Another Form of Modified, Highly-Active 6-Phosphofructo-1-Kinase in Cancer Cells

Research Article

Ann Hematol Oncol. 2021; 8(5): 1344.

Another Form of Modified, Highly-Active 6-Phosphofructo-1-Kinase in Cancer Cells

Kristl A, Camernik K, Avbelj Š and Legiša M*

National Institute of Chemistry, Slovenia

*Corresponding author: Matic Legiša, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia

Received: March 08, 2021; Accepted: April 09, 2021; Published: April 16, 2021


Enhanced glycolytic flux is a hallmarks of cancer cells. Posttranslational modification of the key regulatory enzyme of glycolysis, 6-Phosphofructo-1- Kinase (Pfk1) might trigger metabolic flux deregulation. In the cancer cells the human 85 kDa muscle type nPfk-M enzyme can be proteolytically cleaved to form highly-active 47 kDa shorter fragments that retain activity but become resistant to feed-back inhibition.

In several tumorigenic cell lines, no native 85 kDa liver type nPfk-L isoforms could be either found and only 70 kDa shorter fragments were detected by immune-blotting.

To learn more about the cancer-specific modified sfPfk-L enzyme, the truncated human sfPfk-L gene encoding 70 kDa fragments was inserted into the pfk null yeast S.cerevisiae cell. The recombinant modified enzyme showed higher affinity toward the substrate fructose-6-phosphate, reduced sensitivity toward the citrate and ATP inhibition in respect to the recombinant native PFK-L enzyme. Partially purified cancer-specific sfPfk-L fragments lacking the C-portion of the enzyme showed some instability under the diluted conditions in the buffer in respect to the tetrameric native nPfk-L enzyme. Growth characteristics of the yeast transformant encoding short sfPfk-L enzymes were similar to those encoding shorter sfPfk-M enzymes. No growth of the transformant with the sfPfk-L gene was observed on glucose but it grew faster than the transformant with the native human nPfk-L enzyme in a narrow ecological niche with low maltose concentration and 10 mM of ethanol in the medium.

Similar to modified 47 kDa sfPfk-M fragments, also the short 70 kDa nPfk- Lfragments might cause deregulation of the glycolytic flux in the yeast and in the cancer cells. In yeast, deregulated metabolic flux unbalances redox potential that results in reduced growth rate. However, the cancer cells beat the redox unbalance by rapid re-oxidation of redundant NADH that results in lactate formation while the growth rate remains high.

Keywords: Posttranslational modification; 6-Phosphofructo-1-kinase; Deregulated glycolytic flux; Tumorigenic cancer cells; Cancer; Lactate generation; Saccharomyces cerevisiae


Almost a century ago, Otto Warburg noticed that rat liver carcinoma cells showed similar oxygen consumption in relation to normal cells, but neoplastic cells produced lactic acid even under aerobic conditions. This observation was in contrast to normal tissues that ceased to produce lactic acid in a fermentative way in the presence of oxygen (Pasteur effect). Nowadays, it is generally accepted that the cancer glycolytic phenotype is induced by oncogenic mutations that alter growth factor signaling [1]. Activated transcription complex HIF-1a is increased, which, in combination with transcription factor c-Myc, enhances the synthesis of the majority of glycolytic enzymes [2]. However, glycolytic flux in eukaryotic organisms is tightly controlled by allosteric enzymes; therefore, some modifications of the kinetics of regulatory enzymes must be involved in the metabolic changes during the transformation of normal cells into cancer cells.

The most complex control of glycolytic flux in mammalian cells is attributed to allosteric regulation of 6-Phosphofructo-1-Kinase (Pfk1), the last enzyme of the preparatory stage of glycolysis. Pfk1 catalyzes the phosphorylation of Fructose-6-Phosphate (F6P) to fructose-1, 6-bisphosfate, using MgATP as a phosphoryl donor [3]. Pfk1 is stimulated by fructose-2, 6-bisphosphate (F-2, 6-BP), ADP/ AMP, and ammonium ions, whereas citrate and ATP act as strong inhibitors [3]. During evolution, eukaryotic Pfk1 enzymes developed by duplication, tandem fusion, and the divergence of catalytic and effector binding sites of a prokaryotic ancestor [4]. However, the active site of the eukaryotic enzyme is located only at the N-terminal portion, while the allosteric ligand binding sites that developed during evolution by mutations and enable the fine-tuning of the regulatory enzyme are scattered at both the N and C termini.

We were the first to show that the human Pfk1 enzyme is subjected to posttranslational modification in cancer cells [5]. The C-terminal portion of the native 85-kDa enzyme is cleaved by a specific protease to form a highly active 47-kDa fragment. Newly formed, modified enzymes were resistant to the feedback inhibition by citrate and ATP, while F-2, 6-BP increased their activities to a level higher than that of the native enzymes. In several tumorigenic cell lines, only the shorter 47-kDa Pfk1 fragments and no native Pfk1 enzymes of approximately 85-kDa were detected using immunostaining against the muscle type nPfk-M isoenzyme [5].

In cancer cells, the enhanced synthesis of glycolytic enzymes by the transcription complex Hif-1a, in collaboration with transcription factor c-Myc, was proposed to cause enhanced glycolytic flux [2]. However, posttranslational modification of Pfk1 enzymes might be a more important phenomenon for the deregulation of glycolytic flux in tumors that, in combination with altered signaling mechanisms, essentially supports the fast proliferation of cancer cells.

In mammalian genomes, three different Pfk1 genes are present that enable the synthesis of proteins; they have the following molecular masses: muscle type (nPfk-M), 85,051 Da [6]; liver type (nPfk-L), 84,917 Da [7]; and platelet type (nPfk-P), 85, 596 Da [8]. In different human tissues, Pfk1 isoenzymes are differently expressed and different proportions of all three isoenzymes have also been found in different tumorigenic cell lines [9,10].

A different type of shorter fragment was observed in cancer cells. Several tumorigenic cell lines that originate from the metastatic phases of tumors were immune-stained with monoclonal antibodies raised against liver type nPfk-L. Only the 70-kDa fragments were observed, while 47-kDa fragments were absent. Again, the 85-kDa native protein could not be detected either. In the present paper, the presence of the shorter 70-kDa sfPfk-L fragments in some tumorigenic cell lines is described, and the changed kinetic characteristics in respect to the native enzyme are discussed.

Materials and Methods


The tumorigenic cell lines UOK 262, Colo 829, Jurkat, MDAMB- 231, Caco-2, UT-7 and Raji were either purchased from American Type Culture Collection (Manassas, Virginia, USA) or obtained through the courtesy of various investigators at National Institute of Biology (Ljubljana, Slovenia) or Josef Stefan Institute (Ljubljana, Slovenia). Mycoplasma testing has been performed of all cell lines before used in the experiments.

Before immunostaining, tumorigenic cell lines were incubated in the following media: Colo 829, Jurkat, and MDA-MB-231, and Raji cells were grown in RPMI 1640-Glutamax medium supplemented with 10% FBS, while UOK 262 and UT-7 were grown in DMEM with 10% or 20% FBS, respectively. Inoculation started with 1.105 cells/ mL and culture plates were incubated at 37°C and 5% CO2. The cells were harvested when the total number reached approximately 1.106 cells/mL. For the extraction, the cells were collected by centrifuging at 1500 rpm for 5 minutes and washed twice with cold PBS buffer. The cells were lysed by RIPA buffer that was left on ice for 20 minutes. Finally, cell debris was collected by centrifugation and supernatantcontaining solubilized proteins were preserved at -20°C until needed. Protein concentrations of the samples taken from different tumorigenic cell lines were determined by Coomassie Protein Assay kit (Pierce, Thermo Fisher; USA). For western blotting, samples of the cell line homogenates containing 20 μg of protein were separated on SDS-PAGE polyamide gel with 0.1 sodium dodecyl sulfate. The transfer of proteins to a nitrocellulose membrane was confirmed by Ponceau Red. The membrane was blocked with I-Block reagent (Tropix Inc.; Bedford, MA, USA), and washed and incubated with primary PFKL antibody (H-36) sc-292523 (Santa Cruz Biotech; Santa Cruz, CA, USA). Rabbit polyclonal IgG antibody was prepared after inoculation of the epitope corresponding to the mapping of amino acids 46-81 near the N-terminus of the nPfk-L of human origin. Subsequently, goat anti-rabbit-HPS (Abcam; Cambridge, UK) secondary antibody was used for detection. For the loading control, the lysates were analyzed using anti-β-actin antibody (C4)-HRP sc- 47778 (Santa Cruz, CA, USA).

Recombinant genes construction

The yeast strain HD56-5A (MATa ura3-52, leu2-3, 112his3-11, 15 MAL3 SUC2 GAL) was used as a wild-type strain, and its isogenic Pfk1, pfk2 null derivative, HD114-8D (MATa Pfk1::HIS3 pfk2::HIS3 ura3-52, leu2-3, 112his3-11, 15 MAL3 SUC2 GAL; [11], was used as a recipient of recombinant human genes.

The nPFKL gene encoding native human liver type nPfk-L and the sfPfk-L gene encoding the shorter 70-kDa sfPfk-L fragments were synthesized using the Gibson Assembly technique [12], after the codon usage of the human PFKL nucleotide sequence (CCDS33582.1) was adjusted for the expression in yeast cells. For the insertion of the genes, the p416-GPD vector [13] was first amplified by PCR using oligonucleotide 5’-TCA TGT AAT TAG TTA TGT CAC GCT TAC- 3’ as a forward primer and 5’-TCT TTA TCC GTC GAA ACT AAG TTC-3’ as a reverse primer.

Yeast transformants encoding native human nPfk-L and shorter 70-kDa fragments were designated for the nPFKL and sfPfk-L strains, respectively. Another transformant was constructed by inserting the vector carrying the sfPfk-L gene into the wild-type strain (HD56-5A). As a negative control, the HD114-8D strain was transformed using an empty p416 plasmid.

Growth of yeast transformants

Transformants were grown on Supplemented Minimal Medium (SMM), as reported previously [14]. The substrate contained synthetic dropout medium without uracil (Sigma-Aldrich; St. Louis, MO, USA) containing 2% glycerol and 2% ethanol as non-fermentable carbon sources. As a nitrogen source, yeast nitrogen base without amino acids or ammonium sulfate (Sigma-Aldrich; St. Louis, MO, USA) was used with the addition of glutamine (0.25 g/L).

Partial purification of recombinant PFKL proteins

Yeast transformants encoding nPfk-L and sfPfk-L enzymes as the only form of Pfk1 were grown in SMM-GE until an OD600 value of 0.6 was reached. The cells were harvested by centrifugation at 5000 x g for 5 min, and then washed with 50 mL of ice-cold 50 mM sodium phosphate buffer. The precipitate was frozen under liquid nitrogen and stored at -80°C until needed.

Frozen yeast cells were disrupted in a Mikro-Dismembrator (Sartorius AG; Gottingen, Germany). Cell-free homogenate was extracted with 10 mL of cold 50 mM sodium phosphate buffer (pH 7.8) containing 0.15 M glycerol, 1 mM DTE, 1mM PMSF, 1 mM EDTA, and 10 μL/mL of protease inhibitor cocktail (Sigma-Aldrich; Steinheim, Germany). The same buffer was used throughout the whole isolation procedure. Dissolved proteins in the supernatant formed after centrifugation at 16,000 x g for 20 min at 4°C and were precipitated with ammonium sulfate, and a fraction (between 45% and 75%) of the saturation was taken for further purification. After dissolving the precipitated proteins and desalting the sample on a SephadexTM G-25 column (GE Health Care; Piscataway, NJ, USA), the proteins were loaded onto an affinity column containing 1 mL of aminophenyl-ATP-Sepharose (Jena Bioscience; Jena, Germany) that had been previously equilibrated with extraction buffer. After the sample was applied to the column, unbound proteins were removed by extensive washing. Recombinant nPfk-L enzyme was eluted from the column with 1.5 mL of buffer containing 6 mM F6P and 1 mM ADP. Eluted enzyme was dialyzed overnight against a buffer containing 20% (v/v) glycerol and stored at 4°C. Due to the extreme instability of the sfPfk-L fragments, the affinity chromatography part of the purification process was omitted and enzyme kinetics were measured immediately after the desalting of the sample.

Enzyme assays

Pfk1 activity was measured spectrophotometrically at 340 nm (Lambda25 UV/VIS spectrophotometer, Perkin Elmer) as reported previously [15] using a coupled reaction system. Unless otherwise stated, in a final volume of 1 mL, the assay mixture contained 50 mM HEPES buffer (pH 7.8), 1 mM DTE, 100 mM KCl, 5 mM MgCl2, 0.2 mM NADH, 0.025 to 2 mM F6P, 10% v/v polyethylene glycol (PEG 6000), 0.9 U/mL aldolase (Sigma-Aldrich; Steinheim, Germany), 15 U/mL triosephosphate isomerase, and 15 U/mL glycerol-3-phosphate dehydrogenase (Sigma-Aldrich; Steinheim, Germany). Before use, the auxiliary enzymes were dialyzed overnight at 4°C against 50 mM HEPES buffer (pH 7.8) containing 1 mM DTE, with one change of buffer after 8 h. Different concentrations of citrate were added to the assay mixture just before the reaction was started by the addition of ATP to achieve a final concentration of 0.5 mM (if not specified differently). ATP concentration used enabled optimal Pfk1 activities, but was below the limit to induce inhibition. Concentration of the enzyme used in the kinetic assays was 0.2 μg/mL. All presented kinetic data are averages obtained from a minimum of three replicate measurements. Total protein concentrations of the samples were determined using a Bio-Rad protein assay (Bio-Rad; Hercules, CA, USA) with bovine γ-globulin as a standard.

Growth rate coefficients

To follow growth kinetics, the transformants were grown in 500 mL baffled Erlenmeyer flasks with 100 mL medium in a rotary shaker at 100 rpm and 30°C. The media were inoculated with a single-cell colony pre-grown on SMM with Glycerol-Ethanol (SMM-GE). For the different carbon source tests, identical SMM were used, but the nonfermentable sugars were replaced with glucose or maltose. Growth kinetics were monitored by measuring optical density (OD600) with a spectrophotometer (Lambda 25, Perkin-Elmer; Boston, MA, USA) and growth coefficients were determined as previously described [14].

Statistics analysis

Analyses were performed using GraphPad Prism version 6.00 for Windows (GraphPad Software, San Diego, CA) and the data were presented as the mean ± SD (n=3). Unpaired two tailed Student’s T-test was used for comparison between two groups. P<0.05 was considered to indicate a statistically significant difference.


Detecting modified liver type sfPfk-L isoenzymes in metastatic tumor cell lines by immunoblotting

Previously, we found that the muscle type nPfk-M isoform can be predominantly cleaved into shorter 45 to 47-kDa fragments [5]. However, the presence of weak shorter 70-kDa fragments could be detected as well in some cell lines using polyclonal rabbit antibody raised against the specific epitope (CKDFREREGRLAA). A matching sequence of the nPfk-L isoform differs in four amino acid residues in respect to the nPfk-M epitope. To examine how liver type nPfk-L enzymes are modified in different neoplastic cell lines isolated from several metastatic tumors, polyclonal antibody raised against known epitope of the human nPfk-L enzyme were used for immunoblotting. The cell lines causing the following cancer types were tested: UOK 262, renal cell carcinoma; Colo 829, melanoma; Jurkat, acute T-cell leukemia; MDA-MB-231, mammary breast adenocarcinoma; Caco- 2, colorectal adenocarcinoma; UT-7 acute myeloid leukemia; and Raji, B-lymphocyte (Burkitt’s) lymphoma.

In the homogenate of all tested tumorigenic cell lines, the level of native 85-kDa nPfk-L was below the detection limit of the method. However, strong signals of approximately 70-kDa proteins were present in all tested cell lines. Faint 45-kDa fragments could be observed only in MDA-MB-231, UT-7, and Raji cells. Most importantly, no native nPfk-L enzymes with a molecular mass of 85- kDa could be detected in the extracts (Figure 1).