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Pyruvate kinase M2 regulates gene transcription by acting as a protein kinase - PubMed

  • ️Sun Jan 01 2012

Pyruvate kinase M2 regulates gene transcription by acting as a protein kinase

Xueliang Gao et al. Mol Cell. 2012.

Abstract

Pyruvate kinase isoform M2 (PKM2) is a glycolysis enzyme catalyzing conversion of phosphoenolpyruvate (PEP) to pyruvate by transferring a phosphate from PEP to ADP. We report here that PKM2 localizes to the cell nucleus. The levels of nuclear PKM2 correlate with cell proliferation. PKM2 activates transcription of MEK5 by phosphorylating stat3 at Y705. In vitro phosphorylation assays show that PKM2 is a protein kinase using PEP as a phosphate donor. ADP competes with the protein substrate binding, indicating that the substrate may bind to the ADP site of PKM2. Our experiments suggest that PKM2 dimer is an active protein kinase, while the tetramer is an active pyruvate kinase. Expression of a PKM2 mutant that exists as a dimer promotes cell proliferation, indicating that protein kinase activity of PKM2 plays a role in promoting cell proliferation. Our study reveals an important link between metabolism alteration and gene expression during tumor transformation and progression.

Copyright © 2012 Elsevier Inc. All rights reserved.

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Figures

Figure 1
Figure 1. PKM2 regulates MEK5 transcription

(A) Nuclear localization of PKM2 in different cancer cells. (Left) Immunoblot analyses of PKM2 using the antibody PabPKM2 (IB:PKM2) in nuclear extracts (NE), cytoplasmic extracts (Cyto), and whole cell lysates (WCL) of ten cell lines (indicated). Blot of GAPDH (IB:GAPDH) in nuclear extracts is a control indicate no cytoplasmic protein contaminations. (Right) Quantization of the nuclear and cytoplasmic PKM2 in different cell lines based on the immunoblots. The results are presented as the ratio of nuclear vs cytoplasmic (NE/Cyto) levels. (B) (Left) RT-PCR analyses of cellular MEK5 mRNA in two pairs of cancer cells (indicated) in which PKM2 was expressed (HAPK) using the adeno-viral expression kit. The results are presented as fold increase in PCR products before and 48 hours after PKM2 expression in the same cells. The level of PCR product from each cell line before PKM2 expression was defined as 1. (Right) The levels of expressed HA-PKM2 (HAPK) in the nuclear (NE) and cytoplasm (Cyto) in four cell lines (indicated) were analyzed by immunoblot of nuclear or cytoplasmic extracts using anti-HA antibody (IB:HA). Vec means that the cells express vector alone. (C) (Left) Expressions of MEK5 (IB:MEK5) in SW480 and SW620 cells in which PKM2 was knocked down (PK) were analyzed by immunoblot using antibody against MEK5. The cellular levels of PKM2 were analyzed by immunoblot of PKM2 (IB:PKM2). NT means the cells were transfected with non-target siRNA. (Middle) RT-PCR analyses of cellular MEK5 mRNA levels and (Right) immunoblot analyses of the cellular PKM2 levels (IB:PKM2) in cancer cells (indicated) in which PKM2 was knocked down (PK). The results of mRNA levels are presented as fold increase in PCR products before and 48 hours after PKM2 expression in the same cells. The level of PCR product from each cell line before PKM2 expression was defined as 1. (D) ChIP of the MEK5 promoter (MEK5 promoter) using antibody against PKM2 in SW620 cells (αPK). ChIP using rabbit IgG was a negative control. ChIP using PCR primer pair targeting a region of exon 1 (MEK5 exon 1) of MEK5 gene (nt 342 – nt 542) and targeting GAPDH promoter (GAPDH promoter) using antibody against PKM2 were negative controls. The primer pair positions are indicated. Inputs were PCR products from DNA extracts without ChIP. Error bars in (B) and (C) are standard deviations of three measurements. In (A) (B), and (C), Immunoblot of β-actin (IB:β-actin), immunoblots of H2B (IB:H2B), and immunoblots of Lamin A/C (IB:Lamin A/C) are loading controls.

Figure 2
Figure 2. PKM2 regulates MEK5 transcription via activation of stat3

(A) Co-immunoprecipitation of PKM2 with stat3. PKM2 was immunoprecipitated from nuclear extracts of SW620 and SW480 cells using the antibody PabPKM2 (IP:αPK). The immunoprecipitates were analyzed by immunoblot using antibodies against stat3 (IB:Stat3) or PKM2 (IB:PKM2). Ponceau S staining of IgG heavy chain (IgG HC) in the immunoprecipitates is the loading control. Immunoprecipitation using rabbit IgG (IP:IgG) was the control immunoprecipitation. (B) & (D) Expressions of MEK5 (IB:MEK5) in SW620/SW480 (B) and WM115/WM266 cells (D) were analyzed by immunoblot of the cell lysate (WCL) of the cells in which stat3 was knocked down by RNAi (RNAi-Stat3, IB:Stat3). HA-PKM2 (HAPK) was expressed in the stat3 knockdown cells (IB:HA). (C) & (E) RT-PCR analyses of cellular MEK5 mRNA levels in SW620/SW480 (C) and WM266/WM115 cells (E) in which stat3 was knocked down (RNAi-Stat3, IB:Stat3). HA-PKM2 (HAPK) was expressed in the stat3 knockdown cells (IB:HA). The results are presented as fold changes in PCR products before and 48 hours after stat3 knockdown and HA-PKM2 expression in the same cells. The level of PCR products from each cell line that was treated with non-target RNAi was defined as 1. In (B), (C), (D), and (E), NT represents the cells treated with non-target RNAi. Vec were the cells infected with virus that carry the empty vector. Error bars in (C) and (E) are standard deviations of three measurements. In (B) and (D), Immunoblots of β-actin (IB:β-actin) are loading controls.

Figure 3
Figure 3. PKM2 upregulates MEK5 transcription by promoting stat3 DNA interaction and phosphorylation of stat3

(A) (Upper panels) ChIP of the MEK5 promoter (MEK5 promoter) using antibody against stat3 in SW620 cells (αStat3). The cells were treated non-target RNAi (left, NT) or with RNAi target PKM2 (right, RNAi-PK), or HA-PKM2 was expressed in the cells (middle, HAPK). Inputs were PCR products from DNA extracts without ChIP. The primer pair positions are indicated. ChIP using rabbit IgG (IgG) was a negative control. ChIP targeting GAPDH promoter (GAPDH promoter) using antibody against stat3 was another negative control. (Lower panels) the cellular PKM2 (right) and HA-PKM2 (left) levels in SW620 cells that were treated with RNAi target PKM2 (PK) or with non-target RNAi (NT), or infected with virus that carry HA-PKM2 expression vector (HAPK) or vector alone (Vec) were analyzed by immunoblots using anti-HA antibody (IB:HA) or anti-PKM2 antibody (IB:PKM2). (B) DNA-protein complex (Complex) assembled on a 32P-labeled oligo containing the stat3 targeting sequence in nuclear extracts of SW620 cells was detected by gel-shift. Free probe indicates the 32P-labeled oligo probe without addition of nuclear extracts. The antibodies against PKM2 (αPK), stat3 (αStat3), or no antibody (NoAb) was added to the complex to create supershift (Supershift). (C) & (D) Supershift complex assembled with the 32P-labeled oligo and anti-stat3 antibody (αStat3) in the nuclear extracts of SW620 cells in which (C) HA-PKM2 was expressed (HAPK) or (D) PKM2 was knocked down (PK) was detected by gel-shift. Probe only (Probe) is the free probe without addition of nuclear extracts. The free probe (low exposure) is the loading control with 1/10 of exposure time in autoradiography. The quantification of the assembled complex and super shift complex were presented as percentage (Shifted/total ×102) of probe in the shift complex calculated by intensities of complex [complex (grey bars) or supershift (open bars)] divided by Intensities of total [free probe + complex + supershift). Immunoblots at bottom of each panel indicate levels of HA-PKM2 (IB:HA) and endogenous PKM2 (IB:PKM2) in the cells from which the extracts were prepared for the above gel shift experiments. (E) & (F) The levels of Y705 phosphorylated stat3 (IB:pY-Stat3) in the cell nucleus were analyzed by immunoblot of nuclear extracts (NE) of SW620 cells in which HA-PKM2 was expressed (E. HAPK) or the PKM2 was knocked down (F, PK). The total cellular stat3 levels were analyzed by immunoblot analyses of stat3 (IB:Stat3) in whole cell lysate (WCL). In (E), immunoblot of HA-tag (IB:HA) indicates the HA-PKM2 expression levels in the cells. In (F), immunoblot of PKM2 (IB:PKM2) represents cellular PKM2 levels in the cells. Immunoblot of lamin A/C in (E) and (F), and β-actin in (A), (C), (D), (E), and (F) are the loading controls. NTs in (A), (C), (D), (E), and (F) mean the cells were treated with non-target RNAi. Vec in (A), (C), (D), (E), and (F) means the cells were infected with virus that carry the empty vector.

Figure 4
Figure 4. Phosphorylation of GST-stat3 by the rPKM2

Phosphorylation of GST-stat3 by the rPKM2 (A) and HA-PKM2 (HAPK(NE)) immunopurified from nuclear extracts of SW620 (B) in the presence of 5 mM ATP (ATP) or 5 mM PEP (PEP) was revealed by immunoblot assays using antibody against Y705 phosphorylated stat3 (IB:pY-stat3). Immunoblot analyses using antibody against stat3 (IB:Stat3) indicates the amounts of GST-stat3 used in each reaction. The bottom panels in (A) & (B) are the quantitative analyses of immunoblot signals. The error bars represent the standard deviations of four measurements. Phosphorylation of GST-stat3 by the HA-PKM2 immunopurified from the nuclear (HAPK (NE) in C) and the cytoplasmic (HAPK(Cyto) in D) extracts of SW620 cells in the presence of 5 mM ATP (ATP) or 5 mM PEP (PEP) was revealed by immunoblots using antibody against Y705 phosphorylated stat3 (IB:pY-stat3). The reactions were also carried out in the presence/absence of 5 mM FBP (FBP), or 5 mM ADP (ADP). The immunoblot of HA (IB:HA) indicates the amounts of HA-PKM2 used in each reaction. IgG HC is the ponceau S stain of antibody heavy chain, representing the amounts of antibody used in immunopurification of HA-PKM2. Coomassie blue staining (CBS) indicates the amounts of GST-stat3 and BSA used in each phosphorylation reaction. Vec were the cells infected with virus that carry an empty vector.

Figure 5
Figure 5. Dimer and tetramer PKM2

(A) Chromatography fractionation of nuclear (NE, right) and cytoplasmic (Cyto, left) extracts of SW620 cells. The fractions were collected at 300 μl per fraction. The fractions 6 to 17 from cytoplasmic extracts and 9 to 18 from nuclear extracts were immunoblotted using the antibody PabPKM2. Fraction 11 (F11) co-eluted with 240 kDa and fraction 14 (F14) co-eluted with 120 kDa on the same column under identical conditions determined by chromatography molecular weight calibration standard (see Fig. S3 C&D). (B) Chromatography fractionation of the wild-type rPKM2 and R399E mutant. The fractions F10 – F18 were collected. The fractions were analyzed by immunoblot using the antibody PabPKM2 (IB:PKM2). (C) Effects of FBP on the dimer/tetramer status of PKM2 in nuclear extracts. Chromatography fractionation of nuclear (NE) extracts of SW620 cells treated (bottom panel) or untreated (upper panel) with FBP (5mM). The fractions were collected at 300 μl per fraction. The fractions 6 to 20 from nuclear extracts were immunoblotted using the antibody PabPKM2 (IB:PKM2). Immunoblots of BSA are the loading controls. 2 μg of BSA was added to each fraction. After SDS-PAGE separation of 20 μl of each fraction, the BSA was analyzed by immunoblot (BSA).

Figure 6
Figure 6. Dimeric PKM2 is active protein kinase and expression of the R399E mutant promotes cell proliferation

(A) Part of x-ray crystal structure of human PKM2. The structure was obtained from PDB bank DOI: 10.1021/bi0474923. The residue R399 and its interactive residues E418, D357, and E396 are highlighted in color. (B) Phosphorylation of GST-stat3 by 10 μg/ml of rPKM2 (rPKM2) and R399E mutant (R399E) in the presence of 5 mM ATP (ATP), 5 mM PEP (PEP), 5 mM FBP (FBP), and/or 5 mM ADP (ADP) was revealed by immunoblot assays using antibody against Y705 phosphorylated stat3 (IB:pY-stat3). Immunoblot analyses using antibody against stat3 (IB:Stat3) indicates the amounts of GST-stat3 used in each reaction. (C) Pyruvate kinase activity of the rPKM2 or R399E (5 μg/ml) was analyzed by the method described by Christofk and coworkers. The pyruvate kinase activity was expressed as relative pyruvate kinase activity by define the activity in the rPKM2 as 100. (D) Phosphorylation of GST-stat3 by 10 μg/ml of rPKM2, the rPKM1, or the R399E in the presence of 32P-PEP (~0.002 μCi) and unlabeled PEP (5 mM). The reaction mixture were separated by SDS-PAGE and subjected to autoradiograph. (E) Interaction of GST-stat3 and R399E in the presence of FBP (5 mM), PEP (5 mM), or various concentrations of ADP (inducated) was analyzed by GST-pull-down. The co-precipitation of R399E with GST-stat3 was detected by immunoblot using the antibody against PKM2 (IB:PKM2). Immunoblots of precipitates using antibody against stat3 (IB:Stat3) indicate the amounts of GST-stat3 that was pulled-down by the glutathione beads. (F) Phosphorylation of GST-stat3 by the PKM2 (10 μg/ml) purified from nuclear extracts of SW620 cells in the presence of 200 μM PEP (PEP) and 200 μM ADP (ADP) was revealed by immunoblot assays using antibody against Y705 phosphorylated stat3 (IB:pY-stat3). Immunoblot analyses using antibody against stat3 (IB:Stat3) indicates the amounts of GST-stat3 used in each reaction. (G) Phosphorylation of stat3 in SW480 cells was examined by immunoblot analyses of the nuclear extracts (NE) using antibody against the Y705 phosphorylated stat3 (IB:pY-Stat3). PKM2 (HAPK) or the R399E (R399E) was expressed in the cells. Immunoblots using anti-HA antibody (IB:HA) and anti-stat3 antibody (IB:Stat3) in the whole cell lysate (WCL) indicate the levels of stat3 and HA-PKM2/HA-R399E in the cells. Immunoblot of lamin A/C (IB:Lamin A/C) and β-actin (IB:β-actin) are loading controls. (H) Phosphorylation of GST-stat3 by the HA-p68 and the HA-R399E that were immunopurified from cell lysate of SW480 cells in the presence of 5 mM PEP was revealed by immunoblot analyses using antibody against the Y705 phosphorylated stat3 (IB:pY-Stat3). The reactions were also carried out in the presence/absence of 5 mM FBP (FBP), or mM of ADP (ADP). The immunoblot of HA (IB:HA) indicates the amounts of HA-PKM2 or HA-R399E used in each reaction. (I) Expression of MEK5 mRNA in SW480 cells was analyzed by RT-PCR. HA-PKM2 (HAPK) or HA-R399E (R399E) was expressed in the cells. The results are presented as fold changes in PCR products before and 48 hours after HA-PKM2 or HA-R399E expression. (J) Cell proliferations of SW480 cells were measured using a proliferation kit. Proliferations were presented as fold changes in BrdU incorporation before and 48 hours after HA-PKM2 (HAPK) or HA-R399E (R399E) expression. The BrdU incorporation of the cells that were transfected with the empty vector (Vec) was defined as 1. In (F) and (H), IgG HC is the ponceau S stain of antibody heavy chain, representing the amounts of antibody used in immunopurification of HA-PKM2 from the extracts. Error bars in (C), (I), and (J) are standard deviations of three independent measurements. Vec in (G), (H), (I), and (J) are the cells transfected with the empty vector.

Figure 7
Figure 7. The effects of PKM2 protein kinase activity on tumor growth

(A) PKM2 (HAPK) or R399E (R399E) was stably expressed in SW480 cells (IB:HA) and stat3 was phosphorylated in R399E expressing cells (IB: pY-stat3). (B) The representative examples of tumor-bearing mice and excised tumors from the derived (indicated) cell lines. (C) Tumor growth was monitored by measuring tumor volumes every two days. (D) At end of four weeks growth, tumors were sliced out and weighted. (E) (Left) Tissue sections were immune fluorescence stained with antibodies against Ki67 (Ab: Ki67, Red) and HA (Ab:HA, Green). The blue is DAPI stain of cell nucleus. (Right) Quantization of Ki-67 staining signals (Upper) and HA-staining signals (Lower) of the tissue sections. The proliferation index was percentage of the positive nucleus staining by counting the nucleus number with ki-67 positive staining in randomly selected three fields in each slide and randomly selected three slides from each tumor. The HA-stains are quantified by image-J software by manually define the areas of interest (e.g. nuclear or cytoplasmic based on the DAPI blue staining) with randomly selected five fields from randomly selected three slides of each tumor. In (A), (B), (C), (D), and (E), the Vec means the vector alone was expressed in the cells. The error bars in (C) and (E) are standard deviations from the measurements of six mice. The p values in (C) and (D) are calculated using pairwise student t-test. (F) A hypothetic model that illustrates the functional role of PKM2 in gene transcription.

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References

    1. Aaboe M, Birkenkamp-Demtroder K, Wiuf C, Sorensen FB, Thykjaer T, Sauter G, Jensen KM, Dyrskjot L, Orntoft T. SOX4 expression in bladder carcinoma: clinical aspects and in vitro functional characterization. Cancer Res. 2006;66:3434–3442. - PubMed
    1. Altenberg B, Greulich KO. Genes of glycolysis are ubiquitously overexpressed in 24 cancer classes. Genomics. 2004;84:1014–1020. - PubMed
    1. Ashizawa K, Willingham MC, Liang CM, Cheng SY. In vivo regulation of monomer-tetramer conversion of pyruvate kinase subtype M2 by glucose is mediated via fructose 1,6-bisphosphate. J Biol Chem. 1991;266:16842–16846. - PubMed
    1. Bradbury DA, Simmons TD, Slater KJ, Crouch SP. Measurement of the ADP:ATP ratio in human leukaemic cell lines can be used as an indicator of cell viability, necrosis and apoptosis. J Immunol Methods. 2000;240:79–92. - PubMed
    1. Brosnan MJ, Chen L, Van Dyke TA, Koretsky AP. Free ADP levels in transgenic mouse liver expressing creatine kinase. Effects of enzyme activity, phosphagen type, and substrate concentration. J Biol Chem. 1990;265:20849–20855. - PubMed

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