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.     

Crystal Structures of S120G Mutant and Wild Type of Human Nucleoside Diphosphate Kinase A in Complex with ADP

Nm23 was the first metastasis suppressor gene identified. This gene encodes a NDP kinase that also exhibits other properties like histidine protein kinase and interactions with proteins and DNA. The S120G mutant of NDPK-A has been identified in aggressive neuroblastomas and has been found to reduce the metastasis suppressor effect of Nm23. In order to understand the differences between the wild type and the S120G mutant, we have determined the structure of both mutant and wild type NDPK-A in complex with ADP. Our results reveal that there are no significant changes between the two enzyme versions even in the surroundings of the catalytic histidine that is required for NDP kinase activity. This suggests that the S120G mutation may affect an other protein property than NDP kinase activity.     

Nm23-H1/NDP kinase folding intermediates and cancer: a hypothesis

The Nm23-H1/nucleoside diphosphate (NDP) kinase A is a metastasis suppressor, besides its enzymatic activity. The mutant S120G has been found in high-grade neuroblastomas. The mutant protein, once denatured in urea, is unable to refold in vitro. A size-exclusion chromatography analysis of the folding/association pathway showed that recombinant wild-type and S120G mutant human Nm23-H1/NDP kinase A unfold and refold passing through a molten globule state while typical hexameric NDP kinases unfold without dissociated species and refold through a native monomeric intermediate. A survey of the recent literature showed that several proteins involved in cancer, and their mutants, are marginally stable, like the wild-type Nm23-H1/NDP kinase A, or are misfolded, like its S120G mutant. We therefore suggest that the low thermodynamic stability and the folding intermediate of the Nm23-H1/NDP kinase A may be necessary for its regulatory properties.     

A Nucleoside Diphosphate Kinase from Paramecium tetraurelia with Protein Kinase Activity

Nucleoside diphosphate kinase (NDP kinase) from Paramecium was purified to homogeneity. The native enzyme was 80 kDa (by gel filtration), with subunits of 18 and 20 kDa. Near the amino terminus, 15 of 20 residues were identical with those in human NDP kinase, and 17 of 20 with the awd gene product from Drosophila. NDP kinase bound α-labeled ATP and GTP, and a photoreactive GTP analog labeled both subunits. Purified NDP kinase underwent autophosphorylation on a histidine and a serine residue using either ATP or GTP as a substrate. The enzyme also catalyzed acid-stable phosphorylation of casein and phosvitin. This protein kinase activity is distinct from the histidine phosphorylation that is part of the NDP kinase catalytic cycle. Antiserum against the purified protein from Paramecium cross-reacted with 16- to 20-kDa proteins in most species tested, and with a larger protein (44 kDa) in Paramecium, Xenopus, and two human lines. The multiple forms (20 and 44 kDa) of the NDP kinase in Paramecium and its protein kinase activity, suggest that the protein is more than a housekeeping enzyme; it may have regulatory roles such as those of the NDP kinase-like awd protein of Drosophila and Nm23 protein of humans.     

Isolation of a mRNA encoding a nucleoside diphosphate kinase from tomato that is up-regulated by wounding

A cDNA clone (TAB2) encoding a nucleoside diphosphate (NDP) kinase has been isolated from a tomato (Lycopersicon esculentum Mill. cv. Ailsa Craig) cDNA library. The clone is 590 bp long and exhibits a high degree of sequence identity with spinach NDP kinases I and II, Pisum sativum NDP kinase I, Arabidopsis thaliana NDP kinase, Drosophila melanogaster NDP kinase, Dictyostelium discoideum NDP kinase and human Nm 23-H1 and Nm23-H2. Northern analysis has revealed that the mRNA encoded by TAB2 is up-regulated in both leaf and stem tissue in response to wounding. The increase is apparent within 1 h of wounding and is not further elevated by application of ethylene. Southern blot analysis indicates that TAB2 is a member of a small gene family.     

Expression of a dominant negative allele of cdc2 prevents activation of the endogenous p34cdc2 kinase

Summary The cdc2 gene of the fission yeast Schizosaccharomyces pombe encodes a 34 kDa phosphoprotein with serine/threonine protein kinase activity that acts as the key component in regulation of the eukaryotic cell cycle. We used a repressible promoter fused to the cdc2 cDNA to isolate conditionally dominant negative mutants of cdc2. One of these mutants, DL5, is described in this paper. Overexpression of the mutant protein in a wild-type cdc2 background is lethal and confers cell cycle arrest with a typical cdc phenotype. Sequencing of the mutant cdc2 gene revealed a single amino acid substitution in a region highly conserved in cdc2-like proteins. The mutant protein exhibits no protein kinase activity, but is able to bind a component(s) required for an active protein kinase complex and thereby prevents binding of this component(s) to the co-existing wild-type cdc2 protein. We also demonstrate that S. pombe p34^cdc2 contains no phosphoserine.     

Subcellular Localization and Characterization of Nucleoside Diphosphate Kinase in Rat Retina: Effect of Diabetes

Nucleoside diphosphate kinase (NDP kinase) catalyzes the transfer of terminal phosphate from nucleotide triphosphates (e.g. ATP) to nucleotide diphosphates (e.g. GDP) to yield nucleotide triphosphates (e.g. GTP). Since guanine nucleotides play critical role(s) in GTP-binding protein (G-protein)-mediated signal transduction mechanisms in retina, we quantitated NDP kinase activity in subcellular fraction-derived from normal rat retina. A greater than 85% of the total specific activity was present in the soluble fraction, which was stimulated (up to 7 fold) by 2 mM magnesium. NDP kinase exhibited saturation kinetics towards di- and tri-phosphate substrates, and was inhibited by known inhibitors of NDP kinase, uridine diphosphate (UDP) or cromoglycate (CRG). We have previously reported significant abnormalities in the activation of G-proteins in streptozotocin (STZ)-diabetic rat retina (Kowluru et al. Diabetologia 35:624–631, 1992). Since NDP kinase hasbeen implicated in direct interaction with and/or activation of various G-proteins, we quantitated both basal and magnesium-stimulated NDP kinase activity in soluble and particulate fractions of retina derived from STZ-diabetic rats to examine whether abnormalities in G-protein function in diabetes are attributable to alterations in retinal NDP kinase. There was no effect of diabetes either on the basal or the magnesium-activated retinal NDP kinase activity. This study represents the first characterization of NDP kinase activity in rat retina, and suggests that in diabetes, this enzyme may not be rate-limiting and/or causal for the observed alterations in retinal G-protein functions.     

Quaternary Structure of Nucleoside Diphosphate Kinases

Nucleoside (NDP) diphosphate kinases are oligomeric enzymes. Most are hexameric, but somebacterial enzymes are tetrameric. Hexamers and tetramers are constructed by assemblingidentical dimers. The hexameric structure is important for protein stability, as demonstratedby studies with natural mutants (the Killer-of-prune mutant ofDrosophila NDP kinase andthe S120G mutant of the human NDP kinase A in neuroblastomas) and with mutants obtainedby site-directed mutagenesis. It is also essential for enzymic activity. The function of the tetrameric structure is unclear.     

Identification of a genomic region that complements a temperature-sensitive, high CO2-requiring mutant of the cyanobacterium, Synechococcus sp. PCC7942

Summary In a temperature-sensitive, high CO₂-requiring mutant of Synechococcus sp. PCC7942, the ability to fix intracellularly accumulated inorganic carbon was severely impaired at non-permissive temperature (41° C). In contrast, inorganic carbon uptake and ribulose-1,5-bisphosphate carboxylase activity in the mutant were comparable to the respective values obtained with the wild-type strain. The mutant was transformed to the wild-type phenotype (ability to form colonies at non-permissive temperature under ordinary air) with the genomic DNA of the wild-type strain. A clone containing a 36 kb genomic DNA fragment of the wild-type strain complemented the mutant phenotype. The complementing activity region was associated with internal 17 kb SmaI, 15 kb HindIII, 3.8 kb BamHI and 0.87 kb Pstl fragments. These 4 fragments overlapped only in a 0.4 kb HindIII-PstI region. In the transformants obtained with total genomic DNA or a plasmid containing the 3.8 kb BamHI fragment, the ability to fix intracellular inorganic carbon was restored. Southern hybridization and partial nucleotide sequence analysis indicated that the cloned genomic region was located approximately 20 kb downstream from the structural genes for subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase. The cloned region was transcribed into a 0.5 kb mRNA. These results indicate that the cloned genomic region of Synechococcus sp. PCC7942 is involved in the efficient utilization of intracellular inorganic carbon for photosynthesis.     

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.     

Neuroblastoma specific effects of DR-nm23 and its mutant forms on differentiation and apoptosis

DR-nm23 belongs to a gene family which includes nm23-H1, originally identified as a candidate metastasis suppressor gene. Nm23 genes are expressed in different tumor types where their levels have been alternatively associated with reduced or increased metastatic potential. Nm23-H1, -H2, DR-nm23 and nm23-H4 all possess NDP kinase activity. Overexpression of DR-nm23 inhibits differentiation and promotes apoptosis in hematopoietic cells. By contrast, it induces morphological and biochemical changes associated with neural differentiation in neuroblastoma cells. In this study, we show that mutations in the catalytic domain and in the serine 61 phosphorylation site, possibly required for protein-protein interactions, impair the ability of DR-nm23 to induce neural differentiation. Moreover, neuroblastoma cells overexpressing wild-type or mutant DR-nm23 are less sensitive to apoptosis triggered by serum withdrawal. By subcellular fractionation, wild-type and mutant DR-nm23 localize in the cytoplasm and prevalently in the mitochondrial fraction. In co-immunoprecipitation experiments, wild-type DR-nm23 binds other members of nm23 family, but mutations in the catalytic and in the RGD domains and in serine 61 inhibit the formation of hetero-multimers. Thus, the integrity of the NDP kinase activity and the presence of a serine residue in position 61 seem essential for the ability of DR-nm23 to trigger differentiation and to bind other Nm23 proteins, but not for the anti-apoptotic effect in neuroblastoma cells. These studies underline the tissue specificity of the biological effects induced by DR-nm23 expression.     

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 toa nucleoside diphosphate, is ubiquitously found in organisms from bacteria to human. Overthe past 10 years, eight human genes of the nm23/NDP kinase family have been discoveredthat can be separated into two groups based on analysis of their sequences. In addition tocatalysis, which may not be exhibited by all isoforms, evidence for regulatory roles has comerecently from the discovery of the genes nm23 and awd, which encode NDP kinases and areinvolved in tumor metastasis and Drosophila development, respectively. Current work showsthat the human NDP kinase genes are differentially expressed in tissues and that their productsare targeted to different subcellular locations. This suggests that Nm23/NDP kinases possessdifferent, but specific, functions within the cell, depending on their localization. The roles ofNDP kinases in metabolic pathways and nucleic acid synthesis are discussed.     

YopH of Yersinia pseudotuberculosis interrupts early phosphotyrosine signalling associated with phagocytosis

The PTPase YopH of Yersinia is essential to the ability of these bacteria to block phagocytosis. Wild-type Yersinia pseudotuberculosis, but not the yopH mutant strain, resisted phagocytosis by J774 cells. Ingestion of a yopH mutant was dependent on tyrosine kinase activity. Transcomplementation with wild-type yopH restored the anti-phagocytic effect, whereas introduction of the gene encoding the catalytically inactive yopH_C403A was without effect. The PTPase inhibitor orthovanadate impaired the anti-phagocytic effect of the wild-type strain, further demonstrating the importance of bacteria-derived PTPase activity for this event. The ability to resist phagocytosis indicates that the effect of the bacterium is immediately exerted when it becomes associated with the phagocyte. Within 30 s after the onset of infection, wild-type Y. pseudotuberculosis caused a YopH-dependent dephosphorylation of phosphotyrosine proteins in J774 cells. Furthermore, interaction of the cells with phagocytosable strains led to a rapid and transient increase in tyrosine phosphorylation of paxillin and some other proteins, an event dependent on the presence of the bacterial surface-located protein invasin. Co-infection with the phagocytosable strain and the wild-type strain abolished the induction of tyrosine phosphorylation. Taken together, the present findings demonstrate an immediate YopH-mediated dephosphorylation of macrophage phosphotyrosine proteins, suggesting that this PTPase acts by preventing early phagocytosis-linked signalling in the phagocyte.     

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.     

Mitochondrial NDP kinase from Dunaliella tertiolecta (Chlorophyceae, Chlorophyta)

A cDNA (DtNDK1) encoding a nucleoside diphosphate (NDP) kinase with a putative mitochondrial targeting signal sequence was previously isolated from the halo‐tolerant green alga Dunaliella tertiolecta. When expressed in Saccharomyces cerevisiae, the processed DtNDK1 enzyme was specifically localized to mitochondria. The present study reports several biochemical characteristics of the mitochondrial NDP kinase from D. tertiolecta. The mature protein was expressed as either N‐ or C‐terminal hexahistidine‐tagged protein and purified to homogeneity by affinity chromato‐graphy. Native gel electrophoresis and sedimentation velocity in sucrose density gradients showed that the active enzyme consisted of a hexamer. The enzyme, with a pH optimum of 7, showed the highest specificity to dCDP (K_m= 50 μmol/L) and the highest turnover towards the synthesis of UTP (up to 140‐fold higher). The present study also provides evidence that purified DtNDK1 proteins are capable of transferring a phosphate group to another protein.     

Activation of G-proteins by receptor-stimulated nucleoside diphosphate kinase in Dictyostelium.

Recently, interest in the enzyme nucleoside diphosphate kinase (EC2.7.4.6) has increased as a result of its possible involvement in cell proliferation and development. Since NDP kinase is one of the major sources of GTP in cells, it has been suggested that the effects of an altered NDP kinase activity on cellular processes might be the result of altered transmembrane signal transduction via guanine nucleotide-binding proteins (G-proteins). In the cellular slime mould Dictyostelium discoideum, extracellular cAMP induces an increase of phospholipase C activity via a surface cAMP receptor and G-proteins. In this paper it is demonstrated that part of the cellular NDP kinase is associated with the membrane and stimulated by cell surface cAMP receptors. The GTP produced by the action of NDP kinase is capable of activating G-proteins as monitored by altered G-protein-receptor interaction and the activation of the effector enzyme phospholipase C. Furthermore, specific monoclonal antibodies inhibit the effect of NDP kinase on G-protein activation. These results suggest that receptor-stimulated NDP kinase contributes to the mediation of hormone action by producing GTP for the activation of GTP-binding proteins.     

Production of flavin mononucleotide by metabolically engineered yeast Candida famata

Recombinant strains of the flavinogenic yeast Candida famata able to overproduce flavin mononucleotide (FMN) that contain FMN1 gene encoding riboflavin (RF) kinase driven by the strong constitutive promoter TEF1 (translation elongation factor 1α) were constructed. Transformation of these strains with the additional plasmid containing the FMN1 gene under the TEF1 promoter resulted in the 200-fold increase in the riboflavin kinase activity and 100-fold increase in FMN production as compared to the wild-type strain (last feature was found only in iron-deficient medium).Overexpression of the FMN1 gene in the mutant that has deregulated riboflavin biosynthesis pathway and high level of riboflavin production in iron-sufficient medium led to the 30-fold increase in the riboflavin kinase activity and 400-fold increase in FMN production of the resulted transformants. The obtained C. famata recombinant strains can be used for the further construction of improved FMN overproducers.     

Molecular characterization of polyphosphate kinase (ppk) gene from Serratia marcescens

To understand the mechanism of phosphate accumulation, a gene encoding polyphosphate kinase (PPK) was cloned from the genomic library of Serratia marcescens by Southern hybridization. From the nucleotide sequence of a 4 kb DNA fragment, an open reading frame of 2063 nucleotides was identified encoding a protein of 686 amino acids with molecular mass of 70 kDa. The potential CRP binding site and pho box sequence were found upstream of the putative promoter in the regulatory region. The expression of PPK resulted in the formation of inclusion bodies and the product was active at low temperature. The E. coli strain harboring plasmid pSPK5 with ppk gene increased enzyme activity of polyphosphate kinase, resulting in increased accumulation of polyphosphate in E. coli.     

Overexpression of genes encoding tRNA<Superscript>Tyr</Superscript> and tRNA<Superscript>Gln</Superscript> increases the viability of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains with nonsense mutations in the <Emphasis Type="Italic">SUP45</Emphasis> gene

At present, the machinery supporting the viability of organisms possessing nonsense mutations in essential genes is not entirely understood. Nonsense mutants of Saccharomyces cerevisiae yeast containing a premature translation termination codon in the essential SUP45 gene are known. These strains are viable in the absence of mutant suppressor tRNAs; hence, the existence of alternative mechanisms providing nonsense suppression and mutant viability is conjectured. Analysis of clones obtained by transformation of a strain bearing a nonsense-mutant allele of SUP45 with a multicopy yeast genomic library revealed three genes encoding wild-type tRNA^Tyr and four genes encoding wild-type tRNA^Gln, which increased nonsense mutant viability. Moreover, overexpression of these genes leads to an increase in the amount of the full-length eRF1 protein in cells and compensates for heat sensitivity in the nonsense mutants. Probable ways of tRNA^Tyr and tRNA^Gln influence on the increase in the viability of strains with nonsense mutations in SUP45 are discussed.