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.     

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.     

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.     

Nm23/NDP kinases in hepatocellular carcinoma

One of the most aggressive cancers is hepatocellular carcinoma, which is associated with a very poor patient outcome due to a high recurrence rate and metastatic spread. NM23, the first metastasis suppressor gene to be identified, has been widely studied in human cancers. However, conflicting results have been obtained depending on the tumor type and the evaluation protocol. The current knowledge of NM23 as a diagnostic and/or prognostic marker in hepatocellular carcinoma is reviewed herein. Most studies demonstrate an inverse association between the expression of NM23-H1 and the metastatic potential, which is not observed with the closely related NM23-H2 isoform. Transfection of metastatic hepatoma cells with NM23 reduced their metastatic potential, as for other tumor cell lines. The demonstration of a causative role of NM23 in metastatic dissemination in a mouse model of hepatocarcinoma suggests that hepatocarcinoma-derived cells could be good models for the analysis of the molecular mechanisms involved in NM23 action.     

Nm23-M5 mediates round and elongated spermatid survival by regulating GPX-5 levels

Nucleoside diphosphate (NDP) kinases are involved in numerous regulatory processes associated with proliferation, development, and differentiation. Previously, we cloned a new member of the NDPK family from mouse, Nm23-M5, which encodes a 211-amino acid protein and has 86% identity to the human Nm23-H5 [Hwang, K.C., Ok, D.W., Hong, J.C., Kim, M.O. and Kim, J.H. (2003) Cloning, sequencing, and characterization of the murine Nm23-M5 gene during mouse spermatogenesis and spermiogenesis. Biochem. Biophys. Res. Commun. 306, 198-207]. To better understand Nm23-M5 function, we generated transgenic mice with reduced Nm23-M5 levels in vivo using a short hairpin RNA (shRNA) knock-down system. Nm23-M5 expression was markedly reduced, as indicated by Northern and Western blot analysis. Nm23-M5 shRNA transgenic mice exhibited reduced numbers of haploid cells. Furthermore, the antioxidant enzyme glutathione peroxidase 5 (GPX-5) is regulated by Nm23-M5 at the level of both expression and activity. These results reveal that expression of Nm23-M5 plays a critical role in spermiogenesis by increasing the cellular levels of GPX-5 to eliminate reactive oxygen species.     

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.     

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.     

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.     

NM23-NDP kinase

NM23 belongs to a large family of structurally and functionally conserved proteins consisting of 4–6 identically folded subunits of approximately 16–20 kDa. These oligomeric proteins exhibit nucleoside diphosphate kinase (NDPK) activity that catalyzes nonsubstrate specific conversions of nucleoside diphosphates to nucleoside triphosphates. Many NM23 proteins bind DNA. In vivo, NM23–NDPKs regulate a diverse array of cellular events including growth and development. They are also implicated in the pathogenesis and metastasis of tumors. The mechanism whereby NM23 regulates gene expression is proposed to entail DNA-binding and subsequent alterations in promoter DNA structure. Accordingly, NM23 has the potential to become a useful reagent for gene manipulations.     

Identification of the tumor metastasis suppressor Nm23-H1/Nm23-R1 as a constituent of the centrosome

Processes like cell proliferation, differentiation, and tumor metastasis require a flexible adaptation of cell shape and cell plasticity. A regulator of cell structure and shape is the centrosome and its associated microtubules. Recently, oncogenes like p53, pRB, and the tumor suppressor BRCA1 have been characterized as members of the centrosome. In this communication, we identified rat Nm23-R1/NDPKbeta, a homologue of the human tumor metastasis suppressor Nm23-H1 and a regulator of cell proliferation and differentiation, as a component of the centrosomal complex. We used confocal laser scanning microscopy on different cell types and biochemical analysis of purified centrosomes to demonstrate that Nm23-R1 is located in the centrosome of dividing and nondividing cells. We also showed that the centrosomal enzyme is catalytically active and able to transfer the gamma-phosphate from a nucleoside triphosphate to a nucleoside diphosphate. In addition, Nm23-R1 coimmunoprecipitated with gamma-tubulin, a core centrosomal protein essential for microtubule nucleation. In addition, human Nm23-R1/-H1 was also shown to be present in the centrosome of different human and rat cell types, demonstrating that the presence of Nm23-H1 homologues in the latter organelle is a general event.     

地黄核苷二磷酸激酶基因的克隆及生物信息学分析

核苷二磷酸激酶(NDPK)是一种高度保守的多功能蛋白,具有催化底物磷酸化的作用,能够参与植物的生长发育、非生物胁迫、感病应激、光合作用和能量代谢等过程。为了解地黄核苷二磷酸激酶基因(RgNDPKⅠ)的结构、功能和性质,该研究利用地黄转录组学数据,通过电子克隆的方法获得了RgNDPKⅠ基因的全长cDNA序列,长度为765 bp。生物信息学分析结果表明,RgNDPKⅠ基因的开放阅读框长度为447 bp,编码148个氨基酸,具有典型的核苷二磷酸激酶活性结构域和其他磷酸化活性位点。RgNDPKⅠ基因编码的蛋白质定位于细胞质,是无跨膜区域的亲水性蛋白,该蛋白质与芝麻、紫花风铃的核苷二磷酸激酶相似性较高,分别为97%和96%。在多种生物中已经克隆得到了核苷二磷酸激酶基因,且不同植物核苷二磷酸激酶氨基酸序列中存在多个相似的保守结构域,推测RgNDPKⅠ基因所编码的蛋白质为核苷二磷酸激酶超家族成员。该研究结果为进一步探明RgNDPKⅠ的性质、结构、功能及表达机制提供了重要的理论依据。     

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.     

Localization and characterization of the mitochondrial isoform of the nucleoside diphosphate kinase in the pancreatic beta cell: evidence for its complexation with mitochondrial succinyl-CoA synthetase

Nucleoside diphosphate kinase (NDPK) catalyzes the transfer of terminal phosphates from nucleoside triphosphates to nucleoside diphosphates to yield nucleotide triphosphates. The present study was undertaken to localize and characterize the mitochondrial isoform of NDPK (mNDPK) in the pancreatic beta cell since it could contribute to the generation of mitochondrial nucleotide triphosphates and, thereby, to the mitochondrial high-energy phosphate metabolism of the pancreatic beta cell. Mitochondrial fractions from the insulin-secreting beta cells were isolated by differential centrifugation. mNDPK activity was assayed as the amount of [(3)H]GTPgammaS formed from ATPgammaS and [(3)H]GDP. Incubation of isolated mitochondrial extracts with either [gamma-(32)P]ATP or GTP resulted in the formation [(32)P]NDPK, which could be immunoprecipitated by an anti-NDPK serum. mNDPK exhibited saturation kinetics with respect to its nucleoside diphosphate acceptors and nucleoside triphosphate donors and sensitivity to known inhibitors of NDPK (e.g., uridine diphosphate and cromoglycate). By Western blot analyses, at least three isoforms of NDPK were identified in various subcellular fractions of the beta cell. The nm23-H1 (NDPK-A) was predominantly soluble whereas nm23-H2 (NDPK-B) was associated with the soluble as well as membranous fractions. The mitochondrial isoform of NDPK, nm23-H4, was uniformly distributed in the beta cell mitochondrial subfractions. A significant amount of NDPK (as determined by the catalytic activity and immunological methods) was recovered in the immunoprecipitates of mitochondrial fraction precipitated with an antiserum directed against succinyl-CoA synthetase (SCS), suggesting that NDPK might remain complexed with SCS. We provide the first evidence for the localization of a mitochondrial isoform of the NDPK in the islet beta cell and thus offer a potential mechanism for the generation of intramitochondrial GTP which, unlike ATP, is not transported into mitochondria via the classical nucleotide translocase. Further work will be required to determine the importance of the NDPK/SCS complex to normal beta cell function in the secretion of insulin.     

The regulatory alpha subunit of phosphorylase kinase may directly participate in the binding of glycogen phosphorylase

The yeast two-hybrid screen has been used to identify potential regions of interaction of the largest regulatory subunit, , of phosphorylase kinase (PhK) with two fragments of its protein substrate, glycogen phosphorylase b (Phb). One fragment, corresponding to residues 17-484 (PhbN"), contained the regulatory domain of the protein, but in missing the first 16 residues was devoid of the sole phosphorylation site of Phb, Ser14; the second fragment corresponded to residues 485-843 (PhbC) and contained the catalytic domain of Phb. Truncation fragments of the subunit were screened for interactions against these two substrate fragments. PhbC was not found to interact with any constructs; however, PhbN" interacted with a region of (residues 864-1014) that is near the phosphorylatable region of that subunit. PhbN" was also screened for interactions against a variety of fragments of the catalytic subunit of PhK; however, no interactions were detected, even with fulllength . Our results support the idea that amino acid residues proximal to the convertible serine of Phb are important for its specific interaction with the catalytic subunit of PhK, but that regions distinct from the convertible serine residue of Phb and from the catalytic domain of PhK may also be involved in the interaction of these two proteins.     

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.     

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.