Regulation of dynamin by nucleoside diphosphate kinase

Nucleoside diphosphate (NDP) kinase is required for multiple cellular functions, including cell growth, motility, and differentiation, and its loss is associated with pathologies including tumor metastasis. A recent study has revealed a previously unknown function for NDP kinase as positive regulator of dynamin, a GTPase essential for endocytosis. In this review we describe the evidence that NDP kinase function is essential for endocytosis and also elaborate on a mechanism for NDP kinase regulation of dynamin. Recently documented interactions between endocytosis and cell signaling have revealed new insights into potential mechanisms of cancer. In this context, we discuss the possible relevance of NDP kinase and dynamin interaction for tumor suppression.     

Phosphorylation of anti-HIV nucleoside analogs by nucleoside diphosphate kinase.

The reaction of NDP kinase with antiviral nucleoside triphosphates used in antiviral therapies was studied at the presteady state by fluorescence stopped-flow and compared with the steady-state parameters. The affinity of the analogs was determined by fluorescence titration of a mutated enzyme with an inserted Trp in the binding site. The lack of the 3' hydroxyl in analogs is shown to decrease the kcat more than the KD.     

The microtubule-associated nucleoside diphosphate kinase

Microtubule protein prepared by cycles of assembly-disassembly contains a nucleoside diphosphate kinase (NDP kinase) activity. We have isolated the NDP kinase responsible for this activity from twice-polymerized bovine brain microtubule protein by a five-step chromatographic procedure. The molecular weight of this enzyme was 103,000 +/- 7,000 daltons as determined by sedimentation equilibrium experiments performed with a Beckman Airfuge. A doublet of subunit bands with molecular masses of about 18,000 daltons was detected by silver staining after gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this preparation. We conclude that the enzyme is a hexamer, although we cannot identify the mix of subunits. We were able to isolate only nanogram quantities of this enzyme, too little for extensive studies, so we isolated the enzyme directly from bovine brain without a preliminary microtubule protein isolation. The whole-brain NDP kinase was isolated by the same chromatographic steps as the enzyme from microtubule protein preparations. Both enzymes had a doublet of subunits at the same molecular weights and both were the same isozyme, chromatofocusing at a pH of 8.0. Both enzymes had similar kinetic properties and similar thermal inactivation profiles. These similar properties of the two enzymes suggest that they are identical. Both subunits of NDP kinase could be reversibly phosphorylated by ATP. Phosphorylation of the native enzyme created multiple, more acidic forms that retained activity. The isolation of this NDP kinase, which can copurify with microtubule protein through cycles of assembly-disassembly, will facilitate future studies on the role of this enzyme in the mechanism and regulation of microtubule assembly.     

X-ray structure of nucleoside diphosphate kinase.

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.     

Post-translational processing of Drosophila nucleoside diphosphate kinase

Nucleoside diphosphate kinase (NDPK) was purified from Drosophila melanogaster by a combination of anion-exchange, hydroxyapatite, and reversed-phase chromatography. The identity of the purified enzyme was confirmed by sequencing internal peptides (the N-terminus appeared to be blocked). Post-translational modifications were investigated by using protein chemical and mass spectrometric methods. Analysis by nanoelectrospray ionization-mass spectrometry revealed that the mass of the enzyme was considerably smaller than that predicted from its amino acid sequence. Although its open-reading frame predicts a 153-residue polypeptide, the mature enzyme was found to comprise 152 amino acids, being modified by proteolytic removal of the initiator Met and N-acetylation of Ala2. This explains why the observed pI of the Drosophila enzyme is more acidic than that predicted from its amino acid sequence. No additional post-translational modifications such as glycosylation or O-phosphorylation, which have been identified on homologous NDPKs from other organisms, were detected on the Drosophila enzyme.     

Conversion of GDP into GTP by nucleoside diphosphate kinase on the GTP-binding proteins

A direct interaction of alpha beta gamma trimeric GTP binding proteins (G proteins; G0 and Gs) with nucleoside diphosphate kinase (NDP kinase) was investigated with homogeneously purified proteins. There was a progressive release of 32Pi from [gamma-32P]ATP when GDP-bound G0 was incubated together with NDP kinase. The Pi release induced by the interaction of G0 with NDP kinase was not accompanied by the dissociation of GDP bound to the alpha-subunit of G0. This was a sharp contrast to G protein-catalyzed GTP hydrolysis observed with GTP as the substrate; the dissociation of bound GDP was essentially required for the following binding of the substrate, GTP, to be hydrolyzed. A kinetic analysis displayed different properties for the substrate of NDP kinase between free GDP and G protein-bound GDP. NDP kinase-dependent phosphorylation of GDP on G0 was indeed demonstrated with adenosine 5'-(3-O-thio)triphosphate as the phosphate donor; there was a formation of guanosine 5'-(3-O-thio)triphosphate-bound G0 from the ATP analogue. Moreover, purified Gs was readily ADP-ribosylated by cholera toxin in the presence of NDP kinase, ATP, and an ADP-ribosylation factor, also suggesting that the nucleotide form on Gs was certainly GTP. These results indicate that NDP kinase can transfer the gamma-phosphate of ATP directly to GDP bound to G proteins and that this phosphorylation results in the activation of the signal-coupling proteins. A possible role of the new activation mechanism of G proteins is discussed in comparison with the previously characterized GDP-GTP exchange pathway by the agonist-receptor complex.     

Biochemical characteristics of bovine retina nucleoside diphosphate kinase

Nucleoside diphosphate kinase (NDP kinase; ATP: NDP phosphotransferase; EC 2.7.4.6) was purified from bovine retina. The molecular mass of the native enzyme was found to be 72 kDa, and those of its subunits were 17.5 and 18.5 kDa. Kinetic characteristics of the enzyme were determined. It was shown that NDP kinase exists in retina in both soluble and membrane-bound forms.     

Binding of nucleotides to nucleoside diphosphate kinase monitored by rose Bengal

The binding of nucleotides to nucleoside-diphosphate kinase from pig heart was studied using the dye rose Bengal as an optical probe. By difference absorption spectroscopy and by equilibrium dialysis it was shown that one dye molecule strongly bound per enzyme subunit. By competition with nucleotides it was shown that two nucleotide-binding sites exist on each subunit of either unphosphorylated or phosphorylated enzyme: one of them binds ATP or ADP tightly, the other one binds rose Bengal tightly and ADP loosely. As detected by different affinities for rose Bengal the enzyme exists in two conformations: a 'relaxed' conformation induced by the binding of ADP, and a 'tense' conformation induced by the binding of ATP or by phosphorylation.     

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.     

Molecular interactions involving Escherichia coli nucleoside diphosphate kinase

Nucleoside diphosphate kinase plays a distinctive metabolic role as the enzyme poised between the last reaction of deoxyribonucleoside triphosphate (dNTP) biosynthesis and the DNA polymerization apparatus. In bacteriophage T4 infection, NDP kinase is one of very few enzymes of host cell origin to participate in either dNTP synthesis or DNA replication. Yet NDP kinase forms specific contacts with phage-coded proteins of dNTP and DNA synthesis. This article summarizes work from our laboratory that identifies and characterizes these interactions. Despite these specific interactions, the enzyme appears to be dispensable, both for T4 replication and for growth of the host, Escherichia coli, because site-specific disruption of ndk, the structural gene for NDP kinase, does not interfere with growth of the host cell and only partly inhibits phage replication. However, ndk disruption unbalances the dNTP pools and stimulates mutagenesis. We discuss our attempts to understand the basis for this enhanced mutagenesis.     

Crystallization and preliminary X-ray diffraction studies of nucleoside diphosphate kinase from Dictyostelium discoideum.

Nucleoside diphosphate kinase from the slime mold Dictyostelium discoideum is highly homologous to gene products that are involved in development in Drosophila and in oncogenesis in human cells. The cloned protein expressed in Escherichia coli has been purified and crystallized in a hexagonal space group with a = b = 74.9 A, c = 211.4 A. The asymmetric unit contains either one or two 17,000 Mr subunits of the hexamer.     

Response of nucleoside diphosphate kinase to the adenylate energy charge

The reaction catalyzed by nucleoside diphosphate kinase responds to the energy charge of the adenylate pool. The velocity is maximal at a charge of 1.0, and decreases sharply with a decrease in the charge. This response may control the flow of phosphate from ATP into the other nucleotide pools and thus participate in the regulation of macromolecular synthesis by the energy level of the cell, as reflected in the charge of the adenylate pool.     

Nucleophilic activation by positioning in phosphoryl transfer catalyzed by nucleoside diphosphate kinase

The nonenzymatic reaction of ATP with a nucleophile to generate ADP and a phosphorylated product proceeds via a dissociative transition state with little bond formation to the nucleophile. Consideration of the dissociative nature of the nonenzymatic transition state leads to the following question: To what extent can the nucleophile be activated in enzymatic phosphoryl transfer? We have addressed this question for the NDP kinase reaction. A mutant form of the enzyme lacking the nucleophilic histidine (H122G) can be chemically rescued for ATP attack by imidazole or other exogenous small nucleophiles. The ATP reaction is 50-fold faster with the wild-type enzyme, which has an imidazole nucleophile positioned for reaction by a covalent bond, than with H122G, which employs a noncovalently bound imidazole nucleophile [(kcat/KM)ATP]. Further, a 4-fold advantage for imidazole positioned in the nucleophile binding pocket created by the mutation is suggested from comparison of the reaction of H122G and ATP with an imidazole versus a water nucleophile, after correction for the intrinsic reactivities of imidazole and water toward ATP in solution. X-ray structural analysis shows no detectable rearrangement of the residues surrounding His 122 upon mutation to Gly 122. The overall rate effect of approximately 10(2)-fold for the covalent imidazole nucleophile relative to water is therefore attributed to positioning of the nucleophile with respect to the reactive phosphoryl group. This is underscored by the more deleterious effect of replacing ATP with AlphaTauPgammaS in the wild-type reaction than in the imidazole-rescued mutant reaction, as follows. For the wild-type, AlphaTauPgammaS presumably disrupts positioning between nucleophile and substrate, resulting in a large thio effect of 300-fold, whereas precise alignment is already disrupted in the mutant because there is no covalent bond to the nucleophile, resulting in a smaller thio effect of 10-fold. In summary, the results suggest a catalytic role for activation of the nucleophile by positioning in phosphoryl transfer catalyzed by NDP kinase.     

Reduction of salt-requirement of halophilic nucleoside diphosphate kinase by engineering S-S bond

Nucleoside diphosphate kinase (HsNDK) from extremely halophilic haloarchaeon, Halobacterium salinarum, requires salt at high concentrations for folding. A D148C mutant, in which Asp148 was replaced with Cys, was designed to enhance stability and folding in low salt solution by S-S bond. It showed increased thermal stability by about 10 °C in 0.2 M NaCl over the wild type HsNDK. It refolded from heat-denaturation even in 0.1 M NaCl, while the wild type required 2 M NaCl to achieve the same level of activity recovery. This enhanced refolding is due to the three S-S bonds between two basic dimeric units in the hexameric HsNDK structure, indicating that assembly of the dimeric unit may be the rate-limiting step in low salt solution. Circular dichroism and native-PAGE analysis showed that heat-denatured HsNDK formed partially folded dimeric structure, upon refolding, in the absence of salt and the native-like secondary structure in the presence of salt above 0.1 M NaCl. However, it remained dimeric upon prolonged incubation at this salt concentration. In contrary, heat-denatured D148C mutant refolded into tetrameric folding intermediate in the absence of salt and native-like structure above 0.1 M salt. This native-like structure was then converted to the native hexamer with time.     

Autophosphorylation of nucleoside diphosphate kinase from Myxococcus xanthus.

The nucleoside diphosphate kinase (NDP kinase) from Myxococcus xanthus has been purified to homogeneity and crystallized (J. Munoz-Dorado, M. Inouye, and S. Inouye, J. Biol. Chem. 265:2702-2706, 1990). In the presence of ATP, the NDP kinase was autophosphorylated. Phosphoamino acid analysis was carried out after acid and base hydrolyses of phosphorylated NDP kinase. It was found that the protein was phosphorylated not only at a histidine residue but also at a serine residue. Replacement of histidine 117 with a glutamine residue completely abolished the autophosphorylation and nucleotide-binding activity of the NDP kinase. Since histidine 117 is the only histidine residue that is conserved in all known NDP kinases so far characterized, the results suggest that the phosphohistidine intermediate is formed at this residue during the transphosphorylation reaction from nucleoside triphosphates to nucleoside diphosphates. Preliminary mutational analysis of putative ATP-binding sites is also presented.     

Autophosphorylation of Solanum chacoense cytosolic nucleoside diphosphate kinase on Ser117

NDPK catalyses the interconversion of NTPs and NDPs using a phosphohistidine intermediate as part of its catalytic site. Recombinant Solanum chacoense cytosolic NDPK incubated with [gamma-(32)P]ATP was allowed to autophosphorylate and (32)P-labelled P-Ser was identified in an acid hydrolysate of the protein by two-dimensional TLC. Further analysis of (32)P-labelled recombinant NDPK by tryptic digestion followed by automated Edman sequencing of the radioactive peptide allowed the identification of a single and conserved P-Ser residue at position 117. Analysis of site-directed mutants where Ser117 was substituted to Asp indicated that the presence of a negative charge at position 117 dramatically lowered the enzyme's catalytic efficiency. Ser autophosphorylation was markedly reduced with increasing ADP concentrations in the autophosphorylation assay. These findings provide evidence that autophosphorylation of cytosolic NDPK on Ser117 could constitute a regulatory mechanism for this important enzyme and that autophosphorylation of Ser117 is modulated by NDP availability.