FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1967809512> ?p ?o ?g. }

• W1967809512 endingPage "35812" @default.
• W1967809512 startingPage "35803" @default.
• W1967809512 abstract "The putative tumor metastasis suppressor protein Nm23-H1 is a nucleoside diphosphate kinase that exhibits a novel protein kinase activity when bound to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In this study we show that the glycolytic enzyme phosphoglycerate mutase B (PGM) becomes phosphorylated in the presence of the Nm23-H1·GAPDH complex in vitro. Mutation of His-10 in PGM abolishes the Nm23-H1·GAPDH complex-induced phosphorylation. Nm23-H1, GAPDH, and PGM are known to co-localize as shown by free flow isoelectric focusing. In association with Nm23-H1 and GAPDH, PGM could be activated by dCTP, which is a substrate of Nm23-H1, in addition to the well known PGM activator 2,3-bisphosphoglycerate. A synthetic cell-penetrating peptide (PGMtide) encompassing the phosphorylated histidine and several residues from PGM (LIRHGE) promoted growth arrest of several tumor cell lines, whereas proliferation of tested non-tumor cells was not influenced. Analysis of metabolic activity of one of the tumor cell lines, MCF-7, indicated that PGMtide inhibited glycolytic flux, consistent with in vivo inhibition of PGM. The specificity of the observed effect was further determined experimentally by testing the effect of PGMtide on cells growing in the presence of pyruvate, which helps to compensate PGM inhibition in the glycolytic pathway. Thus, growth of MCF-7 cells was not arrested by PGMtide in the presence of pyruvate. The data presented here provide evidence that inhibition of PGM activity can be achieved by exogenous addition of a polypeptide, resulting in inhibition of glycolysis and cell growth arrest in cell culture. The putative tumor metastasis suppressor protein Nm23-H1 is a nucleoside diphosphate kinase that exhibits a novel protein kinase activity when bound to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In this study we show that the glycolytic enzyme phosphoglycerate mutase B (PGM) becomes phosphorylated in the presence of the Nm23-H1·GAPDH complex in vitro. Mutation of His-10 in PGM abolishes the Nm23-H1·GAPDH complex-induced phosphorylation. Nm23-H1, GAPDH, and PGM are known to co-localize as shown by free flow isoelectric focusing. In association with Nm23-H1 and GAPDH, PGM could be activated by dCTP, which is a substrate of Nm23-H1, in addition to the well known PGM activator 2,3-bisphosphoglycerate. A synthetic cell-penetrating peptide (PGMtide) encompassing the phosphorylated histidine and several residues from PGM (LIRHGE) promoted growth arrest of several tumor cell lines, whereas proliferation of tested non-tumor cells was not influenced. Analysis of metabolic activity of one of the tumor cell lines, MCF-7, indicated that PGMtide inhibited glycolytic flux, consistent with in vivo inhibition of PGM. The specificity of the observed effect was further determined experimentally by testing the effect of PGMtide on cells growing in the presence of pyruvate, which helps to compensate PGM inhibition in the glycolytic pathway. Thus, growth of MCF-7 cells was not arrested by PGMtide in the presence of pyruvate. The data presented here provide evidence that inhibition of PGM activity can be achieved by exogenous addition of a polypeptide, resulting in inhibition of glycolysis and cell growth arrest in cell culture. Phosphoglycerate mutase (PGM, 1The abbreviations used are: PGM, phosphoglycerate mutase type B; NDPK, nucleoside diphosphate kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; 2,3-BPG, 2,3-bisphosphoglycerate; FCS, fetal calf serum; DAPI, 4,6-diamidino-2-phenylindole. E.C. 5.4.2.1) is an enzyme of the glycolytic pathway where it catalyzes the conversion of 3-phosphoglycerate to 2-phosphoglycerate. Although this step is not considered to be rate-limiting in the majority of differentiated cells, there is increasing evidence from tumor cell lines that PGM may regulate the balance between glycolysis and another ATP-producing pathway, glutaminolysis (1Mc Keehan W.L. Cell Biol. Int. Rep. 1982; 6: 635-650Crossref PubMed Scopus (251) Google Scholar, 2Medina M.A. Sanchez-Jimenez F. Marquez F.J. Perez-Rodriguez J. Quesada A.R. Nunez de Castro I. Biochem. Int. 1988; 16: 339-347PubMed Google Scholar, 3Board M. Humm S. Newsholme E.A. Biochem. J. 1990; 265: 503-509Crossref PubMed Scopus (184) Google Scholar, 4Mazurek S. Eigenbrodt E. Failing K. Steinberg P. J. Cell. Physiol. 1999; 181: 136-146Crossref PubMed Scopus (68) Google Scholar, 5Mazurek S. Zwerschke W. Jansen-Dürr P. Eigenbrodt E. Biochem. J. 2001; 356: 247-256Crossref PubMed Scopus (124) Google Scholar). In addition to tumor cells a rate-limiting role of PGM within the glycolytic pathway has also been shown in leukocytes and heart muscle (6Kashiwaya Y. Sato K. Tsuchiya N. Thomas S. Fell D.A. Veech R.L. Passonneau J.V. J. Biol. Chem. 1994; 269: 25502-25514Abstract Full Text PDF PubMed Google Scholar, 7Shalom-Barak T. Knaus U.G. J. Biol. Chem. 2002; 277: 40659-40665Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). PGM is activated by the cofactor 2,3-bisphosphoglycerate (2,3-BPG), which phosphorylates the enzyme at histidine. In tumor cells PGM is regulated by migration out of the glycolytic enzyme complex into the so-called pre-complex. The PGM associated within the glycolytic enzyme complex and within the pre-complex can be separated by free flow isoelectric focusing (4Mazurek S. Eigenbrodt E. Failing K. Steinberg P. J. Cell. Physiol. 1999; 181: 136-146Crossref PubMed Scopus (68) Google Scholar, 5Mazurek S. Zwerschke W. Jansen-Dürr P. Eigenbrodt E. Biochem. J. 2001; 356: 247-256Crossref PubMed Scopus (124) Google Scholar, 8Mazurek S. Hugo F. Failing K. Eigenbrodt E. J. Cell. Physiol. 1996; 167: 238-250Crossref PubMed Scopus (42) Google Scholar, 9Mazurek S. Grimm H. Wilker S. Leib S. Eigenbrodt E. Anticancer Res. 1998; 18: 3275-3282PubMed Google Scholar). The PGM enzyme associated within the glycolytic enzyme complex is fully activated and independent of 2,3-BPG, whereas the PGM enzyme found in the pre-complex is not fully active and can be activated by 2,3-BPG (4Mazurek S. Eigenbrodt E. Failing K. Steinberg P. J. Cell. Physiol. 1999; 181: 136-146Crossref PubMed Scopus (68) Google Scholar, 5Mazurek S. Zwerschke W. Jansen-Dürr P. Eigenbrodt E. Biochem. J. 2001; 356: 247-256Crossref PubMed Scopus (124) Google Scholar, 9Mazurek S. Grimm H. Wilker S. Leib S. Eigenbrodt E. Anticancer Res. 1998; 18: 3275-3282PubMed Google Scholar). In the pre-complex PGM is in close proximity to nucleoside diphosphate kinase type A (E.C. 2.7.4.6; NDPK A) (5Mazurek S. Zwerschke W. Jansen-Dürr P. Eigenbrodt E. Biochem. J. 2001; 356: 247-256Crossref PubMed Scopus (124) Google Scholar, 9Mazurek S. Grimm H. Wilker S. Leib S. Eigenbrodt E. Anticancer Res. 1998; 18: 3275-3282PubMed Google Scholar). Migration of PGM out of the glycolytic enzyme complex into the pre-complex reduces the conversion of glucose to lactate and increases the flux rate through glutaminolysis as well as serine synthesis. In addition, ATP and GTP levels decrease, whereas UTP and CTP levels stay high. A low (ATP + GTP): (UTP + CTP) ratio is correlated with a high proliferation rate (10Ryll T. Wagner R. Biotechnol. Bioeng. 1992; 60: 934-946Crossref Scopus (49) Google Scholar, 11Zwerschke W. Mazurek S. Massimi P. Banks L. Eigenbrodt E. Jansen-Dürr P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1291-1296Crossref PubMed Scopus (217) Google Scholar, 12Mazurek S. Zwerschke W. Jansen-Dürr P. Eigenbrodt E. Oncogene. 2001; 20: 6891-6898Crossref PubMed Scopus (97) Google Scholar, 13Mazurek S. Boschek C.B. Eigenbrodt E. J. Bioenerg. Biomembr. 1997; 29: 315-330Crossref PubMed Scopus (153) Google Scholar). NDPK directly couples ATP levels with other nucleoside triphosphates (5Mazurek S. Zwerschke W. Jansen-Dürr P. Eigenbrodt E. Biochem. J. 2001; 356: 247-256Crossref PubMed Scopus (124) Google Scholar, 9Mazurek S. Grimm H. Wilker S. Leib S. Eigenbrodt E. Anticancer Res. 1998; 18: 3275-3282PubMed Google Scholar, 12Mazurek S. Zwerschke W. Jansen-Dürr P. Eigenbrodt E. Oncogene. 2001; 20: 6891-6898Crossref PubMed Scopus (97) Google Scholar, 13Mazurek S. Boschek C.B. Eigenbrodt E. J. Bioenerg. Biomembr. 1997; 29: 315-330Crossref PubMed Scopus (153) Google Scholar). NDPK demonstrates a “ping-pong” mechanism of catalysis, and thus, upon incubation with nucleoside triphosphate-Mg, the enzyme becomes phosphorylated at a histidine residue, forming a relatively stable phosphohistidine intermediate. The phosphate can then be transferred to a NDP-Mg acceptor. Research on NDPK was stimulated when the cDNA of NDPK A was cloned as a transcript with reduced expression levels in tumor metastatic cells, termed Nm23-H1 gene (14Steeg P.S. Bevilacqua G. Kopper L. Thorgeirsson U.P. Talmadge J.E. Liotta L.A. Sobel M.E. J. Nat'l. Cancer Inst. 1988; 80: 200-204Crossref PubMed Scopus (1299) Google Scholar). Eight related genes have been thus far identified as members of the human Nm23 gene family (15Lombardi D. Lacombe M.-L. Paggi M.G. J. Cell. Physiol. 2001; 182: 144-149Crossref Scopus (111) Google Scholar). The only corresponding gene products that have been well characterized are Nm23-H1 and Nm23-H2, which encode NDPK A and B, respectively. In addition to their main function of phosphorylating nucleoside diphosphates, both isoforms show different additional biochemical activities (16Steeg P.S. Palmieri D. Ouatas T. Salerno M. Cancer Lett. 2003; 190: 1-12Crossref PubMed Scopus (83) Google Scholar, 17Otero A.S. J. Bioenerg. Biomembr. 2000; 32: 269-275Crossref PubMed Scopus (94) Google Scholar, 18Roymans D. Willems R. Van Blockstaele D.R. Slegers H. Clin. Exp. Metastasis. 2002; 19: 465-476Crossref PubMed Scopus (72) Google Scholar). Nm23/NDPK protein isoforms have been reported to interact with a variety of cellular proteins and were found to be associated with different cellular compartments (17Otero A.S. J. Bioenerg. Biomembr. 2000; 32: 269-275Crossref PubMed Scopus (94) Google Scholar, 18Roymans D. Willems R. Van Blockstaele D.R. Slegers H. Clin. Exp. Metastasis. 2002; 19: 465-476Crossref PubMed Scopus (72) Google Scholar). In terms of alternative biochemical functions, Nm23-H2 was found to be a transcriptional activator for the c-myc proto-oncogene (19Postel E.H. Berberich S.J. Flint S.J. Ferrone C.A. Science. 1993; 261: 478-480Crossref PubMed Scopus (483) Google Scholar, 20Berberich S.J. Postel E.H. Oncogene. 1995; 10: 2343-2347PubMed Google Scholar, 21Ji L. Arcinas M. Boxer L.M. J. Biol. Chem. 1995; 270: 13392-13398Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Nm23 has also been demonstrated to exhibit a protein kinase activity in vitro (22Engel M. Véron M. Theisinger B. Lacombe M.-L. Seib T. Dooley S. Welter C. Eur. J. Biochem. 1995; 234: 200-207Crossref PubMed Scopus (92) Google Scholar, 23Engel M. Seifert M. Theisinger B. Seyfert U. Welter C. J. Biol. Chem. 1998; 273: 20058-20065Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 24Wagner P. Vu N.-D. J. Biol. Chem. 1995; 270: 21758-21764Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 25Wagner P.D. Steeg P.S. Vu N.-D. Proc. Nat'l. Acad. Sci. U. S. A. 1997; 94: 9000-9005Crossref PubMed Scopus (126) Google Scholar, 26Wagner P. Vu N.-D. Biochem. J. 2001; 346: 623-630Crossref Google Scholar). In previous studies we have shown that PGM and Nm23-H1 (NDPK A) are associated and that the Nm23-H1 histidine kinase function is activated by association with GAPDH (5Mazurek S. Zwerschke W. Jansen-Dürr P. Eigenbrodt E. Biochem. J. 2001; 356: 247-256Crossref PubMed Scopus (124) Google Scholar, 9Mazurek S. Grimm H. Wilker S. Leib S. Eigenbrodt E. Anticancer Res. 1998; 18: 3275-3282PubMed Google Scholar, 23Engel M. Seifert M. Theisinger B. Seyfert U. Welter C. J. Biol. Chem. 1998; 273: 20058-20065Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). In the present study, PGM was identified as a protein phosphorylated by the Nm23-H1·GAPDH complex in vitro. Based on the phosphorylation site sequence, we developed a cell-permeable synthetic peptide that encompasses the PGM phosphorylation site and inhibits PGM activity as well as glycolysis in MCF-7 cells and other tumor cell lines. Expression of Recombinant Proteins and Purification—The His6-Nm23-H1·GAPDH complex was expressed from a bicistronic baculovirus vector in Sf9 insect cells and purified by affinity chromatography on Ni2+-agarose followed by pseudoaffinity chromatography on reactive yellow-agarose (Sigma) as described earlier (23Engel M. Seifert M. Theisinger B. Seyfert U. Welter C. J. Biol. Chem. 1998; 273: 20058-20065Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). The complete coding region of human PGM B was PCR-amplified from human kidney parenchyma cDNA and cloned into the BamHI/HindIII restriction sites of the pQE30 expression vector (Qiagen) using the primers 5′-taggatccatggccgcctacaaactg-3′ (forward) and 5′-cgaagcttaatgaaaatgggagccactg-3′ (reverse). After sequencing to ensure that the inserted sequence was correct, the plasmid was used to transform Escherichia coli JM109, the bacteria was grown at 37 °C to an A600 of 0.5, and expression was induced by the addition of 0.5 mm isopropyl-1-thio-β-d-galactopyranoside for 3 h. The bacteria were harvested by centrifugation and lysed, and the recombinant PGM was purified by Ni2+ affinity chromatography under exactly the same conditions as described earlier for other His-tagged proteins (23Engel M. Seifert M. Theisinger B. Seyfert U. Welter C. J. Biol. Chem. 1998; 273: 20058-20065Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). In a second step the PGM eluted from the Ni2+-agarose column was dialyzed overnight against 20 mm KH2PO4/K2HPO4 buffer, pH 7.1, 0.5 mm dithiothreitol at 4 °C. The protein was then passed over a hydroxyapatite column (Bio-Rad) previously equilibrated with the same buffer. Bound proteins were eluted using a linear gradient from 20 mm KH2PO4/K2HPO4 buffer to 500 mm KH2PO4/K2HPO4 buffer. The fractions containing PGM were pooled, dialyzed against storage buffer (20 mm Tris/HCl, pH 7.5, 40% glycerol, 40 mm NaCl, 1 mm dithiothreitol, 0.5 mm EDTA), and stored at -20 °C. To generate the PGMH10G mutant protein, site directed mutagenesis was performed using the plasmid carrying the wild type sequence and the mutagenesis primers 5′-gtgctgatccggggcggcgagagcgca-3′ (forward) and 5′-tgcgctctcgccgccccggatcagcac-3′ (reverse). Mutagenesis was performed using the QuikChange™ kit from Stratagene according to the manufacturer's instructions. Expression and purification of the PGMH10G mutant protein were carried out exactly as with the wild type protein. Screening of a cDNA Expression Library by in Situ Phosphorylation—To identify in vitro substrates of the Nm23-H1·GAPDH complex, a λ-TripleEx fetal brain cDNA expression library (Clontech) was screened. The phages were plated in LB top agar containing E. coli host cells and grown until plaques became slightly visible. Phages (10,000 plaque-forming units/plate) were blotted for 4 h at 37 °C onto nitrocellulose filter membranes (Duralose-UV™, Stratagene) previously soaked in 10 mm isopropyl-1-thio-β-d-galactopyranoside. In addition, the plates were left with the membranes at 4 °C overnight. The nitrocellulose sheets were then removed, washed twice in 20 mm Tris/HCl, pH 7.0, 150 mm NaCl, and blocked in 20 mm Tris/HCl, pH 7.0, 150 mm NaCl, 1 mm GTP, 2% bovine serum albumin. Filters were then incubated with recombinant GAPDH·Nm23-H1 complex (0.1 mg/ml) in the presence of 15 μm [γ-32P]GTP (0.37 millibecquerel/ml) in 20 mm Tris/HCl, pH 7.0, 40 mm NaCl, 1 mg/ml bovine serum albumin for 40 min at 30 °C in roller bottles. After washing 3 times with large volumes of ice-cold wash buffer (20 mm Tris/HCl, pH 7.0, 150 mm NaCl, 0.1% Nonidet P-40) for 5 min each, membranes were incubated in the same buffer containing 1 mm GDP for 10 min at room temperature under shaking to attenuate autophosphorylation of library-encoded NDPK isoenzymes. Filter membranes were washed again in ice-cold wash buffer and exposed wet to film for 2–4 days using intensifying screens at -80 °C. After the first autoradiography, nitrocellulose membranes that produced signals were subjected to acidic buffer treatment (0.2 m glycine/HCl, pH 1.5) and exposed to film again. The phage plaques corresponding to radioactive spots were excised from the plates, eluted, and used for a second round of plating and in situ phosphorylation screening. Only phages corresponding to autoradiographic spots that were abolished after the acidic washing step in the first round continued to produce signals in the secondary screenings. Single phage clones were excised from the agar plates, and the cDNA inserts were amplified by PCR using the LD insert screening amplimer set (Clontech, catalog no. 9107-1). PCR products were subcloned into pCR4.1 TOPO cloning vectors (Invitrogen), and the plasmids were sequenced. Phosphorylation of PGM by the Nm23-H1·GAPDH Complex—If not stated otherwise, phosphorylation assays were performed using 1 μg of recombinant PGM and 200 ng of Nm23-H1·GAPDH complex in a final volume of 25 μl in the following buffer: 20 mm Tris/HCl, pH 7.2, 40 mm NaCl, 5 mm MgCl2, 1 mm dithiothreitol. Reactions were started by the addition of [γ-32P]GTP or [γ-32P]ATP to a final concentration of 20 μm (1 μCi per reaction). After incubation at 30 °C for 10 min, 2 mm cold ATP was added to the samples to decrease the content of [32P]histidine-phosphorylated Nm23-H1 followed by SDS sample buffer pH 8.8 after a 5-min incubation. The samples were immediately applied to SDS-polyacrylamide gel electrophoresis without prior heating. These precautions were taken to avoid artificial chemical transfer reactions. The SDS gels were prepared according to Laemmli except that Tris/HCl buffer pH 8.8 was used for both stacking and separating gels. After electrophoresis, the gels were dried without prior fixation at 80 °C under vacuum and autoradiographed overnight without intensifying screens. For the determination of phosphate incorporation into PGM, phosphorylation was stopped after incubation with [γ-32P]GTP by snap-freezing in liquid nitrogen. Before re-thawing, the solution was adjusted to 8 m urea, and paramagnetic beads loaded with Ni2+ were added to the samples to bind His-tagged proteins (PGM and GAPDH). Beads were washed quickly 4 times with a cold buffer consisting of 50 mm NaH2PO4/Na2HPO4, pH 8.5, 6 m urea and counted in a liquid scintillation counter for 32P content. Phosphate incorporation per mol PGM was calculated based on counts of known amounts of [32P]GTP measured in parallel. To calculate possible background phosphorylation of His-tagged GAPDH, samples that had been prepared and incubated exactly as the PGM phosphorylation reactions except that PGM was omitted were also counted. Peptide Synthesis—The PGMtide peptide (MRQIKIWFPNRRMKWKKHHHHHHPWLIRHGE) and the control peptide (MRQIKIWFPNRRMKWKKHHHHHHPWRIEGHL) were synthesized by ThermoHybaid (formerly Interactiva), Germany. N-terminally fluorescein-coupled derivatives of these peptides and of another peptide lacking the Antennapedia domain (HHHHHHPWLIRHGE) were also synthesized. The freeze-dried peptides were dissolved in phosphate-buffered saline containing 5 m urea. In the experiments, the peptides were added to cells by rapid dilution into the fresh culture media. Isolation of the Glycolytic Enzyme Complex by Isoelectric Focusing— Cells were extracted with a homogenization buffer containing 10 mm Tris, 1 mm NaF, and 1 mm mercaptoethanol, pH 7.4. Isoelectric focusing was carried out with a linear gradient of glycerol (50 to 0% (v/v)) and ampholines (pI 3.5–10.5) as described previously (8Mazurek S. Hugo F. Failing K. Eigenbrodt E. J. Cell. Physiol. 1996; 167: 238-250Crossref PubMed Scopus (42) Google Scholar). Measurements of Enzyme Activities—Specific activities of PGM, NDPK, and GAPDH were measured as described earlier (8Mazurek S. Hugo F. Failing K. Eigenbrodt E. J. Cell. Physiol. 1996; 167: 238-250Crossref PubMed Scopus (42) Google Scholar, 23Engel M. Seifert M. Theisinger B. Seyfert U. Welter C. J. Biol. Chem. 1998; 273: 20058-20065Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 27Bergmeyer H.U. 3rd Ed. Methoden der Enzymatischen Analyse. I und II. Verlag Chemie, Weinheim/Bergstrasse, Germany1974Google Scholar). For the heat inactivation assay, PGM was heated to 80 °C in 50 μl of a buffer containing 50 mm Tris/HCl, pH 7.2, 10 mm dithiothreitol, and 0.1% Nonidet P-40, then chilled on ice. Cell Culture—MCF-7 and Tx3095 cell lines, rheumatoid synoviocytes, and amniotic fluid cells were cultivated in Dulbecco's modified Eagle's medium (Sigma) supplemented with 5 mm glucose, 2 mm glutamine, 100 units of penicillin/ml, 100 μg of streptomycin/ml, and 10% fetal calf serum (FCS). The medium also contained 4 mm sodium pyruvate if not otherwise stated. GLC4 cells were grown in RPMI 1640 medium supplemented as described for MCF-7 and Tx3095 cells. Cells were grown in a humidified atmosphere under 95% air, 5% CO2. MDA-MB-453 cells were grown in Leibovitz L15 medium supplemented with 2 mm glutamine, 4 mm pyruvate, 100 units penicillin/ml, 100 μg streptomycin/ml, and 10% FCS. Cells were grown at 37 °C in air-tight closed culture flasks. The breast carcinoma cell lines MCF-7 and MDA-MB-453 were obtained from the ATCC; the small cell lung carcinoma cell line GLC4 was a gift from the Department of Internal Medicine, University Hospital Groningen, The Netherlands. The glioblastoma cell line Tx3095 was cultivated over 68 passages from a resected glioblastoma in the Department of Human Genetics, Homburg, Germany. Human amniotic fluid cells had been cultivated and used before for diagnostic purposes at the Department of Human Genetics, Homburg. Rheumatoid synoviocytes were obtained from a patient who underwent therapeutic synovectomy at the Department of Orthopaedics, University Hospital, Homburg. Fluorescence Microscopy Using Fluorescein-linked Peptides—Fluorescein-linked versions of the peptides were added to the fully supplemented medium of MCF-7 cells grown directly on glass slides. After an incubation for 10 min at 37 °C, the cells were washed 3 times with medium, fixed for 5 min in PBS, 4% paraformaldehyde at room temperature, washed 3 times in PBS, counterstained with DAPI solution, washed again 2 times with PBS, dried, and mounted with antifade solution (Vectorshield; Vector Laboratories, Burlingame, CA) for examination using fluorescein isothiocyanate and DAPI filters. Glycolytic Flux Measurements—Cell culture supernatants were collected at different cell densities and immediately frozen in liquid nitrogen. Glucose, pyruvate, lactate, glutamine, glutamate, serine, and alanine were measured in the thawed supernatants as described previously (27Bergmeyer H.U. 3rd Ed. Methoden der Enzymatischen Analyse. I und II. Verlag Chemie, Weinheim/Bergstrasse, Germany1974Google Scholar, 28Mazurek S. Michel A. Eigenbrodt E. J. Biol. Chem. 1997; 272: 4941-4952Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 29Hugo F. Mazurek S. Zander U. Eigenbrodt E. J. Cell. Physiol. 1992; 153: 539-549Crossref PubMed Scopus (43) Google Scholar). For the determination of the metabolite conversion rates two different calculations were chosen; the calculation in nmol/(h × 105 cells) reflects the conversion rate of a number of cells. The calculation in nmol/(h × dish) was chosen for the correlation of different conversion rates. Identification of Protein(s) Phosphorylated by the Nm23-H1·GAPDH Complex in Vitro—To elucidate possible functions of the Nm23-H1·GAPDH protein kinase activity, an attempt was made to identify potential substrate proteins. For this purpose, a cDNA expression library in λ-phages was transferred to nitrocellulose filters and screened by incubation with the bacterially expressed Nm23-H1·GAPDH protein complex in the presence of [γ-32P]GTP-Mg. After autoradiography of the filters, few isolated radiolabeled spots were detected on the films (see the example in Fig. 1A, marked by an arrow), and the corresponding phage plaques were picked from the primary plates. After the first autoradiography, phosphorylation signals were further analyzed by incubating the membranes in acidic buffer (glycine/HCl pH 1.5) and re-exposing to film. Using this procedure, acid-labile phage phosphorylations could be detected and further discriminated from background spots. No true acid-stable phage phosphorylation signals, indicative of serine, threonine, or tyrosine phosphate, were found. Phage plaques having produced acid labile phosphorylations were picked and used for a second round of plating and screening. On the autoradiograph, the number of phosphorylated spots corresponding to phage plaques had increased (Fig. 1B), suggesting enrichment of phages displaying a phosphorylation substrate protein. The cDNA inserts of selected phage clones from the second-round plates were amplified by PCR and sequenced (marked by arrows in Fig. 1B). A BLAST search with the sequences revealed that among six non-redundant clones obtained from several plates, four contained the full-length coding sequence of human PGM B, whereas the two remaining clones contained 5′-cDNA sequences from PGM B of different length. The consensus coding sequences were identical to the gene bank entry for PGM B cDNA (accession number BC010038.1). Characterization of the Phosphorylation and Identification of the Phosphoamino Acid Phosphorylated by Nm23-H1—The complete coding sequence of PGM B was amplified by PCR. The PCR product was cloned into an expression vector, and the corresponding protein was expressed in E. coli and purified to homogeneity. The purified protein was phosphorylated in the presence of Nm23-H1·GAPDH complex and [γ-32P]ATP or [γ-32P]GTP but not in the presence of [γ-32P]ATP or [γ-32P]GTP alone (Fig. 2A). PGM was phosphorylated as a native protein as well as after denaturation by heat treatment. Because the heat treatment for 2 min at 80 °C was sufficient to completely inactivate PGM, as verified by activity measurements (Table I), this experiment excludes the possibility that PGM activity could be required for the observed phosphorylation.Table IInactivation of PGM by heat treatment PGM was heated to 80 °C for the times indicated, then chilled on ice. Activity was measured after the addition of substrates and coupling enzymes as described under “Experimental Procedures.” The numbers given represent mean values of the specific activity from duplicate experiments. One unit is defined as μmol of 3-phosphoglycerate converted per min-1 at 30 °C.Heating time030 s1 min1.5 min2 min3 minPGM activity (milliunits/mg)287026044200 Open table in a new tab Stoichiometric analysis showed that less than 0.1 mol of phosphate was incorporated per mol of PGM after 1 h of incubation time. However, maximum phosphorylation was achieved already after 1 min of incubation (Fig. 3). To be active, PGM normally requires small amounts of the co-factor 2,3-BPG, which serves as a phosphate donor for the initial activating autophosphorylation of the enzyme at histidine 10 at the active site. To determine whether the same residue might be targeted by Nm23-H1, histidine 10 was replaced by glycine using site-directed mutagenesis, and the mutant protein was bacterially expressed and purified. By this means it was possible to show that the His-10-Gly mutant was not phosphorylated by Nm23-H1·GAPDH (Fig. 2B), suggesting that it may be the phosphorylation site targeted by Nm23-H1·GAPDH in vitro. Dependence of Nm23-H1-induced PGM Activation on a Native Protein Complex Environment—Possible effects of the in vitro phosphorylation of PGM were then investigated. Hypothetically, Nm23-H1-dependent phosphorylation might activate dephosphorylated PGM in a similar manner to 2,3-BPG. However, we were unable to verify any effect of Nm23-H1 or Nm23-H1·GAPDH on purified PGM in vitro, possibly due to the low stoichiometry of phosphorylation. We hypothesized that the correct ratio and/or concentration of Nm23-H1·GAPDH and PGM as well as a defined orientation of the proteins in a complex environment might be essential for an interaction of Nm23-H1·GAPDH with PGM. To investigate Nm23-H1 involvement in PGM activation in the native complex, cytoplasmic proteins were extracted from proliferating MCF-7 cells and fractionated by isoelectric focusing, which allows separation of the so-called “glycolytic enzyme complex,” which contains most of the glycolytic enzymes, and the so-called “pre-complex,” which contains PGM, NDPK, and GAPDH as well as the cytosolic isoenzymes of the transaminases and glutamate dehydrogenase (Fig. 4A). Analysis of the isoelectric focusing fractions of the MCF-7 cells revealed that the PGM enzyme associated within the pre-complex (Fig. 4B, fractions 33–37) could be activated by 2,3-BPG, whereas the PGM enzyme associated within the glycolytic enzyme complex was only weakly activated (Fig. 4B, fractions 38–43). As described earlier (30Stankiewicz P.J. Gresser M.J. Tracey A.S. Hass L.F. Biochemistry. 1987; 26: 1264-1269Crossref PubMed Scopus (61) Google Scholar), the addition of 0.5 mm orthovanadate into the enzyme assay leads to a complete inactivation of PGM in the absence of stoichiometric amounts of 2,3-BPG. Under these conditions, PGM activity is only measurable when the enzyme is permanently re-phosphorylated and, thus, reactivated by 2,3-BPG. In our study, in the presence of vanadate, a strong activation of PGM by 2,3-BPG was detected in the fractions of the pre-complex (Fig. 4C, fractions 33–37), whereas the PGM enzyme associated within the glycolytic enzyme complex was not activated (Fig. 4C, fractions 38–43). Most importantly, in the fractions of the pre-complex PGM was also activated by dCTP, whereas dCTP di" @default.
• W1967809512 created "2016-06-24" @default.
• W1967809512 creator A5017500848 @default.
• W1967809512 creator A5033544096 @default.
• W1967809512 creator A5054508581 @default.
• W1967809512 creator A5084331520 @default.
• W1967809512 date "2004-08-01" @default.
• W1967809512 modified "2023-09-26" @default.
• W1967809512 title "Phosphoglycerate Mutase-derived Polypeptide Inhibits Glycolytic Flux and Induces Cell Growth Arrest in Tumor Cell Lines" @default.
• W1967809512 cites W110739455 @default.
• W1967809512 cites W1488748351 @default.
• W1967809512 cites W1544621467 @default.
• W1967809512 cites W1564991199 @default.
• W1967809512 cites W1594054725 @default.
• W1967809512 cites W1669699198 @default.
• W1967809512 cites W1754310888 @default.
• W1967809512 cites W1966841783 @default.
• W1967809512 cites W1970415954 @default.
• W1967809512 cites W1977632661 @default.
• W1967809512 cites W1978105472 @default.
• W1967809512 cites W1980236164 @default.
• W1967809512 cites W1992663544 @default.
• W1967809512 cites W1993119123 @default.
• W1967809512 cites W2007599495 @default.
• W1967809512 cites W2014332843 @default.
• W1967809512 cites W2014968902 @default.
• W1967809512 cites W2016728012 @default.
• W1967809512 cites W2025590706 @default.
• W1967809512 cites W2035628161 @default.
• W1967809512 cites W2046687522 @default.
• W1967809512 cites W2050991052 @default.
• W1967809512 cites W2051219431 @default.
• W1967809512 cites W2052386120 @default.
• W1967809512 cites W2057234832 @default.
• W1967809512 cites W2060406725 @default.
• W1967809512 cites W2075636558 @default.
• W1967809512 cites W2084347027 @default.
• W1967809512 cites W2091717762 @default.
• W1967809512 cites W2100809274 @default.
• W1967809512 cites W2112371447 @default.
• W1967809512 cites W2162553581 @default.
• W1967809512 cites W2320829700 @default.
• W1967809512 cites W4243213708 @default.
• W1967809512 doi "https://doi.org/10.1074/jbc.m402768200" @default.
• W1967809512 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15181008" @default.
• W1967809512 hasPublicationYear "2004" @default.
• W1967809512 type Work @default.
• W1967809512 sameAs 1967809512 @default.
• W1967809512 citedByCount "59" @default.
• W1967809512 countsByYear W19678095122012 @default.
• W1967809512 countsByYear W19678095122013 @default.
• W1967809512 countsByYear W19678095122014 @default.
• W1967809512 countsByYear W19678095122015 @default.
• W1967809512 countsByYear W19678095122016 @default.
• W1967809512 countsByYear W19678095122020 @default.
• W1967809512 countsByYear W19678095122021 @default.
• W1967809512 countsByYear W19678095122022 @default.
• W1967809512 countsByYear W19678095122023 @default.
• W1967809512 crossrefType "journal-article" @default.
• W1967809512 hasAuthorship W1967809512A5017500848 @default.
• W1967809512 hasAuthorship W1967809512A5033544096 @default.
• W1967809512 hasAuthorship W1967809512A5054508581 @default.
• W1967809512 hasAuthorship W1967809512A5084331520 @default.
• W1967809512 hasBestOaLocation W19678095121 @default.
• W1967809512 hasConcept C155911427 @default.
• W1967809512 hasConcept C178790620 @default.
• W1967809512 hasConcept C181199279 @default.
• W1967809512 hasConcept C185592680 @default.
• W1967809512 hasConcept C20251656 @default.
• W1967809512 hasConcept C2777552656 @default.
• W1967809512 hasConcept C54355233 @default.
• W1967809512 hasConcept C55493867 @default.
• W1967809512 hasConcept C62112901 @default.
• W1967809512 hasConcept C62231903 @default.
• W1967809512 hasConcept C68709404 @default.
• W1967809512 hasConcept C81885089 @default.
• W1967809512 hasConcept C86803240 @default.
• W1967809512 hasConcept C91754966 @default.
• W1967809512 hasConcept C95444343 @default.
• W1967809512 hasConceptScore W1967809512C155911427 @default.
• W1967809512 hasConceptScore W1967809512C178790620 @default.
• W1967809512 hasConceptScore W1967809512C181199279 @default.
• W1967809512 hasConceptScore W1967809512C185592680 @default.
• W1967809512 hasConceptScore W1967809512C20251656 @default.
• W1967809512 hasConceptScore W1967809512C2777552656 @default.
• W1967809512 hasConceptScore W1967809512C54355233 @default.
• W1967809512 hasConceptScore W1967809512C55493867 @default.
• W1967809512 hasConceptScore W1967809512C62112901 @default.
• W1967809512 hasConceptScore W1967809512C62231903 @default.
• W1967809512 hasConceptScore W1967809512C68709404 @default.
• W1967809512 hasConceptScore W1967809512C81885089 @default.
• W1967809512 hasConceptScore W1967809512C86803240 @default.
• W1967809512 hasConceptScore W1967809512C91754966 @default.
• W1967809512 hasConceptScore W1967809512C95444343 @default.
• W1967809512 hasIssue "34" @default.
• W1967809512 hasLocation W19678095121 @default.
• W1967809512 hasOpenAccess W1967809512 @default.
• W1967809512 hasPrimaryLocation W19678095121 @default.

• next