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.     

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.     

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.     

The human nm23-H4 gene product is a mitochondrial nucleoside diphosphate kinase

We demonstrate here the catalytic activity and subcellular localization of the Nm23-H4 protein, product of nm23-H4, a new member of the human nm23/nucleoside diphosphate (NDP) kinase gene family (Milon, L., Rousseau-Merck, M., Munier, A., Erent, M., Lascu, I., Capeau, J., and Lacombe, M. L. (1997) Hum. Genet. 99, 550-557). Nm3-H4 was synthesized in escherichia coli as the full-length protein and as a truncated form missing the N-terminal extension characteristic of mitochondrial targeting. The truncated form possesses NDP kinase activity, whereas the full-length protein is inactive, suggesting that the extension prevents enzyme folding and/or activity. X-ray crystallographic analysis was performed on active truncated Nm23-H4. Like other eukaryotic NDP kinases, it is a hexamer. Nm23-H4 naturally possesses a serine residue at position 129, equivalent to the K-pn mutation of the Drosophila NDP kinase. The x-ray structure shows that the presence of Ser(129) has local structural effects that weaken subunit interactions. Site-directed mutagenesis shows that the serine is responsible for the lability of Nm23-H4 to heat and urea treatment, because the S129P mutant is greatly stabilized. Examination of human embryonic kidney 293 cells transfected with green fluorescent protein fusions by confocal microscopy shows a specific mitochondrial localization of Nm23-H4 that was also demonstrated by Western blot analysis of subcellular fractions of these cells. Import into mitochondria is accompanied by cleavage of the N-terminal extension that results in NDP kinase activity. Submitochondrial fractionation indicates that Nm23-H4 is associated with mitochondrial membranes, possibly to the contact sites between the outer and inner membranes.     

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.     

Nm23/NDP kinases in human male germ cells: role in spermiogenesis and sperm motility?

Nucleoside diphosphate (NDP) kinases, responsible for the synthesis of nucleoside triphosphates and produced by the nm23 genes, are involved in numerous regulatory processes associated with proliferation, development, and differentiation. Their possible role in providing the GTP/ATP required for sperm function is unknown. Testis biopsies and ejaculated sperm were examined by immunohistochemical and immunofluorescence microscopy using specific antibodies raised against Nm23-H5, specifically expressed in testis germinal cells and the ubiquitous NDP kinases A to D. Nm23-H5 was present in sperm extract, together with the ubiquitous A and B NDP kinases (but not the C and D isoforms) as shown by Western blotting. Nm23-H5 was located in the flagella of spermatids and spermatozoa, adjacent to the central pair and outer doublets of axonemal microtubules. High levels of NDP kinases A and B were observed at specific locations in postmeiotic germinal cells. NDP kinase A was transiently located in round spermatid nuclei and became asymmetrically distributed in the cytoplasm at the nuclear basal pole of elongating spermatids. The distribution of NDP kinase B was reminiscent of the microtubular structure of the manchette. In ejaculated spermatozoa, the proteins presented specific locations in the head and flagella. Nm23/NDP kinase isoforms may have specific functions in the phosphotransfer network involved in spermiogenesis and flagellar movement.     

New insights into Nm23 control of cell adhesion and migration

The molecular mechanisms underlying the role of Nm23/NDP kinase in controlling cell migration and metastasis have been investigated. The recent progress in our understanding of cell migration at a molecular level gives us some clues to the putative Nm23 function as a suppressor of metastasis. Screening of the literature indicates that NDP kinases have pleiotropic effects. By modifying cytoskeleton organization and protein trafficking, some NDP kinase isoforms may indirectly promote adhesion to the extracellular matrix in some cell types. Conversely, Nm23 regulates cell surface expression of integrin receptors and matrix metallo-proteases, and thus directly controls the cell adhesion machinery. Finally, the recent discovery of the interaction between Nm23-H2 and the negative regulator of 1 integrin-mediated cell adhesion, ICAP-1, which targets the kinase to lamellipodia and cell protrusions, suggests that the Nm23-H2/ICAP-1 complex plays a role in integrin signaling, and exerts a fine-tuning between migration and spreading.     

Extracellular Nm23H1 stimulates neurite outgrowth from dorsal root ganglia neurons in vitro independently of nerve growth factor supplementation or its nucleoside diphosphate kinase activity

The nucleoside diphosphate (NDP) kinase, Nm23H1, is a highly expressed during neuronal development, whilst induced over-expression in neuronal cells results in increased neurite outgrowth. Extracellular Nm23H1 affects the survival, proliferation and differentiation of non-neuronal cells. Therefore, this study has examined whether extracellular Nm23H1 regulates nerve growth. We have immobilised recombinant Nm23H1 proteins to defined locations of culture plates, which were then seeded with explants of embryonic chick dorsal root ganglia (DRG) or dissociated adult rat DRG neurons. The substratum-bound extracellular Nm23H1 was stimulatory for neurite outgrowth from chick DRG explants in a concentration-dependent manner. On high concentrations of Nm23H1, chick DRG neurite outgrowth was extensive and effectively limited to the location of the Nm23H1, i.e. neuronal growth cones turned away from adjacent collagen-coated substrata. Nm23H1-coated substrata also significantly enhanced rat DRG neuronal cell adhesion and neurite outgrowth in comparison to collagen-coated substrata. These effects were independent of NGF supplementation. Recombinant Nm23H1 (H118F), which does not possess NDP kinase activity, exhibited the same activity as the wild-type protein. Hence, a novel neuro-stimulatory activity for extracellular Nm23H1 has been identified in vitro, which may function in developing neuronal systems.     

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.     

Double knockout Nme1/Nme2 mouse model suggests a critical role for NDP kinases in erythroid development

Nm23/NDP kinases A and B encoded by the Nme1/Nme2 genes are multifunctional enzymes responsible for the majority of NDP kinase activity in mammals. This review summarizes recent studies on their physiological roles using a mouse model in which both Nme1 and Nme2 genes have been deleted. The double knockout mice are stunted in growth and die perinatally. Additionally, these mice display hematologic phenotypes, including severe anemia, abnormal erythroid cell development, loss of the iron transport receptor molecule TfR1, and reduced iron uptake by Nme1 ^?/? /Nme2 ^?/? erythroid cells. We hypothesize that Nm23/NDP kinases regulate TfR1 gene expression in erythroid cells in some manner, and that defective iron transport into these cells is responsible for the anemia and death. This Nme1/Nme2 mouse model also links nucleotide metabolism with erythropoiesis, suggesting alternative or additional mechanisms that may explain the observed phenomena.     

Nm23/Nucleoside Diphosphate Kinase in Human Cancers

Tumor metastasis is the leading cause of death in cancer patients. From a series of tumorcohort studies, low expression of Nm23/NDP kinase has been correlated with poor patientprognosis and survival, lymph node infiltration, and histopathological indicators of highmetastatic potential in a number of cancer types, including mammary and ovarian carcinomas andmelanoma. In other tumor types, no correlation has been established. Transfection ofNm23/NDP kinase cDNA into highly metastatic breast, melanoma, prostrate and squamous cellcarcinomas, and colon adenocarcinoma cells significantly reduced the metastatic competencyof the cells in vivo. In culture, cell motility, invasion, and colonization were inhibited, whereastumorigenicity and cellular proliferation were not affected, indicating that Nm23/NDP kinaseacts as a metastasis suppressor.     

Translational approaches using metastasis suppressor genes

Cancer metastasis is a significant contributor to breast cancer patient morbidity and mortality. In order to develop new anti-metastatic therapies, we need to understand the biological and biochemical mechanisms of metastasis. Toward these efforts, we and others have studied metastasis suppressor genes, which halt metastasis in vivo without affecting primary tumor growth. The first metastasis suppressor gene identified was nm23, also known as NDP kinase. Nm23 represents the most widely validated metastasis suppressor gene, based on transfection and knock-out mouse strategies. The biochemical mechanism of metastasis suppression via Nm23 is unknown and likely complex. Two potential mechanisms include binding proteins and a histidine kinase activity. Elevation of Nm23 expression in micrometastatic tumor cells may constitute a translational strategy for the limitation of metastatic colonization in high risk cancer patients. To date, medroxyprogesterone acetate (MPA) has been identified as a candidate compound for clinical testing.     

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.     

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.     

Structural and catalytic properties and homology modelling of the human nucleoside diphosphate kinase C, product of the DRnm23 gene.

The human DRnm23 gene was identified by differential screening of a cDNA library obtained from chronic myeloid leukaemia-blast crisis primary cells. The over-expression of this gene inhibits differentiation and induces the apoptosis of myeloid precursor cell lines. We overproduced in bacteria a truncated form of the encoded protein lacking the first 17 N-terminal amino acids. This truncated protein was called nucleoside diphosphate (NDP) kinase CDelta. NDP kinase CDelta had similar kinetic properties to the major human NDP kinases A and B, but was significantly more stable to denaturation by urea and heat. Analysis of denaturation by urea, using size exclusion chromatography, indicated unfolding without the dissociation of subunits, whereas renaturation occurred via a folded monomer. The stability of the protein depended primarily on subunit interactions. Homology modelling of the structure of NDP kinase CDelta, based on the crystal structure of NDP kinase B, indicated that NDP kinase CDelta had several additional stabilizing interactions. The overall structure of the two enzymes appears to be identical because NDP kinase CDelta readily formed mixed hexamers with NDP kinase A. It is possible that mixed hexamers can be observed in vivo.     

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.