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Science 329 (5998): 1492-1499

Copyright © 2010 by the American Association for the Advancement of Science

Evidence for an Alternative Glycolytic Pathway in Rapidly Proliferating Cells

Matthew G. Vander Heiden1,2,3,*, Jason W. Locasale2,3, Kenneth D. Swanson2, Hadar Sharfi2, Greg J. Heffron4, Daniel Amador-Noguez5, Heather R. Christofk2, Gerhard Wagner4, Joshua D. Rabinowitz5, John M. Asara2, and Lewis C. Cantley2,3,{dagger}

1 Dana Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA.
2 Beth Israel Deaconess Medical Center, Division of Signal Transduction and Department of Medicine, Harvard Medical School, Boston, MA 02115, USA.
3 Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.
4 Department of Biological Chemistry and Molecular Pharmacology; Harvard Medical School, Boston, MA 02115, USA.
5 Lewis-Sigler Institute for Integrative Genomics and Department of Chemistry, Princeton University, Princeton, NJ 08544, USA.


Figure 1
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Fig. 1. Evidence of PEP-dependent phosphorylation of a 25-kD protein in PKM2-expressing cells with less pyruvate kinase activity. (A) 6xHis-tagged human PKM1 and PKM2 were expressed in Escherichia coli and purified by Ni affinity chromatography. The specific activity of each enzyme was determined in the presence of saturating amounts of PEP and adenosine diphosphate (ADP). The activity of PKM1 and PKM2 in the presence and absence of FBP is shown. Error bars indicate SEM in all figures. (B) H1299 cells were engineered to express equivalent amount of PKM1 or PKM2 protein as described previously (4). Equivalent expression of PKM1 and PKM2 was confirmed by Western blot using an antibody ({alpha}PK) that recognizes an epitope shared by PKM1 and PKM2. (C) As in (A), pyruvate kinase activity was determined by using saturating amounts of PEP and ADP. The relative pyruvate kinase activity observed in the PKM1- or PKM2-expressing cells described in (B), relative to lysis buffer alone, is shown. (D) HEK293 cells were hypotonically lysed and incubated with [32P]ATP or [32P]PEP before analysis by SDS-PAGE and autoradiography. The lysates were incubated with [32P]ATP or [32P]PEP in the presence of 10 µM ATP or PEP, respectively (–), or with the addition of 1 mM nonradioactive competitor ATP or PEP. (E) Cell lysate was incubated with [32P]PEP in the presence of the indicated concentration of nonradioactive competitor PEP before analysis by SDS-PAGE and autoradiography. (F) Cell lysate was incubated with 32P-labeled PEP as above, and the pH of the reaction was changed to pH 1 or pH 13. Reactions were incubated for 2 hours at 65°C before analysis by SDS-PAGE and autoradiography.

 

Figure 2
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Fig. 2. PGAM1 as the target of PEP-dependent phosphorylation through an enolase-independent reaction. (A) The S100 fraction from a HEK293 cell lysates was passed sequentially through a custom column and a strong cation exchange column before incubating with [32P]PEP (S FT). This reaction was then applied to a hydroxyapatite (HAP) column and eluted with 50 mM NaHPO4. The salt elution (E) containing the 32P-labeled species was diluted to <25 mM NaHPO4 and applied to a weak anion exchange (DEAE) column. Elution from the DEAE column was performed with 100 mM and 200 mM NaCl. The 200-mM salt fraction containing the 32P-labeled species was diluted to 50 mM NaCl and applied to a strong anion exchange (Q) column and eluted sequentially with 100 mM and 350 mM NaCl. The 350-mM salt fraction containing the 32P-labeled species was acetone-precipitated for analysis by 2D-IEF and SDS-PAGE. An aliquot of each fraction was analyzed by SDS-PAGE and autoradiography. Flow-through fractions are indicated as FT. (B) The acetone-precipitated 350 mM salt fraction described in (A) was separated by 2D-IEF and SDS-PAGE, and the 32P-labeled species was identified by autoradiography. (C) The acetone-precipitated 350-mM salt fraction prepared as described in (A) was separated by 2D-IEF and SDS-PAGE, and proteins were identified by Coomassie stain. The species corresponding to the 32P-labeled species is indicated with an arrow. (D) HEK293 cells were transiently transfected with control plasmid (Control), a N-terminally FLAG-tagged PGAM1 complementary DNA (cDNA) (N-FLAG PGAM1), or a C-terminal triple FLAG-tagged PGAM1 cDNA (3xC–FLAG PGAM1). Hypotonic lysates from these cells were incubated with [32P]PEP alone (Ctrl) or in the presence of 1 mM cold competitor ATP or PEP. The products of these reactions were separated by SDS-PAGE and analyzed by autoradiography. Protein immunoprecipitated with an antibody to FLAG from the reactions without nonradioactive competitor were also analyzed by SDS-PAGE and autoradiography. (E) Recombinant 6xHis-tagged PGAM1 (rPGAM1) was produced in E. coli and purified by Ni affinity chromatography. Increasing quantities of rPGAM1 were incubated with 10 µg of HEK293 cell lysate and [32P]PEP. The phosphorylation of both the endogenous PGAM1 present in the cell lysate and rPGAM1 was determined by SDS-PAGE and autoradiography. (F) Cell lysates were incubated with [32P]PEP in the absence (Ctrl) or presence of NaF or exogenously added rabbit muscle enolase enzyme (Eno). The labeling of PGAM1 was determined by SDS-PAGE and autoradiography.

 

Figure 3
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Fig. 3. Transfer of the phosphate of PEP to H11 of PGAM1. (A) Recombinant 6xHis-tagged PGAM1 (rPGAM1) was phosphorylated by [32P]PEP in a cell extract and recovered by binding to Ni-agarose beads. The [32P]rPGAM was then digested with trypsin, and the peptides were separated by using HPLC. A chromatograph identifying peptide peaks by absorbance at 208 nm and the presence of 32P determined by in-line scintillation counting is shown. The peptide peak eluting at ~26 min containing 32P is delineated with an arrow. (B) The HCD MS/MS spectrum for the phosphorylated histidine–containing peptide pHGESAWNLENR (A, Ala; E, Glu; G, Gly; L, Leu; N, Asn; R, Arg; S, Ser; W, Trp) acquired by using a hybrid LTQ linear ion trap–Orbitrap XL mass spectrometer (Thermo Fisher Scientific, San Jose, CA). The a1/pHis immonium ion along with the b- and y-series fragment ions are all consistent with the site of phosphorylation localized to the His1 position of the peptide (H11 in PGAM1). Phosphate losses observed are typical of His phosphorylation (21). The His11 phosphorylation site was confirmed by using both Sequest (www.thermofisher.com/global/en/products/home.asp) and Mascot (www.matrixscience.com) database search engines with a statistically significant expectation value of 0.078. (C) Extracts were prepared from HEK293 cells transiently transfected with N-terminally FLAG-tagged PGAM1 (Ctrl) or N-terminally FLAG-tagged PGAM1 where H11 was mutated to N (H11N). Expression of both FLAG-tagged proteins in relation to endogenous PGAM1 was determined by Western blot using anti-PGAM1. The same extracts were incubated with [32P]PEP, and phosphorylation of PGAM1 determined by SDS-PAGE and autoradiography. (D) rPGAM1 was incubated with a cell extract containing [18O]phosphate-labeled PEP and normal isotopic ([16O]phosphate) ATP before recovery of the H11-containing tryptic peptide by HPLC as described in (A). This peptide was analyzed by microcapillary LC/MS using the high mass accuracy of the FT-MS–only scan in a LTQ Orbitrap-XL mass spectrometer at 30,000 resolution obtaining sub–2-parts-per-million mass accuracy. The peaks at mass/charge (m/z) = 697.79, 698.79, and 699.79 represent the doubly charged phosphorylated peptide pHGESAWNLENR that is heavy by two, four, and six mass units corresponding to the incorporation of one, two, and three 18O-labeled oxygen atoms, respectively. The peak at m/z = 696.79 represents the phosphorylated peptide containing unlabeled oxygen atoms.

 

Figure 4
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Fig. 4. Association of PGAM1 phosphorylation with conversion of PEP into pyruvate in the absence of pyruvate kinase. (A) rPGAM1 was added to a HEK293 cell extract in the absence (Ctrl) or presence of PEP (+PEP). The reactions were analyzed by using 2D IEF and SDS-PAGE followed by Western blot using anti-PGAM1. The newly resolved, more acidic species present only in the PEP-containing reaction are indicated by an arrow. (B) A HEK293 cell lysate was centrifuged at 100,000g, and the S100 supernatant fractionated over a weak anion exchange (DEAE) column. The FT and fractions eluted sequentially with 100 mM, 200 mM, and 500 mM NaCl were collected and incubated with rPGAM1 and [32P]PEP. The ability of each fraction to phosphorylate PGAM1 was determined by SDS-PAGE and autoradiography. The amount of enolase and pyruvate kinase (PK) in each fraction was determined by Western blot. (C) The enolase activity was determined in the FT and 500 mM NaCl (D500) fractions described in (B). In addition, the ADP-dependent pyruvate kinase activity in each fraction was determined. (D) The 2,3-[13C]PEP was incubated with a HEK293 cell S100 fraction (Cell lysate) or the 500 mM NaCl fraction described in (B) (D500), which contained the PGAM1-phosphorylating activity. The [13C]PEP was also incubated under the identical reaction conditions in the absence of any protein (Ctrl). Quantification of the conversion of [13C]PEP to [13C]pyruvate was measured by integrating the intensity of the pyruvate peak and dividing by the intensity of the internal standard consisting of 2 mM 4,4-dimethyl-4-silapentane-1-sulfonic acid for each [1H,13C] HSQC spectra collected. This ratio is graphed for each condition.

 

Figure 5
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Fig. 5. Phosphorylation of PGAM1 H11 in cells and tissues expressing PKM2. (A) Lysates from A549 lung cancer cells engineered to express either PKM1 or PKM2 were subjected to 2D IEF and SDS-PAGE and analyzed by Western blot by using anti-PGAM1 ({alpha}PGAM1) as shown. The most acidic species corresponding to H11 phosphorylation is indicated with an arrow. (B) Metabolites were extracted from H1299 cells engineered to express either PKM1 or PKM2 that were untreated or treated with the phosphatase inhibitor pervanadate (PV) for 10 min to acutely inhibit PKM2. PKM2 activity is decreased by PV treatment, whereas PKM1 activity is not changed (5). The levels of 2,3-BPG and PEP in each extract were determined by mass spectrometry, and the changes in 2,3-BPG and PEP levels resulting from PV treatment are shown for both PKM1- and PKM2-expressing cells. (C) Prostate tissue was removed from 12-week-old mice harboring a conditional allele of the Pten tumor suppressor gene that also did (Ptenpc–/–) or did not (Ptenpc+/+) contain a transgene to express Cre recombinase in the prostate to delete Pten. The Ptenpc–/– was confirmed to have high-grade prostate neoplasia by histology. The expression of PKM1 or PKM2 in each tissue was determined by Western blot as shown. (D) Prostate tissue lysates from the same mice described in (C) were subjected to 2D IEF and SDS-PAGE and analyzed by Western blot using anti-PGAM1 as shown. The most acidic species corresponding to H11 phosphorylation is indicated with an arrow. (E) A breast tumor (cancer) was removed from a 9-month-old mouse harboring a conditional allele of the Brca1 tumor suppressor gene and a transgene to express Cre recombinase in the breast to delete Brca1. Normal breast tissue was removed from a mouse not expressing Cre and hence where Brca1 was not deleted in the breast. Normal breast expresses PKM1; breast tumors express PKM2 (4) (fig. S14C). Lysates from the normal breast tissue and the breast tumor were subjected to 2D IEF and SDS-PAGE and analyzed by Western blot using anti-PGAM1 as shown. The most acidic species corresponding to H11 phosphorylation is indicated with an arrow.

 


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