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PubMed2005

Characterization of the 3' --> 5' exonuclease activity found in human nucleoside diphosphate kinase 1 (NDK1) and several of its homologues.

Nucleoside diphosphate kinases (NDKs), an evolutionarily conserved family of proteins, synthesize nucleoside triphosphates from nucleoside diphosphates and ATP. Here, we have characterized the kinase activity and DNA processing functions of eight human proteins that contain at least one domain homologous to Escherichia coli NDK. Not all human proteins with NDK-like domains exhibited NDK activity when expressed as recombinant proteins in E. coli. Human NDK1 (NM23-H1) has been reported to have 3' --> 5' exonuclease activity. In addition to human NDK1, we also find that human NDK5, NDK7, and NDK8 contain 3' --> 5' exonuclease activity. Site-directed mutagenesis, competition assays between wild-type and mutant NDK proteins, and NMR studies confirmed that the DNA-binding and 3' --> 5' exonuclease activity of human NDK1 is an intrinsic activity of the protein. Using double-stranded DNA substrates containing modified bases, human NDK1 efficiently excised nucleotides from the single-strand break produced by APE1 or Nth1. When human cells were treated with various DNA-damaging agents, human NDK1 translocated from the cytoplasm to the nucleus. These results suggest that, in addition to maintenance of nucleotide pool balance, the human NDK-like proteins may have previously unrecognized roles in DNA nucleolytic processing.

Amino Acid Sequence↗
PubMed2000

The human Nm23/nucleoside diphosphate kinases.

Biochemical experiments over the past 40 years have shown that nucleoside diphosphate (NDP) kinase activity, which catalyzes phosphoryl transfer from a nucleoside triphosphate to a nucleoside diphosphate, is ubiquitously found in organisms from bacteria to human. Over the past 10 years, eight human genes of the nm23/NDP kinase family have been discovered that can be separated into two groups based on analysis of their sequences. In addition to catalysis, which may not be exhibited by all isoforms, evidence for regulatory roles has come recently from the discovery of the genes nm23 and awd, which encode NDP kinases and are involved in tumor metastasis and Drosophila development, respectively. Current work shows that the human NDP kinase genes are differentially expressed in tissues and that their products are targeted to different subcellular locations. This suggests that Nm23/NDP kinases possess different, but specific, functions within the cell, depending on their localization. The roles of NDP kinases in metabolic pathways and nucleic acid synthesis are discussed.

Amino Acid Sequence↗
PubMed2002

Nucleoside diphosphate kinase (NDPK/NM23) and the waltz with multiple partners: possible consequences in tumor metastasis.

Tumor metastasis is responsible for a high degree of mortality in cancer patients. One of the genes involved in tumor metastasis is NM23. At present, eight human isoforms, transcribed from different NM23 genes, have been detected. The gene products have been identified as nucleoside diphosphate kinases (NDPKs), most of which catalyse the transfer of the gamma-phosphate of a (deoxy)nucleoside triphosphate to a (deoxy)nucleoside diphosphate. However, the function of NDPK isoforms involved in tumor metastasis cannot be explained on the basis of their phosphotransferase activity alone. At present, several other properties, like transcriptional regulation and protein kinase activity, have been assigned to these proteins. Moreover, it has also been shown that NDPKs interact with several other proteins, and binding partners of NDPKs are identified at an increasing rate. Accumulating evidence indicates that protein-protein interactions modulate the molecular action of NDPKs. In this review we provide a brief overview of how NDPKs are correlated with cancer, and discuss when and how the activities assigned to NDPKs may affect metastasis, with special emphasis on the role of protein-NDPK interactions in this process.

Animals↗
PubMed1975

The formation of mono-N-acetylhexosamine derivatives of dolichol diphosphate by pig liver microsomal fractions.

Incubation of pig liver microsomal preparations with UDP-N[U-14C]acetylglucosamine yields a 14C-labelled lipid. The requirement for Mn2+, the pH optimum, time-dependence and the reversibility by UMP of the transferase are reported. Evidence is presented in favour of the lipid being a mixture of dolichol diphosphate N-[14C]acetylglucosamine and dolichol diphosphate N-[14C]acetylmannosamine. Available data suggest that the epimerization takes place while the hexosamine is bound in this lipid-soluble form. The N-acetylmannosamine appeared not be be released into the medium. The subfractionation of the microsomal fraction to separate transferase activity from membrane-bound beta-N-acetylglucosaminidase activity is also reported.

Animals↗
PubMed2005

A novel physical and functional association between nucleoside diphosphate kinase A and AMP-activated protein kinase alpha1 in liver and lung.

Nucleoside diphosphate kinase (NDPK, NM23/awd) belongs to a multifunctional family of highly conserved proteins (approximately 16-20 kDa) containing two well-characterized isoforms (NM23-H1 and -H2; also known as NDPK A and B). NDPK catalyses the conversion of nucleoside diphosphates into nucleoside triphosphates, regulates a diverse array of cellular events and can act as a protein histidine kinase. AMPK (AMP-activated protein kinase) is a heterotrimeric protein complex that responds to cellular energy status by switching off ATP-consuming pathways and switching on ATP-generating pathways when ATP is limiting. AMPK was first discovered as an activity that inhibited preparations of ACC1 (acetyl-CoA carboxylase), a regulator of cellular fatty acid synthesis. We report that NM23-H1/NDPK A and AMPK alpha1 are associated in cytosol from two different tissue sources: rat liver and a human lung cell line (Calu-3). Co-immunoprecipitation and binding assay data from both cell types show that the H1/A (but not H2/B) isoform of NDPK is associated with AMPK complexes containing the alpha1 (but not alpha2) catalytic subunit. Manipulation of NM23-H1/NDPK A nucleotide transphosphorylation activity to generate ATP (but not GTP) enhances the activity of AMPK towards its specific peptide substrate in vitro and also regulates the phosphorylation of ACC1, an in vivo target for AMPK. Thus novel NM23-H1/NDPK A-dependent regulation of AMPK alpha1-mediated phosphorylation is present in mammalian cells.

AMP-Activated Protein Kinases↗
PubMed2007

Protein phosphorylation corrects the folding defect of the neuroblastoma (S120G) mutant of human nucleoside diphosphate kinase A/Nm23-H1.

Human nucleoside diphosphate (NDP) kinase A is a 'house-keeping' enzyme essential for the synthesis of nonadenine nucleoside (and deoxynucleoside) 5'-triphosphate. It is involved in complex cellular regulatory functions including the control of metastatic tumour dissemination. The mutation S120G has been identified in high-grade neuroblastomas. We have shown previously that this mutant has a folding defect: the urea-denatured protein could not refold in vitro. A molten globule folding intermediate accumulated, whereas the wild-type protein folded and associated into active hexamers. In the present study, we report that autophosphorylation of the protein corrected the folding defect. The phosphorylated S120G mutant NDP kinase, either autophosphorylated with ATP as donor, or chemically prosphorylated by phosphoramidate, refolded and associated quickly with high yield. Nucleotide binding had only a small effect. ADP and the non-hydrolysable ATP analogue 5'-adenyly-limido-diphosphate did not promote refolding. ATP-promoted refolding was strongly inhibited by ADP, indicating protein dephosphorylation. Our findings explain why the mutant enzyme is produced in mammalian cells and in Escherichia coli in a soluble form and is active, despite the folding defect of the S120G mutant observed in vitro. We generated an inactive mutant kinase by replacing the essential active-site histidine residue at position 118 with an asparagine residue, which abrogates the autophosphorylation. The double mutant H118N/S120G was expressed in inclusion bodies in E. coli. Its renaturation stops at a folding intermediate and cannot be reactivated by ATP in vitro. The transfection of cells with this double mutant might be a good model to study the cellular effects of folding intermediates.

Adenosine Triphosphate↗
PubMed1996

Adenylate kinase complements nucleoside diphosphate kinase deficiency in nucleotide metabolism.

Nucleoside diphosphate (NDP) kinase is a ubiquitous nonspecific enzyme that evidently is designed to catalyze in vivo ATP-dependent synthesis of ribo- and deoxyribonucleoside triphosphates from the corresponding diphosphates. Because Escherichia coli contains only one copy of ndk, the structural gene for this enzyme, we were surprised to find that ndk disruption yields bacteria that are still viable. These mutant cells contain a protein with a small amount NDP kinase activity. The protein responsible for this activity was purified and identified as adenylate kinase. This enzyme, also called myokinase, catalyzes the reversible ATP-dependent synthesis of ADP from AMP. We found that this enzyme from E. coli as well as from higher eukaryotes has a broad substrate specificity displaying dual enzymatic functions. Among the nucleoside monophosphate kinases tested, only adenylate kinase was found to have NDP kinase activity. To our knowledge, this is the first report of NDP kinase activity associated with adenylate kinase.

Adenosine Diphosphate↗
PubMed1997

A point mutation of human nucleoside diphosphate kinase A found in aggressive neuroblastoma affects protein folding.

The point mutation serine 120 to glycine in the human nucleoside diphosphate kinase A has been identified in several aggressive neuroblastomas (Chang, C. L., Zhu, X. X., Thoraval, D. H., Ungar, D., Rawwas, J., Hora, N., Strahler, J. R., Hanash, S. M. & Radany, E. (1994) Nature 370, 335-336). We expressed in bacteria and purified wild-type and S120G mutant nucleoside diphosphate kinase A. The mutant enzyme had enzymatic and structural properties similar to the wild-type enzyme, whereas its stability to denaturation by heat and urea was markedly reduced. More importantly, upon renaturation of the urea-denatured mutant protein, a folding intermediate accumulated, having the characteristics of a molten globule. It had no tertiary structure, as shown by near UV circular dichroism, whereas the secondary structure was substantially recovered. The hydrophobic probe 8-anilino-1-naphthalene sulfonate bound to the intermediate species with an increase in fluorescence intensity and a blue shift. The hydrodynamic size was between that expected for a folded and an unfolded monomer. Finally, electrophoresis in a transverse urea gradient displayed no renaturation curve, and the protein showed the tendency to aggregate at the lowest urea concentrations. The existence of a molten globule folding intermediates resulting from an altered folding in the mutated protein might be related to the aggressiveness of neuroblastomas.

Antigens, Neoplasm↗
PubMed1990

Dictyostelium nucleoside diphosphate kinase highly homologous to Nm23 and Awd proteins involved in mammalian tumor metastasis and Drosophila development.

Two complementary DNAs (cDNAs) previously isolated, one by functional screening and the other by immunological screening of a Dictyostelium discoideum expression library, encode two proteins, Gip17 and Guk7.2, sharing 71% homology. In the present study, we found that the expression of their messenger RNAs (mRNAs) is developmentally regulated, with a sharp decrease during the first hours of differentiation. The Gip17 protein was purified to homogeneity from D. discoideum amoebas and from recombinant Escherichia coli and was conclusively identified as a nucleoside diphosphate (NDP) kinase. NDP kinases play a major role in synthesis of nucleoside triphosphates and, in many systems, are found associated with guanosine triphosphate (GTP)-binding proteins. We found the Gip17 protein to be 77% homologous to the human Nm23 protein and 75% homologous to the Drosophila melanogaster Awd protein. The levels of murine and human nm23 mRNA and Nm23 protein are significantly reduced in tumor cells of high metastatic potential, suggesting that Nm23 is involved in suppression of mammalian tumor metastasis, and mutants of the awd gene exhibit widespread development abnormalities, suggesting that Awd is involved in D. melanogaster development. The high percentage of homology of the Gip17 and Guk7.2 proteins with the Nm23 and Awd proteins indicates that Nm23 and Awd also have nucleoside diphosphate kinase activity. Possible modulations in the activity of this metabolic enzyme could be related to the altered metabolism of tumor cells and the control of metastatic potential. Our results point to an unexpected role of NDP kinase in development, growth control, and oncogenic transformation.

Amino Acid Sequence↗
PubMed1984

Inhibitors of poly(adenosine diphosphate-ribose) synthesis: effect on other metabolic processes.

3-Aminobenzamide and benzamide, purported to be specific inhibitors of the synthesis of poly(adenosine diphosphate-ribose), were used to elucidate possible functions of this biopolymer. These compounds, at frequently used experimental concentrations, not only inhibited the action of poly(adenosine diphosphate-ribose) synthetase but also affected cell viability, glucose metabolism, and DNA synthesis. Thus, the usefulness of 3-aminobenzamide and benzamide may be severely restricted by the difficulty of finding a dose small enough to inhibit the synthetase without producing additional metabolic effects.

Benzamides↗
PubMed1976

Kinetic properties of Serratia marcescens adenosine 5'-diphosphate glucose pyrophosphorylase.

The regulatory properties of partially purified adenosine 5'-diphosphate-(ADP) glucose pyrophosphorylase from two Serratia marcescens strains (ATCC 274 and ATCC 15365) have been studied. Slight or negligible activation by fructose-P2, pyridoxal-phosphate, or reduced nicotinamide adenine dinucleotide phosphate (NADPH) was observed. These compounds were previously shown to be potent activators of the ADPglucose pyrophosphorylases from the enterics, Salmonella typhimurium, Enterobacter aerogenes, Enterobacter cloacae, Citrobacter freundii, Escherichia aurescens, Shigella dysenteriae, and Escherichia coli. Phosphoenolpyruvate stimulated the rate of ADPglucose synthesis catalyzed by Serratia ADPglucose pyrophosphorylase about 1.5- to 2-fold but did not affect the S0.5 values (concentration of substrate required for 50% maximal stimulation) of the substrates, alpha-glucose-1-phosphate, and adenosine 5'-triphosphate. Adenosine 5'-monophosphate (AMP), a potent inhibitor of the enteric ADPglucose pyrophosphorylase, is an effective inhibitor of the S. marcescens enzyme. ADP also inhibits but is not as effective as AMP. Activators of the enteric enzyme counteract the inhibition caused by AMP. This is in contrast to what is observed for the S. marcescens enzyme. Neither phosphoenolpyruvate, fructose-diphosphate, pyridoxal-phosphate, NADPH, 3-phosphoglycerate, fructose-6-phosphate, nor pyruvate effect the inhibition caused by AMP. The properties of the S. marcescens HY strain and Serratia liquefaciens ADPglucose pyrophosphorylase were found to be similar to the above two S. marcescens enzymes with respect to activation and inhibition. These observations provide another example where the properties of an enzyme found in the genus Serratia have been found to be different from the properties of the same enzyme present in the enteric genera Escherichia, Salmonella, Shigella, Citrobacter, and Enterobacter.

1,4-alpha-Glucan Branching Enzyme↗
PubMed1993

A serine phosphorylation of Nm23, and not its nucleoside diphosphate kinase activity, correlates with suppression of tumor metastatic potential.

We describe a serine phosphorylation of the putative metastasis suppressor protein Nm23, and present evidence of its relevance to the signal transduction and tumor metastatic processes. Nm23 was previously demonstrated to exhibit nucleoside diphosphate kinase (NDPK) activity, which transfers a phosphate among nucleoside tri- and diphosphates via an Nm23-phospho-histidine intermediate. Recent data have dissociated the NDPK activity of Nm23 from its phenotypic effects; therefore we have asked whether Nm23 possesses additional biochemical functions. An acid-stable (nonhistidine) phosphorylation was identified on autophosphorylated purified recombinant Nm23 proteins and [32P]orthophosphate-labeled human breast carcinoma and murine melanoma Nm23. Phosphoamino acid analysis identified serine as the acid-stable phosphorylation and serine 44 as the major site of phosphorylation. The acid stable phosphorylation (serine) of Nm23 was inhibited by cAMP in vitro and forskolin in vivo, suggesting that this phosphorylation pathway is regulated in signal transduction. No effect of cAMP was observed on Nm23 NDPK activity. Once phosphorylated, Nm23-phosphoserine can release free phosphate in vitro. The biological relevance of the novel phosphorylation identified herein is suggested by the direct correlation of in vivo Nm23 acid-stable phosphorylation levels, but not Nm23 NDPK activity, with suppression of tumor metastatic potential among control and nm23-1 transfected murine melanoma cells.

Adenosine Triphosphate↗
PubMed1989

Prevalence of nucleoside diphosphate kinase autophosphorylation in human colon carcinoma versus normal colon homogenates.

The G-regulatory proteins of adenylate cyclase, tubulin, and the ras oncogene protein product require the production of GTP from ATP in order to exert their effects within the cell. This implies that the activity of nucleoside diphosphate kinase (NDPK) plays a major role in the regulation of cellular events requiring GTP and that the level of activity of this enzyme is critical. This report presents a simple method for trapping a specific isozyme of NDPK in its high-energy phosphorylated form (NDPK approximately P) using EDTA and demonstrates that this NDPK approximately P is tenfold higher in malignant colon tumor tissue than in normal colon tissue. This autophosphorylation of the 21,000 and 24,000 Mr subunits of NDPK occurs rapidly at 0 degrees C, will use either [gamma-32P]ATP, [gamma-32P]GTP, or the corresponding 8-azidopurine photoprobes, is intramolecular, displays saturation effects, and is prevented from forming if GTP gamma S is added. Dephosphorylation in the homogenate occurs rapidly upon addition of Mg2+ or any nucleoside-5'-diphosphate. The subunits autophosphorylated in the homogenates are mostly in the soluble phase, and they comigrate with the subunits of pure NDPK from human erythrocytes. Cross-addition of normal and malignant homogenates does not decrease the level of autophosphorylation of NDPK, which indicates that the level of NDPK approximately P may be a quantitative measure of the level of this specific NDPK isozyme form. Assays for NDPK activity show correspondingly elevated levels in the malignant homogenates. Using western blot and photoaffinity labeling techniques, we distinguished the NDPK approximately P subunits from two closely migrating GTP-binding proteins. These were identified as the ras gene protein product and a 20,000 Mr protein, which comigrates identically with ADP-ribosylating factor (ARF). The ARF also comigrates in a tight band that is phosphorylated by [gamma 32P]ATP or [gamma-32P]GTP when Mg2+ is present.

Amino Acids↗
PubMed1995

Candida albicans nucleoside-diphosphate kinase: purification and characterization.

Soluble nucleoside-diphosphate kinase (NDP kinase) from Candida albicans was purified to electrophoretic homogeneity and a partial sequence was determined. The enzyme was kinetically and physically characterized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed a single polypeptide of 17 kDa upon staining, by immunodetection with heterologous and homologous antibodies, and by autoradiography of the phosphorylated enzyme. Furthermore, isoelectric focusing of the purified native enzyme showed a single acidic band (pI 4.5). These results together with a native molecular mass of 98 kDa suggest a hexameric native enzyme composed of identical subunits. Like NDP kinases from other sources, the catalysis involves a phosphoenzyme intermediate that is rapidly formed upon incubation of the enzyme with ATP. The transfer of phosphate from phosphoprotein intermediate to nucleoside diphosphates is equally fast. Kinetic experiments indicated that GTP and ATP had the lowest Km compared to UTP, dTTP, and CTP. GDP acted as a preferred acceptor as assessed by kinetic measurements as well as by competition experiments. Experimental data are presented indicating the existence of a membrane-associated NDP kinase. Preliminary characterization of this enzyme suggests that cytosolic and membrane-associated NDP kinases are similar proteins.

Amino Acid Sequence↗
PubMed1993

The direct assay of kinases and acyl-CoA synthetases by HPLC: application to nucleoside diphosphate kinase and succinyl-CoA synthetase.

A general procedure is described for developing and validating HPLC-based assays for nucleotide-utilizing enzymes. The method is comparable in sensitivity to spectrophotometric, enzyme-coupled assays and is applicable to any enzymatic reaction involving coenzyme A and its esters, or the common nucleoside tri-, di-, and/or monophosphates. Nucleotide products of the enzymatic reactions are directly quantitated from their uv absorbances. A typical HPLC-based assay involves running an enzymatic reaction for a fixed time, stopping the reaction by adding acid, and using HPLC to quantitate the amount(s) of nucleotide reactant(s) converted to product(s). Nucleotides containing the same base can be separated under isocratic conditions using a 5-microns C-18, reversed-phase column, and a mobile phase at pH 5 containing 100 mM potassium phosphate, 20 mM acetate, 5 mM tetrabutylammonium ion as an ion-pairing agent, and sufficient acetonitrile to keep the run time within 10 min. Separation of nucleotides containing different bases can usually be accomplished under isocratic conditions by the proper choice of the pH within the range of 3 to 7. A microcomputer is used to control all system hardware and to automate collection and processing of chromatographic data. Application of the general method to development of assays for nucleoside diphosphate kinase and succinyl-CoA synthetase is described. The assay for nucleoside diphosphate kinase quantitates UTP formed from UDP and ATP as substrates. The assay for succinyl-CoA synthetase measures succinyl-CoA after HPLC separation of reactants and products at pH 4 where succinyl-CoA is stable to chemical hydrolysis.

Animals↗
PubMed1996

Discrimination between acid and alkali-labile phosphorylated residues on Immobilon: phosphorylation studies of nucleoside diphosphate kinase.

We have critically analyzed current methodologies for distinguishing histidine and serine phosphorylated residues in proteins and report a simple technique that assures a reliable discrimination. Electro-transfer of a phosphorylated enzyme to Immobilon membranes and its treatment at pH 1 and 14 in buffers containing 5% methanol allows unambiguous distinction between serine/threonine and histidine phosphorylation (O-phosphomonoesters and phosphoramide, respectively) since under these conditions only one type of residue is dephosphorylated. The addition of 5% methanol to all buffers was indispensable to deplete phosphate from membranes incubated successively under acid and basic conditions. The technique was applied to the study of nucleoside diphosphate kinase (NDP kinase) phosphorylation. In this enzyme, autophosphorylation of active site histidine is an accepted intermediate step in the catalytic phosphate transfer activity of nucleoside diphosphate kinase (NDP kinase). Nonetheless, a significant degree of autophosphorylation on other residues has been reported by several laboratories, and the hypothesis has been advanced that this nonhistidine phosphorylation may play an important role in NDP kinase cellular function, signaling the suppression of metastasis in the case of human NDP kinase A. Using this improved method, we show that human, Escherichia coli and Candida albicans NDP kinases are only autophosphorylated on histidine residues. In addition, we present evidence that the presence of phosphoserine after strong acid hydrolysis of the histidine autophosphorylated enzyme is in fact a nonenzymatic transphosphorylation from phosphohistidine due to the harsh acid treatment. This methodology was also applied to in vivo phosphorylation studies of C. albicans NDP kinase. We believe that the technique will be generally useful in histidine phosphorylation screenings.

Candida albicans↗
PubMed1996

A novel function for nucleoside diphosphate kinase in Drosophila.

Nucleoside diphosphate (NDP) kinase is an enzyme that transfers the gamma-phosphate of nucleoside triphosphates to nucleoside diphosphates. Besides this well-defined role, recent evidence suggests that NDP kinase may be implicated in a wide variety of essential cellular processes. In this paper, we showed that the NDP kinase of Drosophila exhibited protein kinase activity as well as autophosphorylation. Ovalbumin was phosphorylated with guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) or ATP. Protein kinase activity was not detected in NDP kinase mutant, abnormal wing discs (awd). These results suggest that this activity could be one of the functions of NDP kinase essential for normal fly development, since awd gene is lethal.

Animals↗
PubMed1994

Molecular cloning, sequence determination and heterologous expression of nucleoside diphosphate kinase from Pisum sativum.

Protein sequence data derived from the N-terminal region of a 17 kDa polypeptide associated with the microsomal membrane fraction from Pisum sativum was used to design degenerate oligonucleotides which were used to amplify P. sativum cDNA via the polymerase chain reaction (PCR). Amplified cDNA was used as a probe to screen a P. sativum cDNA library and a cDNA clone, NDK-P1 was isolated and sequenced. The protein encoded by NDK-P1 had a calculated molecular mass of 16,485 Da and possessed substantial homology with nucleoside diphosphate kinases (NDKs) isolated and cloned from other sources. High levels of expression of NDK-P1 protein were achieved in Escherichia coli using a T7-driven expression system. Recombinant NDK-P1 protein was shown to possess NDK activity and had similar biochemical characteristics to NDKs isolated from other sources. The Michaelis constants for a variety of nucleoside diphosphate (NDP) substrates were found to be broadly similar to those reported for other NDKs, with thymidine nucleotides being the substrates of greatest affinity.

Amino Acid Sequence↗