Nucleotide affinity for a stable phosphorylated intermediate of nucleoside diphosphate kinase

Nucleoside diphosphate (NDP) kinase is transiently phosphorylated on a histidine of the active site during the catalytic cycle. In the presence of a nucleotide acceptor, the phosphohistidine bond is unstable and the phosphate is transferred to the acceptor in less than 1 msec. We describe the synthesis of an analog of the phosphoenzyme intermediate with an inactive mutant of NDP kinase in which the catalytic histidine is replaced by a cysteine. In two sequential disulfide exchange reactions, a thiophosphate group reacts with the thiol function of the cysteine that had previously reacted with dithionitrobenzoate (DTNB). The thiophosphoenzyme presents a 400,000-fold increased stability in the presence of NDPs compared with the phosphoenzyme. The binding of NDP is studied at the steady state and presteady state. Data were analyzed according to a bimolecular association model. For the first time, the true equilibrium dissociation constants of NDP for the analog of the phosphoenzyme are determined in the absence of phosphotransfer, allowing a better understanding of the catalytic mechanism of the enzyme.     

Purification and characterization of nucleoside diphosphate kinase from the brain of Bombyx mori

Nucleoside diphosphate kinase in the brain of Bombyx mori was purified by ammonium sulfate fractionation, and a sequence of chromatographies on DEAE-Cellulofine, hydroxyapatite, Mono-S, and Mono-Q column. The purified enzyme preparation was found to be electrophoretically homogeneous on SDS-PAGE, and its molecular mass was determined to be 18 kDa. The purified protein was digested and the amino acid sequences of resulting peptides were determined. The enzyme showed high similarity to the amino acid sequences of the Drosophila NDP kinase. The enzyme showed NDP kinase activity and mediated the phosphorylation of myelin basic protein. Gel filtration and Hill plot analysis indicate that the purified NDP kinase forms a tetramer and shows little interaction among substrates. Dephosphorylation of NDP kinase by bacterial alkaline phosphatase increased NDP kinase activity. This result indicates that phosphorylation of NDP kinase represses NDP kinase activity.     

Nucleoside diphosphate kinases in mammalian signal transduction systems: recent development and perspective

The role of nucleoside diphosphate (NDP) kinase with special reference to mammalian signal transduction systems was described. The interaction between NDP kinases and G proteins was reevaluated in view of their protein structural information and its significance was extended further on the basis of recent findings obtained with small molecular weight G proteins such as Rad, menin, and Rac. Meanwhile, observations suggesting involvement of NDP kinases in the regulation of cell growth and differentiation led to the realization that NDP kinases may play a crucial role in receptor tyrosine kinase signal transduction systems. In fact, a number of experimental results, particularly obtained with PC12 cells, implicate that NDP kinases appear to regulate differentiation marker proteins and cell-cycle-associated proteins cooperatively. Consequently, we propose a hypothesis that NDP kinases might act like a molecular switch to determine the cell fate toward proliferation or differentiation in response to environmental signals.     

Post-translational processing of Drosophila nucleoside diphosphate kinase

Nucleoside diphosphate kinase (NDPK) was purified from Drosophila melanogaster by a combination of anion-exchange, hydroxyapatite, and reversed-phase chromatography. The identity of the purified enzyme was confirmed by sequencing internal peptides (the N-terminus appeared to be blocked). Post-translational modifications were investigated by using protein chemical and mass spectrometric methods. Analysis by nanoelectrospray ionization-mass spectrometry revealed that the mass of the enzyme was considerably smaller than that predicted from its amino acid sequence. Although its open-reading frame predicts a 153-residue polypeptide, the mature enzyme was found to comprise 152 amino acids, being modified by proteolytic removal of the initiator Met and N-acetylation of Ala2. This explains why the observed pI of the Drosophila enzyme is more acidic than that predicted from its amino acid sequence. No additional post-translational modifications such as glycosylation or O-phosphorylation, which have been identified on homologous NDPKs from other organisms, were detected on the Drosophila enzyme.     

Knockout mice as model systems for studying nm23/NDP kinase gene functions. Application to the nm23-M1 gene

Mice carrying a homozygous germ-line mutation in the nm23-M1 gene that eliminates its protein expression and drives expression of -galactosidase by nm23-M1 promoter have been generated. nm23-M1 gene inactivation is not teratogenic and the pups can grow to adult age without apparent health problems. However, they undergo a growth retardation and knocked out females cannot feed their pups. Both effects are background dependent. -galactosidase mapping of nm23-M1 promoter activation during embryogenesis shows that the nm23-M1 gene is principally expressed in epithelial layer of tissues which require inductive epithelial–mesenchymal interactions for their formation. In conclusion, invalidated mice could be interesting models to analyze the role of nm23-M1 on signal transduction pathway regulation, or cancer induction and proliferation.     

Molecular cloning and nucleotide sequence cDNA encoding nucleoside diphosphate kinase of rice (Oryza sativa L.)

We isolated a rice cDNA encoding nucleoside diphosphate kinase (NDK, EC 2.7.4.6). The deduced amino acid sequence of the rice NDK shows highest homology to spinach NDK-I. The rice NDK gene exhibits a strong codon bias (73.8% GC) in the third position of the codon. DNA blot analysis indicated that at least single NDK gene is present in rice genome.     

Regulatory roles for nm23/nucleoside diphosphate kinase-like enzymes in insulin secretion from the pancreatic islet beta cell

Recent studies from multiple laboratories, including our own, provided fresh insights into the contributory roles for GTP-binding proteins (G-proteins) in glucose-stimulated insulin secretion (GSIS) from the islet β cell. However, the precise mechanisms underlying the activation of this class of signaling proteins by insulin secretagogues remain only partially understood. We recently proposed that nm23/nucleoside diphosphate kinase (NDPK) catalyzes an alternate, non-receptor-dependent activation of islet endogenous G-proteins. In further support of this proposal, we report, herein, that overexpression of wild type (WT) nm23-H1 mutant in INS cells markedly potentiated GSIS. However, an inactive mutant of nm23-H1(H118F), which is deficient in histidine kinase and NDPK activities, was considerably less effective in potentiating GSIS from these cells, suggesting that both of these activities may be relevant for the potentiating effects of nm23-H1. Potential significance of these findings in relation to contributory roles for nm23/NDPK-like enzymes in the stimulus-secretion coupling of GSIS is discussed.     

Protein interactions provide new insight into Nm23/nucleoside diphosphate kinase functions

Nm23–NDPKs besides contributing to the maintenance of the cellular nucleoside triphosphate pool, exert regulatory properties in a variety of cellular events including proliferation, invasiveness, development, differentiation, and gene regulation. This review focuses on recently discovered protein–protein interactions involving the Nm23 proteins. The findings herein summarized provide new and intriguing suggestions for a more extensive understanding of the biological functions of the Nm23 proteins.     

Putative functions of nucleoside diphosphate kinase in plants and fungi

The putative functions of NDP (nucleoside diaphosphate) kinases from various organisms focusing to fungi and plants are described. The biochemical reactions catalyzed by NDP kinase are as follows. (i) Phosphotransferring activity from mainly ATP to cognate NDPs generating nucleoside triphosphates (NTPs). (ii) Autophosphorylation activity from ATP and GTP. (iii) Protein kinase (phosphotransferring) activity phosphorylating such as myelin basic protein. NDP kinase could function to provide NTPs as a housekeeping enzyme. However, recent works proved possible functions of the NDP kinases in the processes of signal transduction in various organisms, as described below. By use of the extracts of the mycelia of a filamentous fungus Neurospora crassa blue-light irradiation could increase the phosphorylation of a 15-kDa protein, which was purified and identified to be NDP kinase (NDK-1). By use of the etiolated seedlings of Pisum sativum cv Alaska and Oryza sativa red-light irradiation of intact plants increased the phosphorylation of NDP kinase. However, successive irradiation by red–far-red reversed the reaction, indicating that phytochrome-mediated light signals are transduced to the phosphorylation of NDP kinase. NDP kinase localizing in mitochondria is encoded by nuclear genome and different from those localized in cytoplasm. NDP kinase in mitochondria formed a complex with succinyl CoA synthetase. In Spinicia oleraceae two different NDP kinases were detected in the chloroplast, and in Pisum sativum two forms of NDP kinase originated from single species of mRNA could be detected in the choloroplast. However, the function of NDP kinases in the choloroplast is not yet known. In Neurospora crassa a Pro72His mutation in NDP kinase (ndk-1 ^Pro72His ) deficient in the autophosphorylation and protein kinase activity resulted in lacking the light-induced polarity of perithecia. In wild-type directional light irradiation parallel to the solid medium resulted in the formation of the perithecial beak at the top of perithecia, which was designated as light-induced polarity of perithecia. In wild-type in darkness the beak was formed at random places on perithecia, and in ndk ^Pro72His mutant the perithecial beak was formed at random places even under directional light illumination. The introduction of genomic DNA and cDNA for ndk-1 demonstrated that the wild-type DNAs suppressed the mutant phenotype. With all these results except for the demonstration in Neurospora, most of the phenomena are elusive and should be solved in the molecular levels concerning with NDP kinases.     

Molecular interactions involving Escherichia coli nucleoside diphosphate kinase

Nucleoside diphosphate kinase plays a distinctive metabolic role as the enzyme poised between the last reaction of deoxyribonucleoside triphosphate (dNTP) biosynthesis and the DNA polymerization apparatus. In bacteriophage T4 infection, NDP kinase is one of very few enzymes of host cell origin to participate in either dNTP synthesis or DNA replication. Yet NDP kinase forms specific contacts with phage-coded proteins of dNTP and DNA synthesis. This article summarizes work from our laboratory that identifies and characterizes these interactions. Despite these specific interactions, the enzyme appears to be dispensable, both for T4 replication and for growth of the host, Escherichia coli, because site-specific disruption of ndk, the structural gene for NDP kinase, does not interfere with growth of the host cell and only partly inhibits phage replication. However, ndk disruption unbalances the dNTP pools and stimulates mutagenesis. We discuss our attempts to understand the basis for this enhanced mutagenesis.     

A novel human nucleoside diphosphate (NDP) kinase,Nm23‐H6, localizes in mitochondria and affects cytokinesis

Nucleoside diphosphate kinases (NDP kinases) are enzymes known to be conserved throughout evolution and have been shown to be involved in various biological events, in addition to the “housekeeping” phosphotransferase activity. We present the molecular cloning of a novel human NDP kinase gene, termed Nm23‐H6. Nm23‐H6 gene has been mapped at chromosome 3p21.3 and is highly expressed in heart, placenta, skeletal muscle, and some of the cancer cell lines. Recombinant Nm23‐H6 protein has been identified to exhibit functional NDP kinase activity. Immunolocalization studies showed that both endogenous and inducibly expressed Nm23‐H6 proteins were present as short, filament‐like, perinuclear radical arrays and that they colocalized with mitochondria. Cell fractionation study also demonstrated the presence of Nm23‐H6 protein in a mitochondria‐rich fraction. Moreover, induction of overexpression of Nm23‐H6 in SAOS2 cells, using the Cre‐loxP gene activation system, resulted in growth suppression and generation of multinucleated cells. Flow cytometric analysis also demonstrated that the proportion of cells with more than 4N DNA content increased to 28.1% after induction of Nm23‐H6, coinciding with the appearance of multinucleated cells. These observations suggest that Nm23‐H6, a new member of the NDP kinase family, resides in mitochondria and plays a role in regulation of cell growth and cell cycle progression. J. Cell. Biochem. 76:254–269, 1999. © 1999 Wiley‐Liss, Inc.     

Structure of a halophilic nucleoside diphosphate kinase from Halobacterium salinarum

Nucleoside diphosphate kinase from the halophilic archaeon Halobacterium salinarum was crystallized in a free state and a substrate-bound form with CDP. The structures were solved to a resolution of 2.35 and 2.2A, respectively. Crystals with the apo-form were obtained with His6-tagged enzyme, whereas the untagged form was used for co-crystallization with the nucleotide. Crosslinking under different salt and pH conditions revealed a stronger oligomerization tendency for the tagged protein at low and high salt concentrations. The influence of the His6-tag on the halophilic nature of the enzyme is discussed on the basis of the observed structural properties.     

Reversibility of nucleoside diphosphate kinase solubilization from the surface of the outer mitochondrial membrane

It was found that in medium with low ionic strength nucleoside diphosphate kinase (NDPK) solubilization from the outer membrane of liver mitochondria could be partially reversed by the addition of 3.3 mM MgCl₂. Complete rebinding of the enzyme after the addition of MgCl₂ was observed when the mitochondrial washing and storage medium contained leupeptin, an inhibitor of cathepsins. It was demonstrated that leupeptin and another inhibitor of cysteine proteinases, E-64, do not influence the rate of NDPK solubilization as well as its solubilized and membrane-associated activity. We conclude that NDPK becomes sensitive to proteolysis only after its solubilization; proteolysis does not affect the part of the enzyme molecule that is responsible for catalysis. After solubilization of NDPK in the absence of leupeptin, cathepsins damage sites of its binding on the membranes. The rate of the enzyme solubilization is dependent on the pH of the storage medium (pH 6.0–8.0); it decreases with increase in pH. It was shown that in the medium with high ionic strength, MgCl₂ does not reverse pH-dependent NDPK solubilization, but solubilization could be reversed by increase in medium pH in the presence of E-64 and BSA. The physiological importance of these results is discussed. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 5, pp. 710–720.     

Residue 134 determines the dimer-tetramer assembly of nucleoside diphosphate kinase from moderately halophilic bacteria

Halomonas nucleoside diphosphate kinase (HaNDK) forms a dimeric assembly and Pseudomonas NDK (PaNDK) forms a tetrameric assembly. The mutation of Glu134 to Ala in HaNDK resulted in the conversion of the native dimeric structure to the tetramer assembly. Conversely, the mutation of Ala134 to Glu in PaNDK lead to the conversion from the tetramer to the dimer assembly, indicating that a single amino acid substitution at position 134 results in an alteration of the oligomeric structure of NDK. By modeling the structure of HaNDK and PaNDK based on the crystal structure of Myxococcus NDK, we showed that Glu134 exerts sufficient repulsive forces to disrupt the dimer-dimer interaction and prevent the formation of the tetramer.     

In vivo cross-linking of nm23/nucleoside diphosphate kinase to the PDGF-A gene promoter

Human isoforms A and B of nm23/nucleoside diphosphate (NDP) kinase, functionally important in development and cancer, have been reported to bind to DNA, and in particular isoform A to the PDGF-A promoter and isoform B to the c-myc promoter and to telomeric repeats. However, no direct proof of the binding in vivo has yet been obtained. To demonstrate this interaction, human erythroleukemic K562 cells were incubated with two different cross-linking reagents, formaldehyde or cis-diammine dichloro platinum II. The DNA-protein covalent complexes were isolated and analyzed by Western blotting. The positive immunochemical staining showed that in both conditions NDP kinase isoforms A and B were efficiently cross-linked to DNA in vivo. NDP kinase-linked DNA fragments obtained by immunoprecipitation, subjected to hybridization with different probes, showed a definite enrichment in the nuclease-hypersensitive silencer element of the PDGF-A promoter. No conclusive evidence was found by this technique of preferential hybridization with a nuclease-hypersensitive element of the c-myc promoter and with the telomeric TTAGGG repeats. The immunoprecipitated NDP kinase-DNA complexes are a promising material for the detection of other specific DNA sequences interacting with NDP kinase.     

Regulation of Cellular Functions by Nucleoside Diphosphate Kinases in Mammals

The role of nucleoside diphosphate (NDP) kinases in cell growth, differentiation, and tumormetastasis in relation to signal transduction was investigated. The essential role of NDP kinasein cell growth was validated by coupling between reduced NDP kinase levels, induced byantisense oligonucleotides, and the suppression of proliferative activity of a cultured cell line.In addition, because NDP kinase levels are often enhanced with development and differentiation,as has been demonstrated in postmitotic cells and tissues, such as the heart and brain, wefurther examined this possibility using the bone tissue (osteoblasts) and a cultured cell linePC12D. The enhanced NDP kinase accumulation was demonstrated in the matured osteoblastsin vivo and in vitro by immunohistochemistry. In PC12D cells neurite outgrowth took placein NDP kinase -transfected clones without differentiation inducers, which was accompaniedby prolongation of doubling time. Neurite outgrowth, triggered by nerve growth factor and acyclic AMP analog, was down-regulated upon forced expression of inactive mutant NDPkinase by virtue of a dominant negative effect. NDP kinase -transfected rat mammaryadenocarcinoma cells (MTLn3) and nm23-H2-transfected human oral squamous cell carcinomacells (LMF4) manifested reduced metastatic potential and were associated with an alteredsensitivity to environmental factors, such as motility and growth factors. NDP kinase ,compared to NDP kinase , was involved in a wide variety of the cellular phenomena examined.Taken together, NDP kinase isoforms appear to elicit both their own respective and commoneffects. They may have an ability to lead cells to both proliferative and differentiated statesby modulating responsiveness to environmental factors, but their fate seems to depend on theirsurrounding milieu.