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.     

Role of AWD/Nucleoside Diphosphate Kinase in Drosophila Development

The abnormal wing discs gene of Drosophila encodes a soluble protein with nucleosidediphosphate kinase activity. This enzymic activity is necessary for the biological function ofthe abnormal wing discs gene product. Complete loss of function, i.e., null, mutations causelethality after the larval stage. Most larval organs in such null mutant larvae appear to benormal, but the imaginal discs are small and incapable of normal differentiation.Killer-of-prune is a neomorphic mutation in the abnormal wing discs gene. It causes dominant lethalityin larvae that lack prune gene activity. The Killer-of-prune mutant protein may have alteredsubstrate specificity. Null mutant larvae have a low level of nucleoside diphosphate kinaseactivity. This suggests that there may be additional Drosophila genes that encode proteinswith nucleoside dipthosphate kinase activity. Candidate genes have been found in theDrosophila genome.     

小鼠胚泡黏附时子宫内膜nm23-M1／NDPK A的表达

nm23家族除与肿瘤转移抑制有关,它还参与调节正常细胞的发育、增殖、分化及凋亡等过程。运用RT-PCR、Western blot和免疫组织化学技术,分析小鼠胚泡黏附时子宫内膜着床点和着床旁组织nm23-M1／NDPK A的表达,以未交配鼠作对照,为进一步阐明胚泡着床的机制提供有意义的实验依据。RT-pCR结果显示,小鼠胚泡黏附时子宫内膜nm23-M1／NDPK A mRNA表达明显高于对照组,并且着床点明显高于着床旁,Western blot和免疫组织化学分析nm23-M1／NDPK A蛋白表达,也得到一致的结果。提示nm23-M1／NDPK A参与胚泡着床这一重要生命活动过程。     

Phorbol 12-myristate 13-acetate enhances nm23 gene expression in murine melanocytes but not in syngeneic B16-Bl6 melanoma variants

The nm23 gene has been described as a potential metastasis suppressor gene in certain rodent and human tumors. We previously demonstrated that tyrosine and phenylalanine restriction suppresses metastatic heterogeneity of B16-BL6 murine melanoma and selects for tumor variants with decreased metastatic potential. In this study, we investigated nm23 expression in the highly metastatic B16-BL6 (ND) melanoma, its nutritionally derived poorly metastatic (LT) variant, and the syngeneic non-tumorigenic Mel-ab melanocytes. No differences in nm23 expression were observed between ND and LT cells, and nm23 expression varied between different isolates. Previously, we showed that metastatic potential of 1-ND cells decreases and is not altered in 1-LT cells after prolonged in vitro cell passage; however, nm23 expression is equivalently increased by 2-fold. In 2-ND and 2-LT cells, expression of nm23 is not different at higher in vitro cell passage. Expression of nm23 decreased about 2-fold when phorbol 12-myristate 13-acetate (PMA) was removed from Mel-ab cells, which induces these cells to become quiescent. Although membrane-associated protein kinase C (PKC) activity decreased after prolonged PMA treatment in all cells, neither nm23 expression nor proliferation of ND and LT cells was affected by PMA. These data indicate that nm23 expression is related to proliferative activity rather than to the suppression of metastatic potential. © 1996 Wiley-Liss, Inc.     

Menin,the multiple endocrine neoplasia type 1 gene product,exhibits GTP-hydrolyzing activity in the presence of the tumor metastasis suppressor nm23

MEN1, the gene responsible for multiple endocrine neoplasia type 1, is a tumor suppressor gene that encodes a protein called menin, of unknown function with no homology to any known protein. Here we demonstrate that menin interacts with a putative tumor metastasis suppressor nm23H1/nucleoside diphosphate (NDP) kinase A in mammalian cells. Given the roles of nm23 as a multi-functional protein, we searched for the possible function of menin. Menin has no effect on the known activities of nm23; that is, nucleoside diphosphate kinase, protein kinase, or GTPase-activating protein for Ras-related GTPase Rad. However, we found that menin hydrolyzes GTP to GDP efficiently in the presence of nm23, whereas nm23 or menin alone shows little or no detectable GTPase activity. Furthermore, menin contains sequence motifs similar to those found in all known GTPases or GTP-binding proteins and shows low affinity but specific binding to GTP/GDP. These results suggest that menin is an atypical GTPase stimulated by nm23.     

A Drosophila gene that is homologous to a mammalian gene associated with tumor metastasis codes for a nucleoside diphosphate kinase

The product of the abnormal wing discs (awd) gene of Drosophila is 78% identical to the product of the nm23 gene of mammals, which is differentially expressed in certain metastatic tumors. We present evidence that the awd gene codes for a nucleoside diphosphate kinase (NDP kinase) and that this Awd/NDP kinase is microtubule associated. Neuroblasts in Drosophila larvae homozygous for a null mutation in the awd gene are arrested in metaphase, indicating that microtubule-associated Awd/NDP kinase plays a critical role in spindle microtubule polymerization.     

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.     

The Association of the Nm23-M1 Protein and β-Tubulin Correlates with Cell Differentiation

The Nm23 protein is a nucleoside diphosphate kinase (NDPK) and is thought to play a critical role in metastatic behavior. It has been reported that a NDPK activity is present in microtubules assembled in vitro. Since microtubule assembly is determinant in cell growth and differentiation, we investigated whether Nm23-M1 forms molecular complexes with β-tubulin in murine cells either actively proliferating or differentiating. For this purpose a polyclonal antibody against the GST-Nm23-M1 fusion protein was generated and employed to detect Nm23-M1/β-tubulin complexes in murine tumor cells derived from the Lewis lung carcinoma (3LL) and in undifferentiated and differentiated myogenic cells (C2C12). Immunoblotting and immunoprecipitation experiments performed using the anti-fusion protein antibody demonstrated that the Nm23-M1 protein is detectable in in vitro tumor cell lines and in in vivo primary tumors but not in spontaneous lung metastases. These data are in good agreement with data previously reported. Immunoprecipitation experiments demonstrated that the Nm23-M1 protein forms complexes with β-tubulin in in vitro tumor cell lines, but not in primary tumors. Furthermore, the Nm23-M1 protein forms complexes with β-tubulin in myogenic cells prior to and after differentiation. Interestingly, however, the level of the Nm23-M1/β-tubulin complexes is remarkably increased in differentiated myotubes. In conclusion, the results indicate that the Nm23-M1 protein forms molecular complexes with β-tubulin and that the number of complexes increases during the differentiation process of murine cells.     

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 metastasis suppressor Nm23 as a modulator of Ras/ERK signaling

NM23-H1 (also known as NME1) was the first identified metastasis suppressor, which displays a nucleoside diphosphate kinase (NDPK) and histidine protein kinase activity. NDPKs are linked to many processes, such as cell migration, proliferation, differentiation, but the exact mechanism whereby NM23-H1 inhibits the metastatic potential of cancer cells remains elusive. However, some recent data suggest that NM23-H1 may exert its anti-metastatic effect by blocking Ras/ERK signaling. In mammalian cell lines NDPK-mediated attenuation of Ras/ERK signaling occurs through phosphorylation (thus inactivation) of KSR (kinase suppressor of Ras) scaffolds. In this review I summarize our knowledge about KSR’s function and its regulation in mammals and in C. elegans. Genetic studies in the nematode contributed substantially to our understanding of the function and regulation of the Ras pathway (i.e. KSR’s discovery is also linked to the nematode). Components of the RTK/Ras/ERK pathway seem to be highly conserved between mammals and worms. NDK-1, the worm homolog of NM23-H1 affects Ras/MAPK signaling at the level of KSRs, and a functional interaction between NDK-1/NDPK and KSRs was first demonstrated in the worm in vivo. However, NDK-1 is a factor, which is necessary for proper MAPK activation, thus it activates rather than suppresses Ras/MAPK signaling in the worm. The contradiction between results in mammalian cell lines and in the worm regarding NDPKs’ effect exerted on the outcome of Ras signaling might be resolved, if we better understand the function, structure and regulation of KSR scaffolds.     

The yeast Cdc8 exhibits both deoxythymidine monophosphate and diphosphate kinase activities

The existence of multifunctional enzymes in the nucleotide biosynthesis pathways is believed to be one of the important mechanisms to facilitate the synthesis and the efficient supply of deoxyribonucleotides for DNA replication. Here, we used the bacterially expressed yeast thymidylate kinase (encoded by the CDC8 gene) to demonstrate that the purified Cdc8 protein possessed thymidylate-specific nucleoside diphosphate kinase activity in addition to thymidylate kinase activity. The yeast endogenous nucleoside diphosphate kinase is encoded by YNK1, which appears to be non-essential. Our results suggest that Cdc8 has dual enzyme activities and could duplicate the function of Ynk1 in thymidylate synthesis. We also discuss the importance of the coordinated expression of CDC8 during the cell cycle progression in yeast.     

Potential roles of 3′-5′exonuclease activity of NM23-H1 in DNA repair and malignant progression

NM23-H1 is a metastasis suppressor protein that exhibits 3′-5′ exonuclease activity in vitro. As 3′-5′ exonucleases are generally required for maintenance of genome integrity, this activity represents a plausible candidate mediator of the metastasis suppressor properties of the NM23-H1 molecule. Consistent with an antimutator function, ablation of the yeast NM23 homolog, YNK1, results in increased mutation rates following exposure to UV irradiation and exposure to the DNA damaging agents etoposide, cisplatin, and MMS. In human cells, a DNA repair function is further suggested by increased NM23-H1 expression and nuclear translocation following DNA damage. Also, forced expression of NM23-H1 in NM23-deficient and metastatic cell lines results in coordinate downregulation of multiple DNA repair genes, possibly reflecting genomic instability associated with the NM23-deficient state. To assess the relevance of the 3′-5′ exonuclease activity of NM23-H1 to its antimutator and metastasis suppressor functions, a panel of mutants harboring defects in the 3′-5′ exonuclease and other enzymatic activities of the molecule (NDPK, histidine kinase) have been expressed by stable transfection in the melanoma cell line, 1205Lu. Pilot in vivo metastasis assays indicate 1205Lu cells are highly responsive to the metastasis suppressor effects of NM23-H1, thus providing a valuable model for measuring the extent to which the nuclease function opposes metastasis and metastatic progression.     

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.     

Transient Expression of Drosophila melanogaster Deoxynucleoside Kinase Gene Enhances Cytotoxicity of Nucleoside Analogs

The Drosophila melanogaster deoxynucleoside kinase gene was introduced into HeLa cells with cationic lipids to allow its transient expression, and cytotoxic effects of several nucleoside analogs in the transfected cells were examined. Of the analogs tested, cytotoxicities of 1-β-D-arabinofuranosylcytosine (araC), 5-fluorodeoxyuridine (FUdR), and 1-(2-deoxy-2-methylene-β-D-erythro-pentofuranosyl)cytosine (DMDC) were increased by the deoxynucleoside kinase gene. These results suggest that the combination of the transient expression of the Drosophila deoxynucleoside kinase gene and these nucleoside analogs is a candidate for the suicide gene therapy.     

Overexpression of wild-type and mutant NDP kinase in Dictyostelium discoideum

Nucleoside diphosphate (NDP) kinase has a central role in the synthesis of (deoxy-)trinucleotides. In addition, mutations in the gene encoding NDP kinase have been shown to have important consequences for Drosophila development and mammalian tumorogenesis. We have overexpressed, in Dictyostelium discoideum, a genomic clone encoding the enzyme NDP kinase. The concomitant increase in the levels of RNA and enzyme activity identifies a 5′ non-coding genomic region of 0.9 kb as being the complete promoter region. Overexpression of wild-type NDP kinase has no effect on development. This is also true for an inactive mutant H122C that does not have a dominant inhibitor effect. Overexpression of the P105G mutant NDP kinase, which is known to be affected in its stability in vitro, only leads to a small increase in total NDP-kinase activity. Thermal and chemical denaturation experiments demonstrate the formation of hexameric hybrids between wild-type and mutant monomers.     

Lbc proto-oncogene product binds to and could be negatively regulated by metastasis suppressor nm23-H2

Lbc was identified as transforming gene from human leukemic cells and encodes Rho type guanine nucleotide exchange factor with 47kDa molecular weight. We isolated overlapping cDNAs of Lbc from human lung tissue. Full-length Lbc cDNA encodes 309kDa huge protein with Ht31 PKA anchoring motif, Dof domain, C1 domain, and coiled-coil structure. In order to analyze the regulatory mechanism of its activity, we searched for binding proteins. By yeast two-hybrid screening, we identified metastasis suppressor nm23-H2 as binding protein, which interacts with amino-terminal region of Lbc containing Dof domain. nm23 gene family encodes nucleoside diphosphate kinase, however, the binding of nm23-H2 to Lbc was independent of kinase activity. nm23-H1, which binds to Rac-specific GEF Tiam1, could not bind to Lbc suggesting nm23-H2 would be specific regulator for Lbc. Expression of nm23-H2 in cells leads to decrease the amount of GTP-bound Rho and suppress stress fiber formation stimulated by expression of Lbc. Our data suggest that metastasis suppressor nm23-H2 could regulate Lbc negatively by binding to amino-terminal region of Lbc proto-oncogene product.