-. Transcriptional regulation of targets of NFAT was analyzed by qRT-PCR at 6 h post-treatment. Broecker-Preuss M., Becher-Boveleth N., Bockisch A., Dhrsen U., Mller S. Regulation of glucose uptake in lymphoma cell lines by c-MYC- and PI3K-dependent signaling pathways and impact of glycolytic pathways on cell viability. Ho P.-C., Bihuniak J.D., Macintyre A.N., Staron M., Liu X., Amezquita R., Tsui Y.-C., Cui G., Micevic G., Perales J.C., et al. 2013 Jul 15;126(Pt 14):3151-8. doi: 10.1242/jcs.124586. (B and C) Glucose uptake in splenic and intratumoral CD44, (A and B) Control (Ctrl) or HK2-OE Braf/Pten tumors were engrafted into the right and left flanks of C57BL/6 mice. Interestingly, ER-stress up-regulates ATF4-dependent transcription of mitochondrial PEPCK (PEPCK-M; PCK2), an isoform of PEPCK commonly found in tumor cells [15]. Phosphoenolpyruvate is synthesized through two reactions catalyzed, in order, by the enzymes: pyruvate carboxylase (EC 6.4.1.1); Nagasaki T., Hara M., Nakanishi H., Takahashi H., Sato M., Takeyama H. Interleukin-6 released by colon cancer-associated fibroblasts is critical for tumour angiogenesis: Anti-interleukin-6 receptor antibody suppressed angiogenesis and inhibited tumourstroma interaction. Mndez-Lucas A., Duarte J.A.G., Sunny N.E., Satapati S., He T., Fu X., Bermdez J., Burgess S.C., Perales J.C. PEPCK-M expression in mouse liver potentiates, not replaces, PEPCK-C mediated gluconeogenesis. Investigation of the kinetics and mechanism of the mitochondrial phosphoenolpyruvate carboxykinase-catalyzed reaction. Also, the inhibition of PEPCK-M exhibited a reduction of NFAT activity and an increase of cytosolic localization of this transcription factor, in accordance with the observed changes in the cytosolic calcium levels. HHS Vulnerability Disclosure, Help enzyme pyruvate kinase Explanation: The tenth and final reaction of glycolysis involves the conversion of phosphoenolpyruvate (PEP) into pyruvate. One molecule of adenosine triphosphate (ATP) is generated from one molecule of PEP. (B and C) Intracellular Ca, (A) Intracellular calcium levels were measured in Fluo-4- and Fura-Red-labeled CD4+ T cells cultured in Ca, (A) Illustration of the glycolysis pathway and the targets of the indicated glycolytic inhibitors. Figure 1. Kuprat T, Johnsen U, Ortjohann M, Schnheit P. Front Microbiol. Would you like email updates of new search results? The assay was performed by mixing 10 L of media or standard with 200 L of reaction buffer (0.3 M hydrazine sulfate, 0.87 M glycine, 2.5 mM NAD+, and 0.19 M EDTA at pH 9.5). 3. After 20 min of incubation at RT NADH synthesized in LDH, catalyzed reaction was measured and corrected for background. (B) Heat map shows the normalized concentrations of the indicated glycolytic metabolites in CD4+ T cells stimulated with anti-CD3/anti-CD28 mAbs for 1 hr in the absence or presence of the glycolytic inhibitors described in (A) as measured by LC-QE-MS. (C) Intracellular Ca, (A) Illustration of the metabolic function of PCK1 in converting OAA to PEP. The mitochondrial isoform of phosphoenolpyruvate carboxykinase (PEPCK-M) and glucose homeostasis: has it been overlooked? Similarly, loading the metabolite directly into the cell increased cytosolic calcium and NFAT activity. Bookshelf doi: 10.7554/eLife.67358. 2021 Aug 10;13:6291-6307. doi: 10.2147/CMAR.S321433. See this image and copyright information in PMC. Graziano F., Ruzzo A., Giacomini E., Ricciardi T., Aprile G., Loupakis F., Lorenzini P., Ongaro E., Zoratto F., Catalano V., et al. p < 0.01, ***,$$$,### Epub 2012 Jul 31. 2022 May 14;23(10):5490. doi: 10.3390/ijms23105490. Cell Death Dis. Accessibility Glycolysis gene expression analysis and selective metabolic advantage in the clinical progression of colorectal cancer. 2017 Sep 15;292 . Testing biochemistry. (D) Western blot analysis of the phosphorylation status of c-Myc at Ser62, and the activation of several c-Myc target proteins. Regulatory crosstalk of the metabolic network. Mitochondrial Phosphoenolpyruvate Carboxykinase Regulates Metabolic Adaptation and Enables Glucose-Independent Tumor Growth. 2006 Apr;60(2):287-98. doi: 10.1111/j.1365-2958.2006.05098.x. p < 0.01, ### To conclude, we propose a model where glucose and PEPCK-M regulate calcium signaling through PEP (Figure 5). When PEP was loaded onto cells in culture, IL6 and PTGS2 mRNA content augmented in a dose-dependent manner (Figure 2H,K). Increasing extracellular glucose or PEP concentrations produced a similar dose-dependent increase in NFAT activity. In the absence of glucose, the concentration of PEP was found close to the detection limit of our assay in both models but the inhibition of PEPCK-M was effective at further decreasing PEP concentrations (Figure 1H). This scenario indicates that glucose and PEPCK-M are supporting, through the PEP/Ca2+ axis, a proliferative state in tumors, and bring about the importance of this target for cancer metabolism, as exemplified by the efficacy of potent inhibitors of this pathway in pre-clinical studies [22]. The aim of this study was to examine the effects of PEP on hepatic energy metabolism at an early phase after . Quantitative modeling of pentose phosphate pathway response to oxidative stress reveals a cooperative regulatory strategy. cDNA was quantitated using real-time quantitative RT-PCR assays in a 7900HT real-time quantitative RT-PCR system (ThermoFisher Scientific, Waltham, MA USA) using commercial Taqman primers for PTGS2 (Hs00153133-m1), IL6 (Hs00174131_m1), and TBP (Hs99999910_m1). Transcribed image text: In glycolysis, 2-phosphoglycerate is converted to phosphoenolpyruvate through a dehydration reaction. Finally, CsA was able to abolish the up-regulation of IL6 and PTGS2 caused by PEP, demonstrating calcineurin/NFAT-dependent regulation (Figure 2H,K). This work was supported by grants from the Spanish Ministerio de Economia y Competitividad (MINECO; BFU2015-66030-R) to JCP and CE, Generalitat de Catalunya (2017SGR106 and 2017SGR204), and the European Union (European Regional Development Fund, FEDER). Evidence of a triosephosphate isomerase non-catalytic function crucial to behavior and longevity. Pan M.-G., Xiong Y., Chen F. NFAT gene family in inflammation and cancer. We examined here whether cytosolic calcium and the activation of downstream effector pathways important for tumor biology are influenced by the presence of glucose and/or cataplerosis through the phosphoenolpyruvate carboxykinase (PEPCK) pathway, as both are hypothesized to feed the PEP pool. You may notice problems with : glucose; 2-DG: 2-deoxy-D-glucose. Pearce E.L., Poffenberger M.C., Chang C.-H., Jones R.G. 2020 Dec 4;11:604926. doi: 10.3389/fmicb.2020.604926. P.M.G.-R.: discussed the preliminary draft and provided feedback on general and specific details of the manuscript. Regulation of Glycolysis and Gluconeogenesis - Step 1. Both ADP and AMP, indicators of a low-energy state, activate PFK-1. Intricate Regulation of Phosphoenolpyruvate Carboxykinase (PEPCK) Isoforms in Normal Physiology and Disease. Following primary antibodies were used: anti-phospho-Ser62/T58 c-Myc (Santa Cruz, Dallas, TX, USA; sc-377552), anti-c-Myc C-19 (Santa Cruz, Dallas, TX, USA; sc-788), anti-GLS1 (Abcam, Cambridge, UK; ab131554), anti-cSHMT A-2 (Santa Cruz, Dallas, TX, USA; sc-365203), anti-mSHMT F-11 (Santa Cruz, Dallas, TX, USA; sc-390641), anti-HK-2 (Cell Signaling, Danvers, MA, USA; 2867), anti-Glut1 (Santa Cruz, Dallas, TX, USA; sc-277228), anti-LDHA (Santa Cruz, Dallas, TX, USA; sc-137243). PEPCK-M recoups tumor cell anabolic potential in a PKC--dependent manner. Two principal calcium transporters and pumps regulate calcium trafficking between the ER and cytosol, IP3R, which consists on a Ca2+-permeable ER ion channel, and SERCA, a Ca2+/ATPase pump located in the ER membrane that regulate the return of Ca2+ from the cytosol to the ER lumen. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (, phosphoenolpyruvate carboxykinase (PEPCK), PEPCK-M, PCK2, phosphoenolpyruvate carboxykinase, cancer metabolism, calcium, c-Myc, NFAT, CaMK2G, phosphoenolpyruvate, TCA cycle, cataplerosis, sarcoplasmic reticulum Ca, {"type":"entrez-nucleotide","attrs":{"text":"F36206","term_id":"4821831"}}, Glucose availability and PEPCK-M regulates cytosolic calcium through PEP. J Biol Chem. Clipboard, Search History, and several other advanced features are temporarily unavailable. 2021 Jul 12;10(7):48. doi: 10.1038/s41389-021-00339-6. Explain how gluconeogenesis bypasses this irreverisble reaction. This step Direct immunofluorescence for NFATc1 (green) and nuclear associated DAPI (blue) in basal, ionomycin (1 M) and CsA (1 M) conditions (D). 2013. All experiments were performed in the SW480 cell line. Stabilization of the c-Myc Protein by CAMKII Promotes T Cell Lymphoma. Would you like email updates of new search results? Alteration of the biochemical valves in the central metabolism of Escherichia coli. The physiological roles of the two enzymes under various growth conditions are discussed. 2022 Jun 8;11(12):1535. doi: 10.3390/plants11121535. 2007. Our results suggest that NFAT activation is modulated in a glucose- and PEPCK-M-dependent manner. The site is secure. : performed experiments, collected and analyze data, and discussed the preliminary draft and provided feedback on general and specific details of the manuscript. Clipboard, Search History, and several other advanced features are temporarily unavailable. 2012 Sep 21;287(39):32881-96. doi: 10.1074/jbc.M112.357889. (B) Intracellular PEP levels were measured after culturing the RV-transduced cells for 1 hr in the indicated conditions using a fluorescence-based assay. The ATP formed during the conversion of PEP to pyruvate by pyruvate kinase was measured using StayBriteTM kit (Highly stable ATP bioluminescence assay kit K791-1000; Biovision, Milipitas, CA, USA). Epub 2013 Feb 22. The transformation in glycolysis of phosphoenolpyruvate (PEP)1 to pyruvate is coupled with the synthesis of ATP. MeSH PMC 2019 Dec 19;9(1):18. doi: 10.3390/cells9010018. Protein expression of several c-Myc targets also correlated with PEP levels. 2021 Mar 23;203(8):e00690-20. Structure of the triosephosphate isomerase-phosphoenolpyruvate complex. After washing 3 times with blocking buffer, samples were examined using a confocal laser scanning microscopy ZEISS LSM 880 (Carl Zeiss AG, Oberkochen, Germany) and ZEN 2012 (Zeiss, Oberkochen, Germany) was used to collect digital images. The authors declare no conflicts of interest. Disclaimer, National Library of Medicine Global network reorganization during dynamic adaptations of Bacillus subtilis metabolism. (, Drawing depicting a model for the integration of ER-stress mediated up-regulation of PEPCK-M and the control of integrative signals downstream of the PEP/Ca. Bethesda, MD 20894, Web Policies Cancer Manag Res. * p < 0.05, ** p < 0.01, *** p < 0.001 versus DMSO/5.5 mM glucose; or # Tumor Microenvironment Deprives Glucose to Infiltrating CD4 + T Cells, Figure 2. Therefore, with DNA damage or cellular stress, calcium fluxes might determine cellular fate. Lin J.-T., Wang J.-Y., Chen M.-K., Chen H.-C., Chang T.-H., Su B.-W., Chang P.-J. Leithner K., Triebl A., Trtzmller M., Hinteregger B., Leko P., Wieser B.I., Grasmann G., Bertsch A.L., Zllig T., Stacher E., et al. PEP Suppresses SERCA-Mediated ER Calcium, Figure 5. Tie X., Han S., Meng L., Wang Y., Wu A. NFAT1 Is Highly Expressed in, and Regulates the Invasion of, Glioblastoma Multiforme Cells. The next point of regulation in glycolysis is the conversion of fructose 6-phosphate to fructose 1,6-bisphosphate by PFK-1 (Figure 3). We examined here whether cytosolic calcium and the activation of downstream effector pathways important for tumor biology are influenced by the presence of glucose and/or cataplerosis through the phosphoenolpyruvate carboxykinase (PEPCK) pathway, as both are hypothesized to feed the PEP pool. 1966 May . Monteith G.R., Prevarskaya N., Roberts-Thomson S.J. C-Myc phosphorylation at Ser62 was reduced by IPEPCK-2 or by glucose starvation (Figure 4D; quantified in Figure 4E). NADH was determined by fluorescence (excitation 340 nm/emission 460 nm). PCK2 transcription is controlled by the ER stress pathway, downstream of GNC2-PERK-ATF4 [15]. Disclaimer, National Library of Medicine J Cell Sci. Matsubara K, Yokooji Y, Atomi H, Imanaka T. Mol Microbiol. Part of the Ca2+ signaling cascade, calmodulin kinase type II gamma (CaMKII), plays an important role in tumor progression of prostate cancer by activation of AKT in a PI3K-independent manner [30], or in the development of colitis-associated cancer through activation of STAT3 [31]. Chang C.-H., Qiu J., OSullivan D., Buck M.D., Noguchi T., Curtis J.D., Chen Q., Gindin M., Gubin M.M., van der Windt G.J.W., et al. Steps of Glycolysis process 10: Transfer of phosphate from PEP to ADP : This is the last step of glycolysis. Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M) is a pro-survival, endoplasmic reticulum (ER) stress response gene involved in tumor cell adaptation to nutrient availability. The https:// ensures that you are connecting to the Epub 2013 May 2. p < 0.05, ## official website and that any information you provide is encrypted Pharmacology and preclinical validation of a novel anticancer compound targeting PEPCK-M. Vander Heiden M.G., Cantley L.C., Thompson C.B. 35, 220227. ER is quasi constitutive in tumors, where glucose is low. One-way ANOVA, Sidak post-test. Federal government websites often end in .gov or .mil. In addition, glycolytic intermediates, such as glucose-6-phosphate (G6P) or phosphoenolpyruvate (PEP), can regulate SERCA activity in the brain and T cells , independently of ATP. 2021 Jan 7;9(1):1. doi: 10.1186/s40170-020-00236-3. Goetzman E.S., Prochownik E.V. J.M.-F.: performed experiments, collected and analyze data. The last step of the payoff phase, and in turn, the last step of glycolysis, involves the transfer of a phosphate group from phosphoenolpyruvate to ADP, forming pyruvate and ATP ( Figure 6 ). Revving an Engine of Human Metabolism: Activity Enhancement of Triosephosphate Isomerase via Hemi-Phosphorylation. Moreno-Felici J, Hyroov P, Arag M, Rodrguez-Arvalo S, Garca-Rovs PM, Escolano C, Perales JC. Federal government websites often end in .gov or .mil. -, Rimessi A., Bonora M., Marchi S., Patergnani S., Marobbio C.M., Lasorsa F.M., Pinton P. Perturbed mitochondrial Ca2+ signals as causes or consequences of mitophagy induction. For full credit, detail: 1) The enzymes . Gu Y., Zhang J., Ma X., Kim B., Wang H., Li J., Pan Y., Xu Y., Ding L., Yang L., et al. 2014 Apr;1840(4):1313-30. doi: 10.1016/j.bbagen.2013.10.033. (B) PTGS2 and IL6 mRNA expression in the presence of IPEPCK-2 (8.68 M) or vehicle (DMSO) in SW480 cells cultured in media with 5.5 mM glucose or the absence of glucose (0 mM). official website and that any information you provide is encrypted The .gov means its official. In eukaryotes, glycolysis takes place in the cytosol In consequence, we hypothesize that the cataplerotic activity of PEPCK-M fluxing glutamine [17] and/or lactate [18] carbons towards the glycolytic intermediary pool might have a potential role in modulating cytosolic calcium signaling pathways, and hence the metabolic fate of the cell. Raffeiner P., Schraffl A., Schwarz T., Rck R., Ledolter K., Hartl M., Konrat R., Stefan E., Bister K. Calcium-dependent binding of Myc to calmodulin. Co-crystal structure of TPI with bound PEP. The samples were diluted in the PEP assay buffer (gly-gly 0.1 M; KCl 0.2 M; MgCl2 1 mM; reconstituted enzyme from StayBriteTM kit 0.1% (v/v); pH 7). Commun. . 8600 Rockville Pike Bethesda, MD 20894, Web Policies The https:// ensures that you are connecting to the Epub 2011 Jul 21. Scale bar represents 20 m. Intracellular levels of (, Activation of the NFAT pathway by PEP/calcium. Artificial intelligence for quantifying Crohn's-like lymphoid reaction and tumor-infiltrating lymphocytes in colorectal cancer. 2012. Cells. It . Wiel C., Lallet-Daher H., Gitenay D., Gras B., Le Calv B., Augert A., Ferrand M., Prevarskaya N., Simonnet H., Vindrieux D., et al. In principle, both enzymes can catalyse the final step of the modified Embden-Meyerhof (EM) pathway found in Thermococcales, the co Multidisciplinary Digital Publishing Institute (MDPI). Protein expression of several c-Myc targets also correlated with PEP levels. Therefore, we hypothesized that a similar mechanism would take place in the tumor cell to relay the concentration of glucose and other metabolites in the environment with PEP levels and calcium cell signaling, with consequential gene expression changes that are relevant to tumor biology. 2022 Jun 28;25(8):104681. doi: 10.1016/j.isci.2022.104681. Accessibility (A) NFAT activity in the presence of IPEPCK-2 (8.68 M) or vehicle (DMSO) in SW480 cells cultured in media with 5.5 mM glucose or the absence of glucose (0 mM). 2022 Jan 21;10(2):233. doi: 10.3390/microorganisms10020233. Plants (Basel). Consistently, the inhibition of PEPCK-M reduced the amount of PEP and phosphorylated c-Myc in the presence or absence of glucose, accounting for a regulatory axis that is independent of glucose but can promote glucose metabolism. The increment of luminescence was measured with a luminometer (TD 20/20; Turner Designs, San Jose, CA, USA) 2 min after the addition of pyruvate kinase (to a final concentration of 13.5 U/mL), and the results were normalized by protein content. Tjaden B, Plagens A, Drr C, Siebers B, Hensel R. Mol Microbiol. The expression of PTGS2 and IL6 were increased at higher concentrations of glucose (Figure 2G), being highest at 25 mM, and lowest at 0 mM glucose co-treated with CsA (Figure 2G,J). Hurbain J, Thommen Q, Anquez F, Pfeuty B. iScience. : performed experiments, collected and analyze data, and discussed the preliminary draft and provided feedback on general and specific details of the manuscript. . Accessibility All membranes were normalized using mouse monoclonal anti--tubulin antibody (Sigma-Aldrich, Darmstadt, Germany; T-6557). An official website of the United States government. Phosphoenolpyruvate (2-phosphoenolpyruvate, PEP) is the ester derived from the enol of pyruvate and phosphate.It exists as an anion.PEP is an important intermediate in biochemistry.It has the highest-energy phosphate bond found (61.9 kJ/mol) in organisms, and is involved in glycolysis and gluconeogenesis.In plants, it is also involved in the biosynthesis of various aromatic compounds, and . (BD) CD4+ T cells were transduced with control (Ctrl) or PCK-1 overexpressing (PCK1-OE) RVs. Science. NFAT target genes PTGS2 and IL-6 mRNA levels at various glucose concentrations in the culture media (G,J). 8600 Rockville Pike Ann N Y Acad Sci. Before 2017;17:367380. NFAT target genes PTGS2 and IL-6 mRNA levels at different extracellular PEP loads (H,K). Expert Answers: Phosphoenolpyruvate, also known as pep or 2-(phosphonooxy)-2-propenoic acid, is a member of the class of compounds known as phosphate esters. SW480 cells plated on coverslips ( 15 mm) were washed with PBS and fixed with 4% paraformaldehyde in PBS for 10 min. 2-DG: 2-deoxyglucose; IAA: iodoacetate; OXA: oxalate. Biology questions and answers. (, Low TPI activity increases PPP metabolite load and causes oxidant resistance and heat sensitivity. After 3 washes with blocking buffer, cells were incubated with anti-rabbit Alexa Fluor 555 (Invitrogen, Carlsbad, CA, USA, A28175) or anti-mouse Alexa Fluor 488 secondary antibodies (Invitrogen, A27039) for 2 h. During this incubation, DAPI was added to stain the nuclei. : analyzed data, discussed the preliminary draft and provided feedback on general and specific details of the manuscript. However, the physiological role of the endogenous PEPCK pathway was not assessed. The glycerol backbone of phospholipids derives from noncarbohydrate precursors in starved lung cancer cells. In principle, both enzymes can catalyse the final step of the modified Embden-Meyerhof (EM) pathway found in Thermococcales, the conversion of phosphoenolpyruvate (PEP) to pyruvate, with the former utilizing ADP, while the latter is dependent on AMP and phosphate. Bookshelf Identification of a putative regulator of early T cell activation genes. However an even more dramatic effect was observed in the Deltapps strain in that the strain could not grow at all on maltooligosaccharides. government site. Bookshelf Please enable it to take advantage of the complete set of features! FOIA PEP correlated with cytosolic calcium and NFAT activity, together with transcriptional up-regulation of canonical targets PTGS2 and IL6 that was fully prevented by CsA pre-treatment. HHS Vulnerability Disclosure, Help This site needs JavaScript to work properly. Can yeast glycolysis be understood in terms of in vitro kinetics of the constituent enzymes? Therefore, ER-stress could potentiate the PEP/Ca2+ axis in a PEPCK-M dependent manner, especially in conditions of limited nutrients for the cell. Cells were blocked in blocking buffer (PBS with 1% NHS, and 0.1% TritonTM X-100) for 2 h and then treated with NFATc2 (A2) and c-Myc (C-19) primary antibodies (Santa Cruz, sc-514929 and sc-788 respectively) overnight at 4 C. The asymmetric unit contains a homodimer of TPI (figure 1a).Each protomer contains eight -helices on the outside and eight parallel -strands . Curr Mol Med. Hyroov P, Arag M, Moreno-Felici J, Fu X, Mendez-Lucas A, Garca-Rovs PM, Burgess S, Figueras A, Vials F, Perales JC. Calcium/calmodulin-dependent kinase II plays an important role in prostate cancer cell survival. Concurrently with c-Myc phosphorylation rates, several c-Myc targets relevant to metabolism were also regulated by glucose, and this regulation was PEP-dependent. 2018;8:25. doi: 10.1186/s13578-018-0223-5. . Unable to load your collection due to an error, Unable to load your delegates due to an error, Co-crystal structure of TPI with bound PEP. (, Drawing depicting a model for the integration of ER-stress mediated up-regulation of PEPCK-M and the control of integrative signals downstream of the PEP/Ca. This site needs JavaScript to work properly. FOIA You may switch to Article in classic view. doi: 10.1016/j.ccell.2017.08.004. Epub 2009 Jul 27. Print 2021 Mar 23. In a paradigmatic case, T-cells exposed to limiting glucose concentrations cannot activate proper anti-tumoral response [24]. Finally, the participation of PEPCK in this axis was interrogated as it should directly contribute to PEP through cataplerosis from TCA cycle intermediates, especially in glucose starvation conditions. HHS Vulnerability Disclosure, Help 2015 Sep 10;162(6):1206-8. doi: 10.1016/j.cell.2015.08.064. These results support a model in which glycolytic regulation requires direct catalytic inhibition of TPI by the pyruvate kinase substrate PEP, mediating a protective metabolic self-reconfiguration of central metabolism under conditions of oxidative stress. Rokhlin O., Taghiyev A.F., Bayer K.U., Bumcrot D., Kotelianski V.E., Glover R.A., Cohen M.B. The calciumcancer signalling nexus. mSystems. Collectively, these data indicate that NFAT dependent transactivation activity depends on glucose availability and subsequent intracellular changes on the PEP pool. doi: 10.1084/jem.20221140. The Role for Myc in Coordinating Glycolysis, Oxidative Phosphorylation, Glutaminolysis, and Fatty Acid Metabolism in Normal and Neoplastic Tissues. Mndez-Lucas A, Hyroov P, Novellasdemunt L, Vials F, Perales JC. In T cells, extracellular glucose is key to activating cell proliferation, aerobic glycolysis, and anabolism as T cells mount anti-tumoral responses. Analysis, Consequences of PEPCK-M inhibition on the levels of activation of the NFAT and, Drawing depicting a model for the integration of ER-stress mediated up-regulation of PEPCK-M, MeSH A temporal examination of calcium signaling in cancer- from tumorigenesis, to immune evasion, and metastasis. Careers. Cell Biosci. The transfer of the phosphate group from PEP to ADP, catalyzed by pyruvate kinase [10], is also highly exergonic . FEBS Lett. Total RNA was extracted using TRIsureTM reagent. Western blot analysis of mitochondrial glucosamine (mGLS), and cytosolic serine hydroxymethyltransferase (cSHMT), both described as a c-Myc targets, after 4 h in the presence or absence of glucose in the culture media (H). Burgos A, Miranda E, Vilaprinyo E, Meza-Canales ID, Alves R. Front Plant Sci. All these results suggest a role for PEPCK-M in upkeeping the PEP pool, even in the presence of glucose. We have carried out a genetic analysis on pyruvate kinase (PykTk) and phosphoenolpyruvate synthase (PpsTk) in the hyperthermophilic archaeon, Thermococcus kodakarensis. 267, 53135329. We have carried out a genetic analysis on pyruvate kinase (PykTk) and phosphoenolpyruvate synthase (PpsTk) in the hyperthermophilic archaeon, Thermococcus kodakarensis. Subcell Biochem. This is consistent with the need for double inhibition of c-Myc and PI3K signaling pathways to reduce glucose uptake and glycolytic flux in lymphoma cell lines [37]. Moreover, PCK1-overexpressing T cells restricted tumor growth and prolonged the survival of melanoma-bearing mice. 2014 Aug 8;289(32):22090-102. doi: 10.1074/jbc.M114.566927. The calciumcancer signalling nexus. High rates of tumor cell glycolysis suppress intratumoral T cell function by depriving T cells of glucose and the downstream metabolite phosphoenolpyruvate (PEP), which is necessary for maximal Ca2+ -NFAT signaling in T cells. Advances and Hurdles in CAR T Cell Immune Therapy for Solid Tumors. pNFATx3-Luc vector was a gift of Merc Prez-Riba (Medical and Molecular Genetics Center, IDIBELL, LHospitalet del Llobregat, Spain). To study if the presence of glucose, via modulation of PEP levels, can activate the calcineurin/NFAT cascade, we evaluated the activity of a luciferase reporter vector under the control of a chimeric promoter containing 3 adjacent canonical NFAT binding sites. Nifetepimine, a dihydropyrimidone, ensures CD4+ T cell survival in a tumor microenvironment by maneuvering sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA). In addition, we have demonstrated that changes in Ca2+ produced by different glucose and PEP concentrations lead to activation and nuclear translocation of NFAT. J Biol Chem. Tumor-specific CD4 and CD8 T cells could be metabolically reprogrammed by increasing PEP production through overexpression of phosphoenolpyruvate carboxykinase 1 (PCK1), which bolstered effector functions. As the enzyme's name suggests, this reaction involves the transfer of a phosphate group. J Biol Chem. Clipboard, Search History, and several other advanced features are temporarily unavailable. Furthermore, phosphorylation was abrogated by pretreatment with KN-93, an inhibitor of calmodulin (Figure 3A; quantitated in Supplementary Figure S3). Fueling immunity: Insights into metabolism and lymphocyte function. ); ude.bu@onalocsec (C.E. Unable to load your collection due to an error, Unable to load your delegates due to an error, (A) Bar graphs show the glucose concentration in blood and interstitial fluid of tumors and spleens from Braf/Pten melanoma-bearing mice. eCollection 2022. Lane MD. We discovered a new role for the glycolytic metabolite phosphoenolpyruvate (PEP) in sustaining T cell receptor-mediated Ca(2+)-NFAT signaling and effector functions by repressing sarco/ER Ca(2+)-ATPase (SERCA) activity. FOIA Other articles where phosphoenolpyruvate is discussed: metabolism: The formation of ATP: reacts with 2-phosphoglycerate to form phosphoenolpyruvate (PEP), water being lost from 2-phosphoglycerate in the process. Therefore, increased consumption of glucose by CD4 and CD8 T cells translates into raising the pool of PEP, inhibiting SERCA, and increasing cytosolic Ca2+ which in turn signals the activation of NFAT. In this process, ATP is regenerated, just like step 7. Logically, we found that NFAT nuclear localization was mainly observed in the presence of glucose and DMSO and was decreased when cells were glucose starved, or PEPCK-M was inhibited (Figure 4C). This study uncovers new metabolic checkpoints for T cell activity and demonstrates that metabolic reprogramming of tumor-reactive T cells can enhance anti-tumor T cell responses, illuminating new forms of immunotherapy. (, The TPI inhibitor PEP and the TPI substrate DHAP have similar interaction sites. The model was validated using ionomycin as a positive control of activation of NFAT, and cyclosporin A, to abrogate Ca2+ signaling (Figure 2A). In addition, we assayed the subcellular localization of total c-Myc (Figure 3B). Dang C. V c-Myc target genes involved in cell growth, apoptosis, and metabolism. The PEP-pyruvate-oxaloacetate node: variation at the heart of metabolism. Also, the proto-oncogenes of the Myc family of transcription factors involved in several cellular processes, including cell proliferation [7], and cell growth and metabolism [8,9,10], have been shown to respond to calcium both transcriptionally and post-transcriptionally upon CaMKII-dependent phosphorylation at Ser62 [11]. Interestingly, our data show that total c-Myc protein levels were lower when glucose was lacking, pointing to c-Myc stability regulation by calcium. Conceived of the work. Partial nuclear exclusion of c-Myc was observed under glucose deprivation (Figure 3C), in agreement with lowered phosphorylation observed under glucose starvation, a reduction that was mimicked by total c-Myc (Figure 3D; quantified in Figure 3E). NFAT activation levels at different extracellular PEP loads (C). Given the curved arrow mechanism below, identify the mechanism type of the dehydration. Phosphoenolpyruvate from Glycolysis and PEPCK Regulate Cancer Cell Fate by Altering Cytosolic Ca 2. Nat. 3.1. Rimessi A., Bonora M., Marchi S., Patergnani S., Marobbio C.M., Lasorsa F.M., Pinton P. Perturbed mitochondrial Ca. The fluxes through glycolytic enzymes in Saccharomyces cerevisiae are predominantly regulated at posttranscriptional levels. Where does the energy for this process come from? eCollection 2021. Colon cancer mesenchymal stem cells modulate the tumorigenicity of colon cancer through interleukin 6. Altogether, these results indicate that PEPCK-M does not alter PEP levels indirectly through the modulation of glycolysis, and hint for a contribution of PEPCK-M to the maintenance of the PEP pool in the presence of glucose by fluxing glutamine or other anaplerotic carbons from the TCA cycle, at least in certain types of cancer. Triosephosphate isomerase: a highly evolved biocatalyst. Understanding the Warburg effect: The metabolic requirements of cell proliferation. Warburg effect in colorectal cancer: the emerging roles in tumor microenvironment and therapeutic implications. NFAT target genes PTGS2 and IL-6 mRNA levels in the presence of ionomycin (1 M) or ionomycin plus CsA (1 M) (F,I). Triosephosphate isomerase (TPI) facilitates the replication of WSSV in Exopalaemon carinicauda. Details: The final step of glycolysis converts phosphoenolpyruvate into pyruvate with the help of the enzyme pyruvate kinase. In plants, it is also involved in the biosynthesis of various aromatic compounds, and in carbon fixation . Cytosolic Ca2+ concentrations, as determined by relative Fluo4 fluorescence mediated by Ca2+ binding, correlated with the level of PEP and glucose media concentrations (Figure 1B; correlation statistics in Figure 1C). Data are the mean SE, n = 4 (for western blot analysis, 2 replicates were loaded in each experiment, and each experiment was independently repeated at least twice). Singh G., Singh S.K., Knig A., Reutlinger K., Nye M.D., Adhikary T., Eilers M., Gress T.M., Fernandez-Zapico M.E., Ellenrieder V. Sequential Activation of NFAT and c-Myc Transcription Factors Mediates the TGF- Switch from a Suppressor to a Promoter of Cancer Cell Proliferation. Then, cells were incubated for 15 min with sucrose medium with the desired PEP concentration. Epub 2017 Jan 23. Two molecules of pyruvate and ATP are obtained as the . Epub 2021 Jul 27. Densitometric analysis (K) of WB shown in (J). Overexpression of cyclooxygenase-2 in multiple myeloma: Association with reduced survival. The phosphate linkage in phosphoenolpyruvate has very high bond energy. An official website of the United States government. PEP Suppresses SERCA-Mediated ER Calcium Reuptake, (A) Illustration of the glycolysis pathway and, Figure 6. Moreover, nuclear distribution was also observed in the presence of glucose, consistent with the translocation of phosphorylated c-Myc in these situations. Fourteen days later, the CD4, (A) Naive CD4+ T cells from Nur-77-eGFP mice were left unstimulated or stimulated with anti-CD3/anti-CD28 mAbs for 5 hr in the indicated conditions and GFP fluorescence was measured by flow cytometry. 2009 May;58(5):801-8. doi: 10.1007/s00262-008-0635-x. Direct immunofluorescence for c-Myc (red) and DAPI (blue) in the presence and absence of glucose in the media (C). PEP loading increased c-Myc phosphorylation at Ser62 by CaMKII. 8600 Rockville Pike Bethesda, MD 20894, Web Policies The results indicated that both enzymes participated in pyruvate metabolism, but were not essential. Pyruvate kinase triggers a metabolic feedback loop that controls redox metabolism in respiring cells. NFAT is activated by ionomycin (1 M) and inhibited by CsA (1 M) (, Glucose increases c-Myc phosphorylation at Ser62 in a PEP/Ca, Consequences of PEPCK-M inhibition on the levels of activation of the NFAT and c-Myc pathways. Direct immunofluorescence for NFATc1 (green) and nuclear DAPI (blue) in the presence or absence of glucose in the culture media (E). Glycosylation defects, offset by PEPCK-M, drive entosis in breast carcinoma cells. Finally, values were normalized by protein content. Loading known concentrations of PEP into cells caused a dose-dependent increase of Ca2+ (Figure 1D,E; correlation statistics in Figure 1F), providing further evidence that the glycolytic intermediate PEP can directly mediate changes in Ca2+ levels. Glucose-6-phosphate reduces calcium accumulation in rat brain endoplasmic reticulum. Federal government websites often end in .gov or .mil. Careers. The mechanism for this regulated process was found to implicate the glycolytic intermediate PEP, an inhibitor of ER Ca2+-ATP pump SERCA [14]. Before Total c-Myc increased its nuclear localization with ionomycin. Bethesda, MD 20894, Web Policies Cancer Immunol Immunother. p < 0.001. Glucose consumption was determined by colorimetry assay, based upon two enzymatic reactions, catalyzed by glucose oxidase and peroxidase. Bookshelf As calcium signaling can stabilize the c-Myc protein, and glucose controls cytosolic Ca2+ levels through PEP, we next questioned whether the presence of extracellular glucose or PEP at varying concentrations might alter c-Myc phosphorylation and nuclear localization. doi: 10.1038/nrc.2017.18. PEP-stirred cytosolic calcium was also responsible for the calmodulin (CaM) dependent phosphorylation of c-Myc at Ser62, resulting in increased activity, probably through enhanced stabilization of the protein. In addition, glycolytic intermediates, such as glucose-6-phosphate (G6P) or phosphoenolpyruvate (PEP), can regulate SERCA activity in the brain [13] and T cells [14], independently of ATP. Chen YJ, Huang YL, Chen YH, Chang ST, Yeh TF. 2011 Sep 7;14(3):415-27. doi: 10.1016/j.cmet.2011.06.017. Absorbance was measured at 450 nm after 30 min of incubation at 37 C. A metabolomics study on HeLa cells (data not shown) showed that PEPCK-M activity contributed glutamine labeled carbons into PEP only under glucose starvation, suggesting that PEPCK-M might influence PEP levels indirectly, possibly by altering the glycolytic flux in the cell. Arag M., Moreno-Felici J., Abs S., Rodrguez-Arvalo S., Hyroov P., Figueras A., Vials F., Prez B., Loza M.I., Brea J., et al. Glucose Metabolism and Acetate Switch in Archaea: the Enzymes in Haloferax volcanii. Drawing depicting a model for the integration of ER-stress mediated up-regulation of PEPCK-M and the control of integrative signals downstream of the PEP/Ca2+ axis. Bittremieux M., Parys J.B., Pinton P., Bultynck G. ER functions of oncogenes and tumor suppressors: Modulators of intracellular Ca. NFAT and c-Myc produce activation of expression of several genes implicated in inflammation and metastasis (PTGS2), glucose uptake and glycolysis (Glut1 and HKII), glutamine catabolism (GLS1) and serine and glycine synthesis, and transference of carbons to one-carbon metabolism (cSHMT) that allow for NADPH recycling. 2011 Sep;81(5):1300-12. doi: 10.1111/j.1365-2958.2011.07762.x. NFAT is activated by ionomycin (1, Glucose increases c-Myc phosphorylation at, Glucose increases c-Myc phosphorylation at Ser62 in a PEP/Ca 2+ dependent manner. Biochemical and genetic characterization of the three metabolic routes in Thermococcus kodakarensis linking glyceraldehyde 3-phosphate and 3-phosphoglycerate. All intermediates carry phosphate groups to lock them into the cell (stops diffusion). Therefore, we interrogated the implication of PEPCK-M in the regulation of PEP and Ca2+ fluctuations, we measured PEP and Ca2+ at various levels of activation of PEPCK-M more specifically using a potent inhibitor of PEPCK-M prepared in our laboratory, iPEPCK-2 [22], in the presence or absence of glucose (Figure 1G). Biochim Biophys Acta. Glucose metabolism or cataplerosis from carbons such as glutamine, contribute to maintaining PEP levels, leading to an inhibition of the SERCA pump similarly to T-cells [14]. Cells were transfected using polyethyleneimine (linear polyethyleneimine, Mr 25,000, Sigma-Aldrich, Darmstadt, Germany). One-way ANOVA, Sidak post-test. J Biol Chem. Densitometric analysis (I) of WB plotted in (H). Cells were incubated overnight in complete medium before treatment. Data are the mean SE, n = 5. Indeed, our data from two colon carcinoma cell lines confirmed that changes in extracellular glucose concentration exhibited concurrent changes in the levels of PEP and cytosolic calcium. CAM Models: Lessons and Implications for CAM Evolution. Licensee MDPI, Basel, Switzerland. Data are the mean SE, n = 5. Moreover, in vertebrates, there are at least three isozymes of pyruvate kinase, of which the M type . 2022 Sep 21;101(12):102195. doi: 10.1016/j.psj.2022.102195. Epub 2010 Aug 7. M.A. The Warburg effect, one of the hallmarks of cancer, allows cancer cells to switch their metabolism to aerobic glycolysis with the objective to increase energy production rates while maintaining anabolism [23,24]. NFAT activity at various glucose concentrations in the culture media (B). Xu Y, Yang S, Zhu Y, Yao S, Li Y, Ye H, Ye Y, Li Z, Wu L, Zhao K, Huang L, Liu Z. Comput Struct Biotechnol J. It has to be re-oxidized to NAD + so that the Glycolytic pathway can continue to take place. Densitometric analysis (G) of WB shown in (F). Recall again that when cells are in a low-energy state, they want to activate . Please enable it to take advantage of the complete set of features! the display of certain parts of an article in other eReaders. Cancer Cell. As shown in Figure 1, cellular PEP linearly increased when glucose concentration in the media was elevated from 0 to 25 mM, both in HCT116 and SW480 colon carcinoma cells (Figure 1A). A model of yeast glycolysis based on a consistent kinetic characterisation of all its enzymes.