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Science 337 (6097): 975-980

Copyright © 2012 by the American Association for the Advancement of Science

Phosphofructokinase 1 Glycosylation Regulates Cell Growth and Metabolism

Wen Yi1,2, Peter M. Clark1,2, Daniel E. Mason3, Marie C. Keenan4, Collin Hill4, William A. Goddard, III5, Eric C. Peters3, Edward M. Driggers4, and Linda C. Hsieh-Wilson1,2,*

1 Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
2 Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA 91125, USA.
3 Genomics Institute of the Novartis Research Foundation, San Diego, CA 92121, USA.
4 Agios Pharmaceuticals, 38 Sidney Street, Cambridge, MA 02139, USA.
5 Materials and Process Simulation Center, California Institute of Technology, Division of Chemistry and Chemical Engineering, 1200 East California Boulevard, Pasadena, CA 91125, USA.


Figure 1
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Fig. 1. Effects of O-GlcNAcylation on cellular metabolism and glycosylation of PFK1. (A) O-GlcNAcylation in H1299 cell lysates, as determined by immunoblotting for O-GlcNAc after treatment of cells with the OGA inhibitor PUGNAc or OGT overexpression. WB, Western blot. (B) Glycolytic rate, lactate production, and relative ATP levels in untreated (Cont), PUGNAc-treated, and OGT-overexpressing H1299 cells (n = 5 experiments). (C) PFK1 activity in untreated (Cont), PUGNAc-treated, and OGT-overexpressing 293T cells (n = 5 assays). (D) Detection of PFK1 glycosylation by protein immunoblotting after chemoenzymatic labeling of O-GlcNAc residues with UDP-GalNAz and the enzyme GalT, followed by reaction with an alkyne-biotin derivative, streptavidin precipitation, and elution of the biotinylated proteins. GalT or UDP-GalNAz was removed to confirm selective labeling of O-GlcNAc. (E) Detection of glycosylated PFK1 in 293T cells stably expressing Flag-tagged PFK1 by chemoenzymatic labeling with a 5-kD mass tag (18) and immunoblotting with a Flag antibody. Cells were untreated (Cont), PUGNAc-treated, or transfected to overexpress OGT. (F) Induction of PFK1 glycosylation under hypoxic conditions. H1299 cells stably expressing Flag-tagged PFK1 were cultured under 0.5% O2 for the indicated times and rapidly lysed. Glycosylated PFK1 was detected after labeling with a 5-kD mass tag as above. (G) PFK1 glycosylation in human lung tumor (T) tissues compared to matched normal (N) tissues. O-GlcNAc–modified proteins from tissue lysates were biotinylated and detected as above. Relative PFK1 glycosylation was normalized using normal tissue for each patient. Error bars denote the standard error of the mean (SEM). Statistical analysis was performed by one-way analysis of variance (ANOVA) and Bonferroni comparison post-test in (B) and (C) and by Student’s t test in (G) (*P < 0.05, **P < 0.01, ***P < 0.001).

 

Figure 2
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Fig. 2. Inhibition of PFK1 activity and oligomerization by glycosylation. (A) Relative activities of WT and S529A PFK1 (L isoform) purified from transfected 293T cells under normoxic or hypoxic conditions. Activities were measured in the presence of 100 μM F-2,6-BP and 3 mM ATP and were normalized with respect to the activity of WT PFK1 under normoxic conditions (n = 3 assays). (B) Oligomerization state of Flag-tagged PFK1 (L isoform) purified from 293T cells under normoxic or hypoxic conditions and from 293T cells overexpressing OGT or treated with PUGNAc under normoxic conditions. Complexes were resolved by nonreducing SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by Coomassie blue staining. (C) Coimmunoprecipitation (IP) of endogenous PFK1 with Flag-tagged WT or S529A PFK1 (L isoform) after OGT overexpression. Complexes were immunoprecipitated with a Flag antibody conjugated to agarose beads and analyzed by reducing SDS-PAGE, followed by immunoblotting with a PFK1 antibody. Error bars denote SEM. Statistical analysis was performed by Student’s t test (*P < 0.05).

 

Figure 3
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Fig. 3. PFK1 glycosylation at Ser529 regulates glycolysis, increases PPP flux, and protects cells from ROS-mediated cell death. (A) Immunoblotting of H1299 cells stably expressing shRNA and rescue constructs. Flag-PFK1 levels were comparable to endogenous PFK1 levels. (B) Glycolytic rate and lactate production of H1299 cells rescued with WT or S529A PFK1 in the absence (Cont) or presence of OGT overexpression (n = 4 experiments). (C) PPP activity in WT or S529A PFK1 rescue cells in the absence (Cont) or presence of OGT overexpression, as measured by the rate of 14CO2 production from glucose via the PPP (n = 3 assays). (D) Percentage of central carbon flux from glucose to lactate flowing through the PPP in WT or S529A PFK1 rescue cells in the absence (Cont) or presence of OGT overexpression, as measured by reverse-phase triple-quadrupole liquid chromatography mass spectrometry. (E) NADPH and GSH levels in WT or S529A PFK1 rescue cells in the absence (Cont) or presence of OGT overexpression. NADPH and GSH concentrations are shown relative to WT PFK1 Cont and were assessed using a colorimetric assay and the thiol probe monochlorobimane, respectively (n = 4 experiments). (F) NADPH and GSH levels in WT or S529A PFK1 rescue cells under hypoxic conditions (n = 3 experiments). (G) Cellular ROS levels induced by varying concentrations of diamide in untreated H1299 cells (Cont) and H1299 cells overexpressing OGT, as measured by a fluorimetric assay. (H) Percentage of cell death induced by varying concentrations of hydrogen peroxide in untreated (Cont) and OGT-overexpressing H1299 cells, as measured by lactate dehydrogenase levels (n = 4 assays). Error bars denote SEM. Statistical analysis was performed by Student’s t test (*P < 0.05) for all figures.

 

Figure 4
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Fig. 4. PFK1 glycosylation contributes to cell proliferation and tumor growth. (A) Cell proliferation rates under hypoxic conditions of WT and S529A PFK1 H1299 rescue cells with and without OGT overexpression, as measured by the amount of cellular ATP (n = 3 experiments). (B) Cell proliferation rates under hypoxic conditions of H1299 cells infected with lentiviruses containing scrambled or PFK1 shRNA constructs after no treatment (Cont), OGT knockdown, or OGT overexpression (n = 3 experiments). (C) Tumor formation in nude mice injected with WT or S529A PFK1 H1299 rescue cells with and without OGT overexpression. (Left, top) Dissected tumors after 7 weeks of growth in mice injected with WT cells on the left flank and S529A cells on the right flank. (Left, bottom) PFK1 glycosylation in tumor lysates originating from WT or S529A H1299 rescue cells after labeling with a 5-kD mass tag and immunoblotting with Flag antibody. (Right) Masses of the dissected tumors. Each x represents the mass from one mouse; the horizontal red line indicates the mean tumor mass. Error bars denote SEM. Statistical analysis was performed by one-way ANOVA and Bonferroni comparison post-test in (B) and (C) and by Student’s t test in (A) (*P < 0.05, **P < 0.01, ***P < 0.001; N.S., not significant).

 


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