression of PGC-1α, possibly through CRTC2 (35) and CREB (36), can bind and target OGT to FoxOs. Thus, glucose activates inappropriate gluconeogenesis in a two-step process. First, an O-GlcNAc-dependent activation of CRTC2/CREB drives PGC-1α expression. Second, higher levels of PGC-1α increase targeting of OGT to FoxOs. MnSOD, manganese superoxide dismutase.
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