human pkm2 wild type construct  (Addgene inc)

Journal: bioRxiv

Article Title: Fructose 1,6-bisphosphate sensing by pyruvate kinase isozymes M2 (PKM2) controls MyoD stability and myogenic differentiation

doi: 10.1101/2020.12.22.424062

Figure Lengend Snippet: (A) Diagram of glycolysis pathway. (B) Diagram illustrating the regulation of Pyruvate Kinase M gene by alternative splicing in myofibers and myoblasts, and different biochemical properties of PKM1 and PKM2. Myofibers use exon 9 to express PKM1 whereas myoblasts use exon 10 to express PKM2. FBP (fructose 1,6-bisphosphate), DASA-58 and TEPP-46 stabilize PKM2 tetramer. (C) Cells were permeabilized with Streptolysin-O (SLO), and treated with 2DG and indicated metabolites (200μM) for 2 hours. DHAP, dihydroxyacetone phosphate. G3P, glyceraldehyde 3-phosphate. (D) Cells were co-treated with 2DG and PKM2 tetramer stabilizing reagents (100μM) for 4 hours. All results were validated in two or more independent experiments.

Article Snippet: Human PKM2 wild-type construct was purchased from Addgene (44242) and sub-cloned to pCMV-Tag2B plasmid (Agilent Technologies).

Techniques: Alternative Splicing

Journal: bioRxiv

Article Title: Fructose 1,6-bisphosphate sensing by pyruvate kinase isozymes M2 (PKM2) controls MyoD stability and myogenic differentiation

doi: 10.1101/2020.12.22.424062

Figure Lengend Snippet: (A) C2C12 cells were transfected with two different siRNAs against Huwe1 and treated with 2DG for 4 hours. (B) Cells treated with 2DG for indicated time were fractionated to cytosolic and nuclear lysates. Lamin B is a nuclear marker and β-Tubulin is a cytosolic marker. (C) Cells were incubated in proliferation media with different glucose concentrations for one day and fractionated as in (B). (D) After 4 hours treatment with 2DG, cells were immunostained for Huwe1. Scale bar, 20μm. (E) Cells were treated as indicated for 4 hours and fractionated. (F) Control or PKM2 siRNA transfected cells were immunostained for Huwe1. Scale bar, 10μm. (G) Endogenous PKM2 was depleted using PKM2 siRNA (#1 in ) followed by transfection with Flag-tagged human PKM2 WT or PKM2 S437Y mutant. Lysates were immunoprecipitated using antibody against Flag. (H) Model of MyoD regulation by FBP-PKM2-Huwe1 axis. FBP, fructose 1,6-bisphosphate. All results were validated in two or more independent experiments. Scales in (B) and (F), 10μm.

Article Snippet: Human PKM2 wild-type construct was purchased from Addgene (44242) and sub-cloned to pCMV-Tag2B plasmid (Agilent Technologies).

Techniques: Transfection, Marker, Incubation, Control, Mutagenesis, Immunoprecipitation

Journal: bioRxiv

Article Title: Fructose 1,6-bisphosphate sensing by pyruvate kinase isozymes M2 (PKM2) controls MyoD stability and myogenic differentiation

doi: 10.1101/2020.12.22.424062

Figure Lengend Snippet: (A) Huwe1 immunostaining in Huwe1 siRNA transfected cells validate the specificity of the antibody. Scale bar, 20μm. (B) C2C12 cells were transfected with two different siRNAs against PKM2 and treated with 2DG for 4 hours. (C) Samples from (B) were analyzed for MyoD mRNA level by RT-qPCR (n=3).

Article Snippet: Human PKM2 wild-type construct was purchased from Addgene (44242) and sub-cloned to pCMV-Tag2B plasmid (Agilent Technologies).

Techniques: Immunostaining, Transfection, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: CARM1 suppresses de novo serine synthesis by promoting PKM2 activity

doi: 10.1074/jbc.RA118.004512

Figure Lengend Snippet: CARM1 promotes pyruvate kinase activity and suppresses serine synthesis. A, Carm1 knockout (−/−) MEFs exhibit lower PK activity than WT MEFs. In vitro PK assays were performed using total cell lysates of WT and Carm1−/− MEFs. B, re-expression of GFP-CARM1 in Carm1−/− MEFs restores PK activity. The rescued expression of CARM1 was confirmed by Western blot analysis (left panel). PK activity assays (right panel) were performed using total cell lysates from untransfected (−) and vector- or G-CARM1–transfected Carm1−/− MEFs. Actin was used as a loading control. C, expression of PKM1 and PKM2 was examined in WT and Carm1−/− MEFs by Western blot analysis. D, re-expression of FLAG-PKM1 (F-PKM1) in Carm1−/− MEFs restores PK activity. The expression of PKM1 was confirmed by Western blot analysis (left panel). PK activity assays (right panel) were performed using total cell lysates from the indicated cells. E, overexpression of PKM1 in Carm1−/− MEFs suppresses de novo serine synthesis. The level of 13C-labeled serine in the indicated cells was measured using GC-MS. Data represent mean ± S.E., n = 3.

Article Snippet: Plasmids and siRNA Human PKM1 (44241)- and PKM2 (44242)-expressing constructs for His-tagged recombinant protein purification were purchased from Addgene.

Techniques: Activity Assay, Knock-Out, In Vitro, Expressing, Western Blot, Plasmid Preparation, Transfection, Control, Over Expression, Labeling, Gas Chromatography-Mass Spectrometry

Journal: The Journal of Biological Chemistry

Article Title: CARM1 suppresses de novo serine synthesis by promoting PKM2 activity

doi: 10.1074/jbc.RA118.004512

Figure Lengend Snippet: CARM1 methylates PKM2 at Arg-445 and Arg-447 sites. A, PKM2 is methylated by CARM1. In vitro methylation assays were performed by incubating GST-tagged recombinant PRMTs (PRMT1, PRMT3, CARM1, Myc-PRMT5, and PRMT6) with purified His-tagged PKM2 proteins. A, B, and E, arrows indicate PKM2 methylation, and solid dots indicate PRMT automethylation. Membranes were stained with Ponceau S to ensure equal protein loading. B, recombinant human PKM1 (hPKM1) and PKM2 (hPKM2), mouse PKM2 (mPKM2), and tetramerization-deficient mutant mPKM2 (R399E) were subjected to in vitro methylation assays with CARM1. C, LC-MS/MS was performed to identify the arginine methylation sites of in vitro methylated His–mPKM2. Arginines 445 and 447 were found to be both mono- and dimethylated. D, PKM2 arginine methylation sites, Arg-445 and Arg-447, share sequence consensus with several known CARM1 substrates, including poly(A)-binding protein 1 (PABP1), histone H3R17, and cAMP-response element-binding protein-binding protein (CBP). E, in vitro methylation assays were performed using WT and R445K/R447K mutant PKM2, confirming that Arg-445 and Arg-447 are the major sites of methylation by CARM1.

Article Snippet: Plasmids and siRNA Human PKM1 (44241)- and PKM2 (44242)-expressing constructs for His-tagged recombinant protein purification were purchased from Addgene.

Techniques: Methylation, In Vitro, Recombinant, Purification, Staining, Mutagenesis, Liquid Chromatography with Mass Spectroscopy, Sequencing, Binding Assay, Protein Binding

Journal: The Journal of Biological Chemistry

Article Title: CARM1 suppresses de novo serine synthesis by promoting PKM2 activity

doi: 10.1074/jbc.RA118.004512

Figure Lengend Snippet: Arginine methylation PKM2 promotes its PK activity in vitro. A, arginine-methylated PKM2 exhibits significantly greater PK activity than unmethylated PKM2. Recombinant PKM2 was subjected to in vitro methylation by either WT or catalytic-deficient CARM1 (R169A) in the presence or absence of SAM. The methylation products were dialyzed and subjected to in vitro PK activity assays (left panel). In vitro methylation assays were performed to confirm that CARM1 (R169A) is catalytically deficient in methylating PKM2 (right panel). *, p < 0.05. B, CARM1 promotes PKM2 PK activity through arginine methylation. Recombinant WT and methylation-defective PKM2 (R445/447K) were subjected to in vitro methylation by CARM1. The reaction products were dialyzed and subjected to in vitro PK activity assays. NS, not significant. C, structural analysis of PKM2 reveals that methylation of Arg-447 (R447me2a) potentiates intramolecular interactions with distal amino acids, Leu-392 and Phe-421. D, mutations of amino acids involved in the intramolecular interactions of PKM2 abolish methylation-mediated regulation of PKM2 activity. E, recombinant WT, methylation-deficient (R445/447K), and intramolecular interaction-deficient (L392A, F421A, and L392A/F421A) PKM2 were subjected to in vitro methylation by CARM1. The reaction products were dialyzed and subjected to in vitro PK activity assays.

Article Snippet: Plasmids and siRNA Human PKM1 (44241)- and PKM2 (44242)-expressing constructs for His-tagged recombinant protein purification were purchased from Addgene.

Techniques: Methylation, Activity Assay, In Vitro, Recombinant

Journal: The Journal of Biological Chemistry

Article Title: CARM1 suppresses de novo serine synthesis by promoting PKM2 activity

doi: 10.1074/jbc.RA118.004512

Figure Lengend Snippet: Arginine methylation enhances PKM2 tetramerization. A, arginine methylation-deficient PKM2 (R445/447K) expressed in mammalian cells is less active than the WT enzyme. FLAG-tagged WT and R445K/R447K mutant PKM2 were expressed and purified from MCF7 cells (left panel). Eluted proteins were subjected to in vitro PK activity assays (right panel). B, loss of arginine methylation reduces PKM2 intermolecular interactions. Co-immunoprecipitation assays were performed to examine the interactions of endogenous PKM2 with FLAG-tagged WT or arginine methylation-deficient (R445/447K) PKM2 in MCF7 cells. * indicates the IgG heavy chain. Black arrow indicates transfected FLAG-tagged constructs, and white arrow indicates the endogenous PKM2. C, arginine methylation promotes PKM2 tetramerization. MCF7 cells were transfected with FLAG-tagged WT and arginine methylation-deficient PKM2 (R445/447K). Total cell lysates from transfected cells were separated by gel filtration, followed by Western blot analysis using an anti-FLAG antibody to determine the proportion of mono-, di-, and tetramers (indicated with open circles above the gels). D, reduced PKM2 tetramerization in Carm1−/− MEF cells. Total cell lysates from WT and Carm1 knockout (−/−) MEFs were separated by gel filtration, followed by Western blot analysis using an anti-PKM2 antibody.

Article Snippet: Plasmids and siRNA Human PKM1 (44241)- and PKM2 (44242)-expressing constructs for His-tagged recombinant protein purification were purchased from Addgene.

Techniques: Methylation, Mutagenesis, Purification, In Vitro, Activity Assay, Immunoprecipitation, Transfection, Construct, Filtration, Western Blot, Knock-Out

Journal: The Journal of Biological Chemistry

Article Title: CARM1 suppresses de novo serine synthesis by promoting PKM2 activity

doi: 10.1074/jbc.RA118.004512

Figure Lengend Snippet: CARM1 knockout cells are more resistant to serine deprivation than WT cells, likely due to reduced PK activity. A, CARM1 knockout (−/−) MEFs are more resistant to serine deprivation. The proliferation of WT and Carm1−/− MEFs in complete (left panel) and serine-free (right panel) medium was assayed over a 4-day period. B, WT and CARM1−/− MEFs respond similarly to the PKM2 small molecule activator TEPP-46. To activate PK activity, WT and Carm1−/− MEFs were either untreated or treated with TEPP-46 (25 μm) for 24 h. The total cell lysates were subjected to in vitro PK activity assays. C, activating PK activity using PKM2 activator TEPP-46 abolishes the survival advantage of CARM1−/− MEFs in serine-free medium. WT and Carm1−/− MEFs were treated with DMSO or TEPP-46 (25 μm) and subjected to culture in serine-free medium over a 4-day period. The cell proliferation was monitored as described in A.

Article Snippet: Plasmids and siRNA Human PKM1 (44241)- and PKM2 (44242)-expressing constructs for His-tagged recombinant protein purification were purchased from Addgene.

Techniques: Knock-Out, Activity Assay, In Vitro

Journal: PLoS ONE

Article Title: Lapachol inhibits glycolysis in cancer cells by targeting pyruvate kinase M2

doi: 10.1371/journal.pone.0191419

Figure Lengend Snippet: (A) The structure of human muscle Pyruvate kinase M2 [1ZJH—Receptor] docked to shikonin (ligand) using Autodock 4.2 software and ligand receptor interaction diagram generated using Discovery studio 2.5. The predicted free energy of binding for shikonin -7.98 kcal/mol and predicted inhibition constant was 1.42 μM. Inset shows the site pocket with surface charge distribution on donor and acceptor atoms. (B) The structure of human Pyruvate kinase M2 docked to lapachol and ligand receptor interaction diagram generated using Discovery studio 2.5. Inset shows the site pocket with surface charge distribution on donor and acceptor atoms. (C) The 17 amino acid residues of human PKM2 protein predicted to interact with the functional groups in shikonin and the type of interaction Van der waals (pale green), H-bond (bright green), Pi-Sigma (purple) and Pi-Alkyl (pink) are highlighted. (D) The 20 amino acid residues of human PKM2 protein predicted to interact with the functional groups in lapachol and the type interaction are highlighted.

Article Snippet: Human PKM1 (#44241) and PKM2 (#44242) in pET28a vector were purchased from addgene expressed as an N-terminal His 6 tag fusion protein. pET28a-PKM1 and pET28a-PKM2 was transformed into BL2 (DE3) pLysS cells and grown to an absorbance of 0.8 at 600 nm, then induced with 0.5 mM IPTG for 7 h at room temperature.

Techniques: Software, Generated, Binding Assay, Inhibition, Functional Assay

Journal: PLoS ONE

Article Title: Lapachol inhibits glycolysis in cancer cells by targeting pyruvate kinase M2

doi: 10.1371/journal.pone.0191419

Figure Lengend Snippet: (A) Recombinant human PKM1 enzyme was isolated and in vitro PKM1 enzyme activity was measured using a continuous assay coupled to lactate dehydrogenase. Different concentrations of lapachol (vehicle, 5 μM and 10 μM) did not show an inhibition in the activity of recombinant PKM1 invitro. (B) lapachol inhibited the activity of recombinant PKM2 in a dose dependent manner (n = 3/condition) **** P<0.0001 (C) Lapachol inhibited the pyruvate kinase activity invivo in the melanoma cell line MEL103 in a dose dependent manner (n = 3/condition)****P<0.0001.

Article Snippet: Human PKM1 (#44241) and PKM2 (#44242) in pET28a vector were purchased from addgene expressed as an N-terminal His 6 tag fusion protein. pET28a-PKM1 and pET28a-PKM2 was transformed into BL2 (DE3) pLysS cells and grown to an absorbance of 0.8 at 600 nm, then induced with 0.5 mM IPTG for 7 h at room temperature.

Techniques: Recombinant, Isolation, In Vitro, Activity Assay, Inhibition