Nucleoside diphosphate kinase from Escherichia coli.

Nucleoside diphosphate (NDP) kinase from Escherichia coli was purified to homogeneity and was crystallized. Gel filtration analysis of the purified enzyme indicated that it forms a tetramer. The enzyme was phosphorylated with [gamma-32P]ATP, and the pH stability profile of the phosphoenzyme indicated that two different amino acid residues were phosphorylated. Both a histidine residue and serine residues, including Ser-119 and Ser-121, appear to be phosphorylated. A Ser119Ala/Ser121Ala double mutant (i.e., with a Ser-to-Ala double mutation at positions 119 and 121), as well as Ser119Ala and Ser121Ala mutants, was isolated. All of these retained NDP kinase activity; also, both the Ser119Ala and Ser121Ala mutants could still be autophosphorylated. In the case of the double mutant, a slight autophosphorylation activity, which was resistant to acid treatment, was still detected, indicating that an additional minor autophosphorylation site besides His-117 exists. These results are discussed in light of the recent report of N. J. MacDonald et al. on the autophosphorylation of human NDP kinase (J. Biol. Chem. 268:25780-25789, 1993).     

Nucleoside diphosphate kinase and the flagellar movement of glycerinated spermatozoa.

Nucleoside diphosphate kinase [EC 2.7.4.6.] of sperm flagella and Tetrahymena cilia is detected mostly in the outer fiber fraction, suggesting an association of the enzyme with the outer fiber microtubules. The enzyme does not catalyze transphosphorylation between microtubule-bound GDP and exogenous ATP. Even when undulatory movement of glycerinated sperm is induced by MgATP, no phosphorylation is detected in the outer fiber fraction. These facts do not appear to support the hypothesis that the phosphorylation of microtubule-bound GDP is involved in the mechanism of flagellar movement.     

Nucleoside diphosphate kinase required for coleoptile elongation in rice

Although several nucleoside diphosphate (NDP) kinase genes have been cloned in plants, little is known about the functional significance of this enzyme during plant growth and development. We introduced a chimeric gene encoding an antisense RNA of NDP kinase under the control of the Arabidopsis heat shock protein HSP81-1 promoter into rice (Oryza sativa L.) plants using the Agrobacterium tumefaciens transformation system. The expression of antisense RNA down-regulated the accumulation of mRNA, resulting in reduced enzyme activity even under the standard growth temperature (25 degrees C) in transgenic plants. Following heat shock treatment (37 degrees C), NDP kinase activities in some transgenic rice plants were more reduced than those grown under 25 degrees C. The comparison of the coleoptile growth under submersion showed that cell elongation process was inhibited in antisense NDP kinase transgenic plants, suggesting that an altered guanine nucleotide level may be responsible for the processes.     

Nucleoside diphosphate kinase and GTP-binding proteins. Possible mechanisms of coupling

The results of numerous investigations during the last 20 years show that nucleoside diphosphate kinase (NDP kinase) is a multifunctional protein. In this paper, the current data are analyzed indicating that one of the possible mechanisms by which NDP kinase manifests its multifunctional role is its participation in the activation (or regulation) of heterotrimeric GTP-binding proteins (G proteins). We demonstrate that one of the NDP kinase isoforms dynamically interacts with the retinal rod G protein transducin (Gt) and phosphorylates its β-subunit at histidine residue (His 266). It is also shown that it leads to the consecutive transfer of the phosphate group to GDP in the active center of G protein α-subunit and G protein activation. The advantages of this mechanism are considered as compared to the classic G protein activation mechanism, GDP/GTP exchange.     

Nucleoside diphosphate kinase activity in purified cores of vesicular stomatitis virus.

Purified cores of vesicular stomatitis virus contain an enzymatic activity that converts GDP, UDP, and CDP into their corresponding triphosphates using ATP as the phosphate donor. Thus, the virion-associated RNA polymerase can synthesize mRNA normally in vitro even when one of the ribonucleoside triphosphates is replaced by its corresponding diphosphate. RNA synthesis does not proceed if ATP is replaced by ADP. Similarly RNA synthesis is impaired if CDP and UDP are present in the same reaction. The role of the nucleoside diphosphate kinase (NDP kinase, EC 2.7.4.6) in vesicular stomatitis virus mRNA synthesis in vitro is discussed.     

Nucleoside diphosphate kinase does not directly interact with tubulin nor microtubules.

Nucleoside diphosphate kinase has been shown to play a role in proliferation and development. Microtubules have been evoked as a possible target of NDP kinase action; in particular it was proposed that NDP kinase could regulate the cellular pool of polymerizable GTP-tubulin by direct phosphorylation of tubulin bound GDP. We show that this reaction does not occur in vitro and also that NDP kinase does not bind to microtubules both in the presence and absence of MAPs. Thus, any possible physiological effect of NDP kinase on microtubule dynamics is exerted only by modulating the concentrations of free guanine nucleotides in the vicinity of microtubules.     

Nucleoside diphosphate kinase from Myxococcus xanthus. I. Cloning and sequencing of the gene

By photoaffinity labeling with a photolysable analog of GTP, 8-N3GTP, we were able to find at least five distinct GTP-binding proteins in Myxococcus xanthus; two of them located in the membrane and the other three in the soluble fraction. The amino-terminal sequence of the 16-kDa GTP-binding protein from the soluble fraction was determined, and the gene that encodes this protein was isolated and cloned using degenerate oligonucleotides as a probe. The DNA sequence of the gene was determined, which did not show similarity with other known proteins. The gene product was overexpressed in Escherichia coli, by using the lacZ promoter, to a level of 13% of the soluble protein. Attempts to isolate deletion mutants were unsuccessful, although double crossing-over events leading to a deletion mutation of the gene were detected by Southern blot hybridization. This result indicates that this gene is essential for cell growth. In the following paper (Mu?oz-Dorado, J., Inouye, S., and Inouye, M. (1990) J. Biol. Chem. 265, 2707-2712), the gene product was biochemically characterized and identified to be a nucleoside diphosphate kinase.     

Nucleoside diphosphate kinase from brain. Purification and effect on microtubule assembly in vitro

Tubulin strictly requires GTP for its polymerization. Nevertheless, microtubule assembly can be observed in the presence of ATP as the only nucleotide triphosphate, due to the nucleoside diphosphate kinase (NDP kinase) present in microtubule preparations, and which phosphorylates the GDP into GTP. We have purified this enzyme from pig brain to homogeneity, and shown that its relative mass is close to 100 000 in its native state, and 17 000 under denaturing conditions. Therefore it is probably a hexamer, as previously shown for the enzyme from other sources, and also presents a microheterogeneity, with the major isoforms between pI 5.0 and 6.0. The enzyme is transiently phosphorylated during catalysis, as expected within a ping-pong bi-bi mechanism. The effect of the NDP kinase on pure tubulin polymerization was studied: in the presence of NDP kinase, the lag time observed in the kinetics of microtubule assembly was shorter and the final extent of assembly was unchanged. The effect of the enzyme was observed at enzyme concentrations 900-fold lower than tubulin concentration, which shows that the NDP kinase acts catalytically. Kinetic data show that the catalytic effect of the NDP kinase is faster than the rate of nucleotide exchange on tubulin under the same conditions. This result demonstrates that the tubulin-GDP complex itself is a substrate for the enzyme, which may indicate that the GDP bound to tubulin at the E site is exposed on the surface of dimeric tubulin.     

Nucleoside diphosphate kinase at the cell surface of neoplastic human cells in culture

Strong evidence is given that a nucleoside diphosphate kinase is present to some extent on the surface of intact neoplastic cells in culture. Experiments could be performed with cells cultured in a few plates to which an incubation medium was added. The cells were firmly attached to the supporting medium and remained viable during the incubation procedure. Determinations of lactate dehydrogenase were carried out to rule out any possible contamination from the culturing medium as well as from the cell interior. From these analyses, a procedure was developed which easily removed the last traces of the culturing medium and which showed that there was no leakage of intracellular lactate dehydrogenase during the incubation procedure. There was a rather insignificant diffusion of nucleoside diphosphate kinase into the incubation medium. In common with other nucleoside diphosphate kinases, the glioma cell surface enzyme seemed to be nonspecific with regard to nucleotide substrates.     

The human dynamin-related protein OPA1 is anchored to the mitochondrial inner membrane facing the inter-membrane space

We have examined the effect of transforming growth factor β₁ (TGF-β₁) and overexpression of the Smad4 gene on the phenotype of Car C, a ras mutated highly malignant spindle carcinoma cell line. TGF-β₁-treated Car C cells overexpressing Smad4 spread with a flattened morphology with membrane ruffles abundant in vinculin and show a reduction in their invasive abilities. TGF-β₁ treatment and overexpression of Smad4 also enhanced the production of PAI-1 measured by the activation of the p3TP-lux reporter gene containing a PAI-1-related promoter. This activation was abolished with a dominant-negative Smad4 construct. These results lead us to conclude that both TGF-β₁ and Smad4 overexpression reduce the invasive potential of Car C cells, probably via the Smad pathway.     

Nucleoside diphosphate kinase, a source of GTP, is required for dynamin-dependent synaptic vesicle recycling

Nucleoside diphosphate kinase (NDK), an enzyme encoded by the Drosophila abnormal wing discs (awd) or human nm23 tumor suppressor genes, generates nucleoside triphosphates from respective diphosphates. We demonstrate that NDK regulates synaptic vesicle internalization at the stage where function of the dynamin GTPase is required. awd mutations lower the temperature at which behavioral paralysis, synaptic failure, and blocked membrane internalization occur at dynamin-deficient, shi(ts), mutant nerve terminals. Hypomorphic awd alleles display shi(ts)-like defects. NDK is present at synapses and its enzymatic activity is essential for normal presynaptic function. We suggest a model in which dynamin activity in nerve terminals is highly dependent on NDK-mediated supply of GTP. This connection between NDK and membrane internalization further strengthens an emerging hypothesis that endocytosis, probably of activated growth factor receptors, is an important tumor suppressor activity in vivo.     

Nucleoside diphosphate kinase A as a controller of AMP-kinase in airway epithelia

This review integrates recent understanding of a novel role for NDPK-A in two related directions: Firstly, its role in an airway epithelial cell when bound to the luminal (apical) membrane and secondly in the cytosol of many different cells (epithelial and non-epithelial) where an isoform-specific interaction occurs with a regulatory partner, AMPKα1. Thus NDPK-A is present in both a membrane and cytosolic environment but in the apical membrane, its roles are not understood in detail; preliminary data suggest that it co-localises with the cystic fibrosis protein (CFTR). In cytosol, we find that NDPK-A is coupled to the catalytic alpha1 isoform of the AMP-activated protein kinase (AMPKα subunit), which is part of 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. We find that ATP is located within this complex and ‘fed’ from NDPK to AMPK without ever ‘seeing’ bulk solution. Importantly, the reverse can also happen such that AMPK activity can be made to decline when NDPK-A ‘steals’ ATP from AMPK. Thus we propose a novel paradigm in NDPK-A function by suggesting that AMP-kinase can be regulated by NDPK-A, independently of AMP.     

A MAP kinase is activated late in plant mitosis and becomes localized to the plane of cell division.

In eukaryotes, mitogen-activated protein kinases (MAPKs) are part of signaling modules that transmit diverse stimuli, such as mitogens, developmental cues, or various stresses. Here, we report a novel alfalfa MAPK, Medicago MAP kinase 3 (MMK3). Using an MMK3-specific antibody, we detected the MMK3 protein and its associated activity only in dividing cells. The MMK3 protein could be found during all stages of the cell cycle, but its protein kinase activity was transient in mitosis and correlated with the timing of phragmoplast formation. Depolymerization of microtubules by short treatments with the drug amiprophosmethyl during anaphase and telophase abolished MMK3 activity, indicating that intact microtubules are required for MMK3 activation. During anaphase, MMK3 was found to be concentrated in between the segregating chromosomes; later, it localized at the midplane of cell division in the phragmoplast. As the phragmoplast microtubules were redistributed from the center to the periphery during telophase, MMK3 still localized to the whole plane of division; thus, phragmoplast microtubules are not required to keep MMK3 at this location. Together, these data strongly support a role for MMK3 in the regulation of plant cytokinesis.     

Nucleoside diphosphate kinase B (NDKB) scaffolds endoplasmic reticulum membranes in vitro

The mechanisms that structure the mammalian endoplasmic reticulum (ER) network are not fully understood. Here we show that salt extraction of semi-intact normal rat kidney (NRK) fibroblasts and subsequent incubation of the extracted cells with ATP resulted in dramatic ER network retraction. Under these conditions, addition of a single protein, Nucleoside Diphosphate Kinase B (NDKB), was sufficient to reverse the retraction and to promote ER network extension. The underlying mechanism of membrane extension involved direct lipid binding, as NDKB bound phosphatidylinositol (PtdIns)(4)P, PtdIns(4,5)P₂ and phosphatidic acid (PA); binding to these anionic lipids required clusters of basic residues on the surface of the NDKB hexamer; and amino acid changes in NDKB that blocked lipid binding also blocked ER network extension. Remarkably, purified NDKB transformed a uniform population of synthetic lipid vesicles into extensive membrane networks, and this also required its phospholipid-binding activity. Altogether these results identify a protein sufficient to scaffold extended membrane networks, and suggest a possible role for NDKB-like proteins, as well as phosphoinositides and/or acidic phospholipids, in modulating ER network morphogenesis.     

Nucleoside diphosphate kinase from Escherichia coli; its overproduction and sequence comparison with eukaryotic enzymes.

The gene encoding nucleoside diphosphate (NDP) kinase of Escherichia coli was identified by polymerase chain reaction using oligodeoxyribonucleotide primers synthesized on the basis of consensus sequences from Myxococcus xanthus and various eukaryotic NDP kinases. The gene (ndk), mapped at 54.2 min on the E. coli chromosome, was cloned and sequenced. The E. coli NDP kinase was found to consist of 143 amino acid residues that are 57, 45, 45, 42, 43, and 43% identical to the M. xanthus, Dictyostelium discoideum, Drosophila melanogaster, mouse, rat, and human enzymes, respectively. The ndk gene appears to be in a monocistronic operon and, when cloned in a pUC vector, NDP kinase was overproduced at a level of approx. 25% of total cellular proteins. The protein could be labeled with [gamma-32P]ATP and migrated at a 16.5 kDa when electrophoresed in SDS-polyacrylamide gel, which is in good agreement with the Mr of the purified E. coli NDP kinase previously reported.     

Nucleoside mono- and nucleoside diphosphate kinase activities in rat liver and brain in radiation sickness

Activity of nucleoside di- and nucleoside triphosphates metabolism enzymes in tissues of rats gamma-irradiated by a dose of 30 Gy was studied 0.5, 1, 3, 6 and 24 hours after the radiation effect. It is shown that the nucleoside monophosphate kinase activity of the liver and brain is enhanced almost at all stages of the studies and the nucleoside diphosphate kinase activity is inhibited. A significant but reversible decrease of the nucleoside monophosphate kinase activity is observed in the liver 3 h later. By an end of the first day after irradiation the nucleoside mono- and nucleoside diphosphate kinase activities increase significantly both in the liver and brain.     

Human UMP-CMP kinase 2, a novel nucleoside monophosphate kinase localized in mitochondria

Enzyme deficiency in the salvage pathway of deoxyribonucleotide synthesis in mitochondria can cause mtDNA depletion syndromes. We have identified a human mitochondrial UMP-CMP kinase (UMP-CMPK, cytidylate kinase; EC 2.7.4.14), designated as UMP-CMP kinase 2 (UMP-CMPK2). The C-terminal domain of this 449-amino acid protein contains all consensus motifs of a nucleoside monophosphate kinase. Phylogenetic analysis showed that UMP-CMPK2 belonged to a novel nucleoside monophosphate kinase family, which was closer to thymidylate kinase than to cytosolic UMP-CMP kinase. Subcellular localization with green fluorescent protein fusion proteins illustrated that UMP-CMPK2 was localized in the mitochondria of HeLa cells and that the mitochondrial targeting signal was included in the N-terminal 22 amino acids. The enzyme was able to phosphorylate dUMP, dCMP, CMP, and UMP with ATP as phosphate donor, but the kinetic properties were different compared with the cytosolic UMP-CMPK. Its efficacy to convert dUMP was highest, followed by dCMP, whereas CMP and UMP were the poorest substrates. It also phosphorylated the monophosphate forms of the nucleoside analogs ddC, dFdC, araC, BVDU, and FdUrd, which suggests that UMP-CMPK2 may be involved in mtDNA depletion caused by long term treatment with ddC or other pyrimidine analogs. UMP-CMPK2 mRNA expression was exclusively detected in chronic myelogenous leukemia K-562 and lymphoblastic leukemia MOLT-4 among eight studied cancer cell lines. Particular high expression in leukemia cells, dominant expression in bone marrow, and tight correlation with macrophage activation and inflammatory response suggest that UMP-CMPK2 may have other functions in addition to the supply of substrates for mtDNA synthesis.