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Nucleoside diphosphate kinases (Nm23/NDPK) are enzymes functional in cell proliferation, differentiation, development, tumor progression, and metastasis. Nevertheless, no consensus exists about the molecular mechanism by which Nm23/NDPK isoforms exert their role in these processes. We investigated the expression of the rat Nm23-R1/NDPKβ and Nm23-R2/NDPKα isoforms, homologues of the human Nm23-H1/NDPK A and Nm23-H2/NDPK B proteins, respectively, upon cAMP-induced differentiation of rat C6 glioma cells and demonstrated a differential interaction with intermediate filaments. Semiquantitative RT-PCR, immunoblotting, and flow cytometry showed a constitutive expression of both Nm23 isoforms. After induction of differentiation in C6 cells with cAMP analogs or isoproterenol, a dose-dependent 2- and 2.5-fold upregulation of the Nm23-R1 mRNA and protein, respectively, was observed. In contrast, the expression of Nm23-R2 remained unchanged. Localization of both isoforms with confocal laser scanning microscopy demonstrated a punctate reticular staining pattern for both Nm23 isoforms in the cytosol and processes of the cells which was particularly intense in the perinuclear region. In addition, while Nm23-R2 was colocalized and coimmunoprecipitated with vimentin in nondifferentiated cells, both isoforms were associated with GFAP in differentiated cells. The significance of these findings in relation to a possible function of Nm23 isoforms in cell proliferation, differentiation, and tumor-associated mechanisms is discussed.

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.

The X‐ray structure of a point mutant of nucleoside diphosphate kinase (NDP kinase) from Dictyostelium discoideum has been determined to 2.2 A resolution. The enzyme is a hexamer made of identical subunits with a novel mononucleotide binding fold. Each subunit contains an alpha/beta domain with a four stranded, antiparallel beta‐sheet. The topology is different from adenylate kinase, but identical to the allosteric domain of Escherichia coli ATCase regulatory subunits, which bind mononucleotides at an equivalent position. Dimer contacts between NDP kinase subunits within the hexamer are similar to those in ATCase. Trimer contacts involve a large loop of polypeptide chain that bears the site of the Pro‐‐‐‐Ser substitution in Killer of prune (K‐pn) mutants of the highly homologous Drosophila enzyme. Properties of Drosophila NDP kinase, the product of the awd developmental gene, and of the human enzyme, the product of the nm23 genes in tumorigenesis, are discussed in view of the three‐dimensional structure and of possible interactions of NDP kinase with other nucleotide binding proteins.

Overexpression of the nm23H1 gene has been associated with the suppression of metastasis in several solid tumors. However, in colorectal carcinoma and neuroblastoma, increased levels of nm23 H1 nucleoside diphosphate kinase A (NDPKA) mRNA are associated with tumorigenesis. To determine the role of nm23 H1/NDPKA in the prostate, normal and/or malignant tissue samples from 29 consecutive patients were studied. Levels of nm23 H1/NDPKA mRNA and nm23 H1/NDPKA mRNA protein were determined in tissue from 18 and 27 patients, respectively. In all, 16 of the 18 tumor samples expressed increased levels of nm23 H1/NDPKA mRNA as compared with those measured in normal tissue. The level of nm23 H1/NDPKA mRNA was >10-fold higher in a metastatic lymph node than in normal prostate tissue. All cancer specimens and areas of prostatic intraepithelial neoplasia showed immunoreactivity with the nm23 H1/NDPKA antibody; however, normal prostatic tissue was unreactive. These findings suggest that overexpression of the nm23 H1/NDPKA gene occurs frequently in adenocarcinomas of the prostate and may be an early event in prostate cancer tumorigenesis.

The X‐ray structure of a point mutant of nucleoside diphosphate kinase (NDP kinase) from Dictyostelium discoideum has been determined to 2.2 A resolution. The enzyme is a hexamer made of identical subunits with a novel mononucleotide binding fold. Each subunit contains an alpha/beta domain with a four stranded, antiparallel beta‐sheet. The topology is different from adenylate kinase, but identical to the allosteric domain of Escherichia coli ATCase regulatory subunits, which bind mononucleotides at an equivalent position. Dimer contacts between NDP kinase subunits within the hexamer are similar to those in ATCase. Trimer contacts involve a large loop of polypeptide chain that bears the site of the Pro‐‐‐‐Ser substitution in Killer of prune (K‐pn) mutants of the highly homologous Drosophila enzyme. Properties of Drosophila NDP kinase, the product of the awd developmental gene, and of the human enzyme, the product of the nm23 genes in tumorigenesis, are discussed in view of the three‐dimensional structure and of possible interactions of NDP kinase with other nucleotide binding proteins.

The point mutation serine 120 to glycine in the human nucleoside diphosphate kinase A has been identified in several aggressive neuroblastomas (Chang, C. L., Zhu, X. X., Thoraval, D. H., Ungar, D., Rawwas, J., Hora, N., Strahler, J. R., Hanash, S. M. & Radany, E. (1994) Nature 370, 335-336). We expressed in bacteria and purified wild-type and S120G mutant nucleoside diphosphate kinase A. The mutant enzyme had enzymatic and structural properties similar to the wild-type enzyme, whereas its stability to denaturation by heat and urea was markedly reduced. More importantly, upon renaturation of the urea-denatured mutant protein, a folding intermediate accumulated, having the characteristics of a molten globule. It had no tertiary structure, as shown by near UV circular dichroism, whereas the secondary structure was substantially recovered. The hydrophobic probe 8-anilino-1-naphthalene sulfonate bound to the intermediate species with an increase in fluorescence intensity and a blue shift. The hydrodynamic size was between that expected for a folded and an unfolded monomer. Finally, electrophoresis in a transverse urea gradient displayed no renaturation curve, and the protein showed the tendency to aggregate at the lowest urea concentrations. The existence of a molten globule folding intermediates resulting from an altered folding in the mutated protein might be related to the aggressiveness of neuroblastomas.

The X-ray structure of the nucleoside diphosphate kinase (NDP kinace) from Dictyostelium discoideumhas been refined at 1.8 Å resolution from a hexagonal crystal form with a 17 kDa monomer in its asymmetric unit. The atomic model was derived from the previously determined structure of a point mutant of the protein. It contains 150 amino acid residues out of 155, and 95 solvent molecules. The R-factor is 0.196 and the estimated accuracy of the average atomic position, 0.25 Å. The Dictyoteliumstructure is described in detail and compared to those of Drosophilaand Myxococcua xanthusNDP kinases. The protein is a hexamer with D3 symmetry. Residues 8 to 138 of each subunit form a globular α/βdomain. The four-stranded β-sheet is antiparallel; its topology is different from other phosphate transfer enzymes, and also from the HPr protein which, like NDP kinase, carries a phosphorylated histidine. The same topology is nevertheless found in several other proteins that bind mononucleotides, RNA or DNA. Strand connections in NDP kinase involve α-helices and a 20-residue segment called the Kpn loop. The β-sheet is regular except for a β-bulge in edge strand β2 and a γ-turn at residue Ile120 just preceding strand β4. The latter may induce strain in the main chain near the active site His122. The α1β2motif participates in forming dimers within the hexamer, helices α1, and α3, the Kpn loop and C terminus, in forming trimers. The subunit fold and dimer interactions found in Dictyosteliumare conserved in other NDP kinases. Trimer interactions probably occur in all eukaryotic enzymes. They are absent in the bacterial Myxococus xanthusenzyme which is a tetramer, even though the subunit structure is very similar. In Dictyostelium, contacts between Kpn loops near the 3-fold axis block access to a central cavity lined with polar residues and filled with well-defined solvent molecules. Biochemical data on point mutants highlight the contribution of the Kpn loop to protein stability. In Myxococcus, the Kpn loops are on the tetramer surface and their sequence is poorly conserved. Yet, their conformation is maintained and they make a similar contribution to the substrate binding site.

We have previously isolated cDNA clones for the gip17 gene encoding the cytosolic nucleoside diphosphate (NDP) kinase from Dictyostelium discoideum, and partial cDNAs for guk, a second member of the NDP kinase gene family (Wallet, V., Mutzel, R., Troll, H., Barzu, O., Wurster, B., Véron, M., and Lacombe, M. L. (1990) J. Natl. Cancer Inst. 80, 1199-1202). We now characterize genomic DNA clones for both NDP kinase genes, and we show that guk defines a nuclear-encoded mitochondrial NDP kinase. Isolated D. discoideum mitochondria contain 3% of the total cellular NDP kinase activity. Antibodies which specifically recognize and inhibit the activity of either cytosolic or mitochondrial NDP kinase unambiguously distinguish between these activities. The nascent mitochondrial NDP kinase contains a presequence of 57 amino acids that is removed during import into the organelle as shown by determination of the NH2 terminus of the mature protein from mitochondria. The genes for mitochondrial and cytosolic NDP kinases contain four and two introns, respectively. The positions of the of the introns in the gene for the cytosolic enzyme match exactly the positions of the second and fourth introns in the coding region of its mitochondrial homologue. From these results we conclude that the isozymes diverged from a common ancestor, and we discuss possible phylogenetic pathways for the evolution of cytosolic and organelle NDP kinases.

Alanine dehydrogenase from Bacillus cereus, a non-allosteric enzyme composed of six identical subunits, was purified to homogeneity by chromatography on blue-Sepharose and Sepharose 6B-CL. Like other pyridine-linked dehydrogenases, alanine dehydrogenase is inhibited by Cibacron blue, competitively with respect to NADH and noncompetitively with respect to pyruvate. The enzyme was inactivated by 0.1 M glycine/HCl (pH 2) and reactivated by 0.1 M phosphate (pH 8) supplemented with NAD+ or NADH. The reactivation was characterized by sigmoidal kinetics indicating a complex mechanism involving rate-limiting folding and association steps. Cibacron blue interfered with renaturation, presumably by competition with NADH. Chromatography on Sepharose 6B-CL of the partially renatured alanine dehydrogenase led to the separation of several intermediates, but only the hexamer was characterized by enzymatic activity. By immobilization on Sepharose 4B, alanine dehydrogenase from B. cereus retained 66% of the specific activity of the soluble enzyme. After denaturation of immobilized alanine dehydrogenase with 7 M urea, 37% of the initial protein was still bound to Sepharose, indicating that on the average the hexamer was attached to the matrix via, at most, two subunits. The ability of the denatured, immobilized subunits to pick up subunits from solution shows their capacity to fold back to the native conformation after urea treatment. The formation of “hybrids” between subunits of enzyme from B. cereus and Bacillus subtilis demonstrates the close resemblance of the tertiary and quaternary structures of alanine dehydrogenases from these species.

Nucleotide analogs are widely used in antiviral therapy and particularly against AIDS. Delivered to the cell as nucleosides, they are phosphorylated into their active triphospho derivative form by cellular kinases from the host. The last step in this series of phosphorylations is performed by nucleoside diphosphate (NDP) kinase, an enzyme that can use both purine or pyrimidine and oxy- or deoxynucleotides as substrates. Using pure recombinant human NDP kinase type B (product of the gene nm23-H2), we have characterized the kinetic parameters of several nucleotide analogs for this enzyme. Contrary to what is generally assumed, diphospho- and triphospho- derivatives of azidothymidine as well as of dideoxyadenosine and dideoxythymidine are very poor substrates for NDP kinase. The rate of phosphorylation of these analogs varies between 0.05% and 0.5%, as compared to the corresponding natural nucleotide, a result that is not due to the inability of the analogs to bind to the enzyme. Using the data from the high resolution crystal structure of NDP kinase, we provide an interpretation of these results based on the crucial role played by the 3'-OH moiety of the nucleotide in catalysis.

The eukaryotic nucleoside diphosphate (NDP) kinases are hexamers, while the bacterial NDP kinases are tetramers made of small, single domain subunits. These enzymes represent an ideal model for studying the effect of subunit interaction on protein stability. The thermostability of NDP kinases of each class was studied by differential scanning calorimetry and biochemical methods. The hexameric NDP kinase from Dictyostelium discoideum displays one single, irreversible differential scanning calorimetry peak (Tm 62 degrees C) over a broad protein concentration, indicating a single step denaturation. The thermal stability of the protein was increased by ADP. The P105G substitution, which affects a loop implicated in subunit contacts, yields a protein that reversibly dissociates to folded monomers at 38 degrees C before the irreversible denaturation occurs (Tm 47 degrees C). ADP delays the dissociation, but does not change the Tm. These data indicate a "coupling" of the quaternary structure with the tertiary structure in the wild-type, but not in the mutated protein. We describe the x-ray structure of the P105G mutant at 2.2-A resolution. It is very similar to that of the wild-type protein. Therefore, a minimal change in the structure leads to a dramatic change of protein thermostability. The NDP kinase from Escherichia coli behaves like the P105G mutant of the Dictyostelium NDP kinase. The detailed study of their thermostability is important, since biological effects of thermolabile NDP kinases have been described in several organisms.

We have investigated phosphorylation of human nucleoside diphosphate kinase (NDPK) and of homologous NDPK from different species by human casein kinase 2 (CK-2). The human NDPK isotypes A and B were phosphorylated by CK-2 in vitro both when the purified proteins and total lysate of HL-60 leukemia cells were used. The homologous NDPK′s from Yeast and E. coli were also substrates for CK-2 in vitro, but not Drosophila NDPK. Phosphorylation of all NDPK types by the CK-2 holoenzyme was entirely polyamine-dependent. The CK-2 phosphorylation site in human NDPK A, that was about 2.5 times stronger phosphorylated than was the B isotype, was tentatively assigned to Ser-122. The location of the corresponding residue in the 3D-structure of the 80% homologous Drosophila NDPK suggests that its phosphorylation may directly influence substrate binding and/or catalysis.

NM23 is a putative metastasis-suppressor gene for some human cancers. Here we have studied NM23 expression during melanoma progression using Northern blotting and immunocytochemistry. There was no significant difference in the average amounts of NM23 mRNA between cell lines derived from metastatic and primary melanomas. The level of NM23 mRNA was also determined for three pairs of poorly metastatic parental (P) and their highly metastatic variant (M) cell lines; the ratios for M/P were 1.2, 0.98 and 0.80. Next we used immunocytochemistry to study NM23 protein in normal skin, benign naevi and primary and metastatic melanomas. Melanocytes in all normal skin and benign samples were positive for NM23; however most primary melanomas (7/11) were not stained by the antibody. All metastatic melanoma samples (5/5) were positively stained. Findings were similar with an antiserum reactive with both forms of NM23 (H1 and H2), and with an antibody specific for NM23-H1. No relationship was apparent between NM23 immunoreactivity in primary tumours and their aggressiveness or prognosis. Hence, in contrast to the situation described for murine melanoma, the amount of NM23 mRNA or protein in human melanoma did not correlate inversely with metastasis.

A sensitive assay for nucleoside diphosphate kinase which utilizes microtiter plates is described. ATP, formed in the reaction between dGTP and ADP, is trapped by the glycerol kinase reaction. A colored compound is generated by glycerol-3-phosphate oxidase and peroxidase. This assay is useful for testing a large number of samples generated by chromatographic techniques or for screening purposes.Copyright 1993, 1999 Academic Press