Dynamin superfamily molecular motors use guanosine triphosphate (GTP) as a source of energy for membrane-remodeling events. We found that knockdown of nucleoside diphosphate kinases (NDPKs) NM23-H1/H2, which produce GTP through adenosine triphosphate (ATP)-driven conversion of guanosine diphosphate (GDP), inhibited dynamin-mediated endocytosis. NM23-H1/H2 localized at clathrin-coated pits and interacted with the proline-rich domain of dynamin. In vitro, NM23-H1/H2 were recruited to dynamin-induced tubules, stimulated GTP-loading on dynamin, and triggered fission in the presence of ATP and GDP. NM23-H4, a mitochondriaspecific NDPK, colocalized with mitochondrial dynamin-like OPA1 involved in mitochondria * Corresponding author. mathieu.boissan@inserm.fr (M.B.); philippe.chavrier@curie.fr (P.C. Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts inner membrane fusion and increased GTP-loading on OPA1. Like OPA1 loss of function, silencing of NM23-H4 but not NM23-H1/H2 resulted in mitochondrial fragmentation, reflecting fusion defects. Thus, NDPKs interact with and provide GTP to dynamins, allowing these motor proteins to work with high thermodynamic efficiency.The 100-kD dynamin guanosine triphosphatase (GTPase) promotes uptake of cell-surface receptors both by clathrin-dependent and -independent pathways (1, 2). Dynamin polymerizes into helix around the neck of endocytic pits and induces guanosine triphosphate (GTP) hydrolysis-driven membrane fission (3-7). Typical of molecular motors, dynamin has a low affinity for GTP and a high basal GTP-hydrolysis rate, which can be further stimulated by dynamin polymerization (8,9). This maximizes chemical energy gain and kinetics of hydrolysis, respectively, which in vivo depend on high concentration ratios of adenosine triphosphate/adenosine diphosphate (ATP/ADP) or GTP/guanosine diphosphate (GDP). The cellular concentrations of GTP and GDP are at least a factor of 10 lower than those of ATP and ADP, and GTP/GDP ratios could thus decrease much more rapidly at elevated workload, both of which make GTP not an ideal substrate for high-turnover, energy-dependent enzymes. Paradoxically, dynamin GTPases are among the most powerful molecular motors described (7).Studies in Drosophila identified a genetic interaction between dynamin and Awd (10-12). Awd belongs to the family of nucleoside diphosphate kinases (NDPKs), which catalyze synthesis of nucleoside triphosphates, including GTP, from corresponding nucleoside diphosphates and ATP (13). The most abundant human NDPKs are the highly related cytosolic proteins NM23-H1 and -H2. NM23-H4, another NDPK-family member, localizes exclusively at the mitochondrial inner membrane (14, 15). Mitochondrial membrane dynamics require dynamin-related GTPases (16). We hypothesized that NDPKs could influence the function of dynamin family members in membrane-remodeling events through spatially controlled GTP production and availability.Knockdown of NM23-H1 and -H2 (fig. S1, A to E) reduced clathrin-dependent endocyt...

Loss of NM23-H1 expression correlates with the degree of metastasis and with unfavorable clinical prognosis in several types of human carcinoma. However, the mechanistic basis for the metastasis suppressor function of NM23-H1 is obscure. We silenced NM23-H1 expression in human hepatoma and colon carcinoma cells and methodologically investigated effects on cell-cell adhesion, migration, invasion, and signaling linked to cancer progression. NM23-H1 silencing disrupted cell-cell adhesion mediated by E-cadherin, resulting in β-catenin nuclear translocation and T-cell factor/lymphoid-enhancing factor-1 transactivation. Further, NM23-H1 silencing promoted cellular scattering, motility, and extracellular matrix invasion by promoting invadopodia formation and upregulating several matrix metalloproteinases (MMP), including membrane type 1 MMP. In contrast, silencing the related NM23-H2 gene was ineffective at promoting invasion. NM23-H1 silencing activated proinvasive signaling pathways involving Rac1, mitogen-activated protein kinases, phosphatidylinositol 3-kinase (PI3K)/Akt, and src kinase. Conversely, NM23-H1 was dispensable for cancer cell proliferation in vitro and liver regeneration in NM23-M1 null mice, instead inducing cellular resistance to chemotherapeutic drugs in vitro. Analysis of NM23-H1 expression in clinical specimens revealed high expression in premalignant lesions (liver cirrhosis and colon adenoma) and the central body of primary liver or colon tumors, but downregulation at the invasive front of tumors. Our findings reveal that NM23-H1 is critical for control of cell-cell adhesion and cell migration at early stages of the invasive program in epithelial cancers, orchestrating a barrier against conversion of in situ carcinoma into invasive malignancy. Cancer Res; 70(19); 7710-22. ©2010 AACR.

Nucleoside diphosphate kinases (NDPK) are encoded by the NME genes, also called NM23. They catalyze the transfer of gamma-phosphate from nucleoside triphosphates to nucleoside diphosphates by a ping-pong mechanism involving the formation of a high energy phospho-histidine intermediate [1, 2]. Besides their known functions in the control of intracellular nucleotide homeostasis, they are involved in multiple physiological and pathological cellular processes such as differentiation, development, metastastic dissemination or cilia functions. Over the past 15 years, ten human genes have been discovered encoding partial, full length, and/or tandemly repeated Nm23/NDPK domains, with or without N-or C-terminal extensions and/or additional domains. These genes encode proteins exhibiting different functions at various tissular and subcellular localizations. Most of these genes appear late in evolution with the emergence of the vertebrate lineage. This review summarizes the present knowledge on these multitalented proteins.

Mitophagy is critical for cell homeostasis. Externalization of the inner mitochondrial membrane phospholipid, cardiolipin (CL), to the surface of the outer mitochondrial membrane (OMM) was identified as a mitophageal signal recognized by the microtubuleassociated protein 1 light chain 3. However, the CL-translocating machinery remains unknown. Here we demonstrate that a hexameric intermembrane space protein, NDPK-D (or NM23-H4), binds CL and facilitates its redistribution to the OMM. We found that mitophagy induced by a protonophoric uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP), caused externalization of CL to the surface of mitochondria in murine lung epithelial MLE-12 cells and human cervical adenocarcinoma HeLa cells. RNAi knockdown of endogenous NDPK-D decreased CCCP-induced CL externalization and mitochondrial degradation. A R90D NDPK-D mutant that does not bind CL was inactive in promoting mitophagy. Similarly, rotenone and 6-hydroxydopamine triggered mitophagy in SH-SY5Y cells was also suppressed by knocking down of NDPK-D. In situ proximity ligation assay (PLA) showed that mitophagy-inducing CL-transfer activity of NDPK-D is closely associated with the dynamin-like GTPase OPA1, implicating fission-fusion dynamics in mitophagy regulation.

Exogenous overexpression of the metastasis suppressor gene Nm23-H1 reduces the metastatic potential of multiple types of cancer cells and suppresses in vitro tumor cell motility and invasion. Mutational analysis of Nm23-H1 revealed that substitution mutants P96S and S120G did not inhibit motility and invasion. To elucidate the molecular mechanism of Nm23-H1 motility suppression, expression microarray analysis of an MDA-MB-435 cancer cell line overexpressing wild-type Nm23-H1 was done and cross-compared with expression profiles from lines expressing the P96S and S120G mutants. Nine genes, MET, PTN, SMO, FZD1, L1CAM, MMP2, NETO2, CTGF, and EDG2, were down-regulated by wild-type but not by mutant Nm23-H1 expression. Reduced expression of these genes coincident with elevated Nm23-H1 expression was observed in human breast tumor cohorts, a panel of breast carcinoma cell lines, and hepatocellular carcinomas from control versus Nm23-M1 knockout mice. The functional significance of the down-regulated genes was assessed by transfection and in vitro motility assays. Only EDG2 overexpression significantly restored motility to Nm23-H1-suppressed cancer cells, enhancing motility by 60-fold in these cells. In addition, silencing EDG2 expression with small interfering RNA reduced the motile phenotype of metastatic breast cancer cells. These data suggest that Nm23-H1 suppresses metastasis, at least in part, through downregulation of EDG2 expression. [Cancer Res 2007;67(15):7238-46]

Nucleoside diphosphate kinases (NDPK), 3 encoded by NME genes (also called NM23), catalyze the exchange of ␥-phosphate between di-and triphosphonucleosides and participate in the regulation of intracellular nucleotide homeostasis. They mainly utilize ATP formed by oxidative phosphorylation to synthesize the other triphosphonucleosides, in particular GTP (1). Given the poor substrate selectivity of NDPKs, it is assumed that specificity could arise from the presence of different isoforms at different subcellular localizations. Associated in networks with other nucleotide-metabolizing enzymes such as adenylate kinases, creatine kinases, and glycolytic enzymes, NDPKs participate in high energy phosphoryl transfer and signal communication in the cell (2). Up to now nine genes encoding NDPK or NDPK-like proteins have been identified (3, 4), but little is known about their respective role within the cell. The most studied, NDPK-A and -B, encoded by NME1 and NME2 genes, respectively, play a key role in tumor progression and metastasis dissemination (5, 6).NDPK activity has been found associated with different cellular compartments, such as cytosol, nucleus, plasma membrane, and mitochondria. Precise localization in the latter organelles has been a matter of debate. Depending on species and tissue examined, NDPK activity was reported in both the matrix and the intermembrane/cristae space (7), including the so-called contact sites between inner and outer membrane (8 -10). In mammalian liver (rat and rabbit), the NDPK activity was mainly associated with an extra-matrix compartment, probably the intermembrane/cristae space, whereas in heart activity was more abundant in the matrix (11). For mitochondrial NDPK in matrix, many functions have been proposed ranging from nucleotide supply for mitochondrial nucleic acid and protein synthesis to functional interaction with the Krebs * This work was supported by the Germaine de Stael Program for FrancoSwiss collaboration (to U. S. and M.-L. L.), the Agence Nationale de la Recherche (Chaire d'Excellence (to U. S.)), the Marie Curie Intraeuropean Fellowship of the European Community (to M. T.-S.), INSERM, and grants from the Groupement des Entreprises Françaises contre le Cancer and from the Association pour la Recherche contre le Cancer (to M.-L. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Background: Nm23-H4 is a mitochondrial nucleoside diphosphate kinase that binds mitochondrial membranes. Results: Nm23-H4 interaction with GTPase OPA1 provides a local GTP supply; its interaction with anionic phospholipids inhibits kinase activity but allows intermembrane cardiolipin transfer and sensitizes for apoptosis. Conclusion: Nm23-H4 is a bifunctional switch operated by cardiolipin. Significance: The cardiolipin transfer property has various implications, e.g. for lipid metabolism and apoptosis.

The lack of NM23-M1 expression promotes metastasis in the SV40 animal model of liver carcinogenesis.