Subcellular localization of A and B Nm23/NDPK subunits

The human Nm23-H1/NDPK A and Nm23-H2/NDPK B encode for two subunits of nucleoside diphosphate kinase--a ubiquitous enzyme that transfers the terminal phosphates from ATP to (d)NDPs. Although having an 88% amino acid sequence identity and an already assigned biochemical role in the cell, the two subunits appear to have additional and distinctive cell functions. In particular, both subunits have been reported to be involved in tumor progression and metastasis. The aim of this study was to determine the specific, and potentially distinct, localizations of both subunits in tumor cells of different origin and differentiation and therefore to search for a possible link between their localization and the stage of disease. We used the GFP reporter system to analyze the ectopic expression of GFP-Nm23 proteins in head and neck tumor cell lines by fluorescent microscopy techniques. Our experiments revealed that GFP-fused Nm23-H1 and -H2 proteins display the same localization in transfected cells, regardless of their origin and differentiation status. The proteins are principally found in the cytosol and the endoplasmic reticulum. Moreover, some cells exhibit nuclear staining, which appears to be cell cycle-dependent.     

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.     

Subcellular localization of Nm23/NDPK A and B isoforms: a reflection of their biological function?

ABCG2 is a half-transporter initially described in multidrug-resistant cancer cells and lately identified as an important factor in the pharmacokinetics of its substrates. Q141K is by far the most intensively studied single nucleotide polymorphism of ABCG2 with potential clinical relevance. Here we used stably transfected HEK cells to study the Q141K polymorphism together with the deletion of amino acids 315–316, which were recently reported to coexist in two cancer cell lines (A549 and SK-OV-3). Functional studies confirmed our previous report that when normalized to surface expression, Q141K has impaired transport of mitoxantrone. This result was extended to include the ABCG2-specific substrate pheophorbide a. While we found no functional consequence of deleting amino acids 315 and 316, we did find that the deletion mutant is no longer recognized by the BXP-21 antibody. We conclude that amino acids 315 and 316 form part of the epitope for the BXP-21 antibody.     

Basic and translational advances in cancer metastasis: Nm23

Cancer metastasis is a significant contributor to breast cancer patient morbidity and mortality. To develop new anti-metastatic therapies, we need to understand the biological and biochemical mechanisms of metastasis. Toward these efforts, we and others have studied metastasis suppressor genes, which halt metastasis in vivo without affecting primary tumor growth. The first metastasis suppressor gene confirmed was nm23, also known as NDP kinase. Using in vitro assays, nm23 overexpression resulted in reduced anchorage-independent colonization in response to TGF-, reduced invasion and motility in response to multiple factors, and increased differentiation. We hypothesize that the mechanism of action of Nm23 in metastasis suppression involves diminished signal transduction, downstream of a particular receptor. We hypothesize that a histidine protein kinase activity of Nm23 underlies its suppression of metastasis, and identify candidate substrates. This review also discusses therapeutic options on the basis of reexpression of metastasis suppressors.     

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.     

Nm23-H1/NDPK-A基因在大肠杆菌中的高效表达及产物纯化的研究

利用聚合酶链反应（ＰＣＲ）技术扩增人二磷酸核苷激酶Ａ亚基（ＮＤＰＫ－Ａ）基因,即ｎｍ２３－Ｈ１／ＮＤＰＫ－Ｋ基因的编码序列,经序列分析后,定向克隆于表达质粒载体ｐＢＶ２２０,在大肠杆菌ＤＨ５α中高效表达出重组人ＮＤＰＫ－Ａ．表达产物为可溶性的非融合蛋白,占菌体总蛋白４２％．斑点ＥＬＩＳＡ法鉴定表明表达产物与ＮＤＰＫ－Ａ标准抗血清呈阳性反应．以ＤＥＡＥ纤维素弱阴离子交换层析、ＣｉｂａｃｒｏｎＢｌｕｅ染料亲和层析结合高效液相排阻色谱技术纯化ｒＮＤＰＫ－Ａ,得纯度为９６．７％的目标蛋白．以反相高效液相色谱法进行酶活性分析,表明纯化的ｒＮＤＰＫ－Ａ能催化ＡＴＰ＋ＵＤＰ＝ＡＤＰ＋ＵＴＰ的反应,比活性为８００Ｕ／ｍｇ蛋白．     

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.     

Double mutant P96S/S120G of Nm23-H1 abrogates its NDPK activity and motility-suppressive ability

The Nm23-H1 gene is a metastasis suppressor gene. However, its biochemical mechanism of suppressing the metastatic potential of cancer cells is still unknown. The previous hypothesis that a histidine protein kinase activity may contributes to the motility-suppressive effect of Nm23-H1 could not explain why the H118F mutant, a kinase-deficient mutant, still had motility-suppressive ability. We conducted a study on the double mutant P96S/S120G of Nm23-H1 and succeeded in introducing the RP-HPLC method in NDPK assay. The results showed that the double mutant P96S/S120G, when expressed in the bacteria, was completely aggregated in inclusion bodies; this mutant abrogated not only its motility-suppressive ability, but also its NDPK activity. Based on previous work and this study, we prompted that the deficiency of motility-suppressive function of S120G, P96S, and P96S/S120G mutants was due to their altered structure, which might deprive Nm23-H1 of most activities including kinase activity or interactions with other proteins.     

Developmental function of Nm23/awd: a mediator of endocytosis

The metastasis suppressor gene Nm23 is highly conserved from yeast to human, implicating a critical developmental function. Studies in cultured mammalian cells have identified several potential functions, but many have not been directly verified in vivo. Here, we summarize the studies on the Drosophila homolog of the Nm23 gene, named a bnormal w ing d iscs (awd), which shares 78% amino acid identity with the human Nm23-H1 and H2 isoforms. These studies confirmed that awd gene encodes a nucleoside diphosphate kinase, and provided strong evidence of a role for awd in regulating cell differentiation and motility via regulation of growth factor receptor signaling. The latter function is mainly mediated by control of endocytosis. This review provides a historical account of the discovery and subsequent analyses of the awd gene. We will also discuss the possible molecular function of the Awd protein that underlies the endocytic function.     

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.     

Epstein-Barr virus nuclear protein EBNA-3C interacts with the human metastatic suppressor Nm23-H1: a molecular link to cancer metastasis

Epstein-Barr virus (EBV) is an oncogenic virus associated with a number of human malignancies including Burkitt lymphoma, nasopharyngeal carcinoma, lymphoproliferative disease and, though still debated, breast carcinoma. A subset of latent EBV antigens is required for mediating immortalization of primary B-lymphocytes. Here we demonstrate that the carboxy-terminal region of the essential latent antigen, EBNA-3C, interacts specifically with the human metastatic suppressor protein Nm23-H1. Moreover, EBNA-3C reverses the ability of Nm23-H1 to suppress the migration of Burkitt lymphoma cells and breast carcinoma cells. We propose that EBNA-3C contributes to EBV-associated human cancers by targeting and altering the role of the metastasis suppressor Nm23-H1.     

Occupy tissue: The movement in cancer metastasis

The critical role of migration and invasion in cancer metastasis warrants new therapeutic approaches targeting the machinery regulating cell migration and invasion. While 2-dimensional (2D) models have helped identify a range of adhesion molecules, cytoskeletal components and regulators that are potentially important for cell migration, the use of models that better mimic the 3-dimensional (3D) environment has yielded new insights into the physiology of cell movement. For example, studying cells in 3D models has revealed that invading cancer cells may switch between heterogeneous invasion modes and thus evade pharmacological inhibition of invasion. Here we summarize published data in which the role of cell adhesion molecules in 2D vs. 3D migration have been directly compared and discuss mechanisms that regulate migration speed and persistence in 2D and 3D. Finally we discuss limits of 3D culture models to recapitulate the in vivo �situation.     

Nm23/nucleoside diphosphate kinase: Toward a structural and biochemical understanding of its biological functions

The nm23 gene, a putative metastasis suppressor gene, was originally identified by its reduced expression in highly metastatic K-1735 murine melanoma cell lines, as compared to related, low metastatic melanoma cell lines. Transfection of nm23 cDNA has been reported to suppress malignant progression in Drosophila and mammalian cells. Highly conserved homologues of nm23 have been found in organisms ranging from the prokaryote Myxococcus xanthus to Drosophila, where the gene is involved in normal development and differentiation. The product of the nm23 gene exhibits a nucleoside diphosphate kinase activity, yet the nucleoside diphosphate kinase activity of Nm23 does not correlate with its apparent biological functions. We review recent cellular, genetic, biochemical and X-ray crystallographic data to formulate and evaluate hypotheses concerning the molecular mechanism of nm23 action.     

From family to nation: Organizing the Mississippi movement

John Dittmer, LOCAL PEOPLE: THE STRUGGLE FOR CIVIL RIGHTS IN MISSISSIPPI, Urbana, IL: University of Illinois Press, 1995, 530 pp., $29.95.

Charles M. Payne, I'VE GOT THE LIGHT OF FREEDOM: THE ORGANIZING TRADITION AND THE MISSISSIPPI FREEDOM STRUGGLE, Berkeley, CA: University of California Press, 1995, xiv + 525 pp, $28.00.     

Studies on reactivated cilia : I. The utilization of various nucleoside triphosphates during ciliary movement

It has been found that the movement of reactivated cilia is prolonged, providing various nucleoside triphosphates (NTP) are present in the reactivation medium in addition to ATP. The results suggest the existence of an axoneme-bound phosphotransferase that regenerates ATP from NTP. A model is proposed for the enzymatic regulation of ATP levels in the cilium.     

The family of Deg/HtrA proteases: from Escherichia coli to Arabidopsis

In the genomic era, an increasing number of protease genes have been identified in various organisms. During the last few years, many of these proteases have been characterized using biochemical as well as molecular biological techniques. However, neither the precise location nor the physiological substrates of these enzymes has been identified in many cases, including the Deg/HtrA proteases, a family of serine-type ATP-independent proteases. This family has become especially interesting for many researchers following the determination of the crystal structures of an Escherichia coli and a human Deg/HtrA protease. A breakthrough in photosynthesis research has revealed that a Deg/HtrA protease of Arabidopsis thaliana is involved in the degradation of the D1 protein of photosystem II following photoinhibition. In this review, the available data on Deg/HtrAs of different organisms are compared with those from the photoautotroph cyanobacterium Synechocystis sp. PCC 6803 and the plant Arabidopsis thaliana .     

TSG101/mammalian VPS23 and mammalian VPS28 interact directly and are recruited to VPS4-induced endosomes

Class E vacuolar protein sorting (vps) proteins are required for appropriate sorting of receptors within the yeast endocytic pathway, and most probably function in the biogenesis of multivesicular bodies. We have identified the mammalian orthologue of Vps28p as a 221- amino acid cytosolic protein that interacts with TSG101/mammalian VPS23 to form part of a multiprotein complex. Co-immunoprecipitation and cross-linking experiments demonstrated that hVPS28 and TSG101 interact directly and that binding requires structural information within the conserved C-terminal portion of TSG101. TSG101 and hVPS28 are predominantly cytosolic. However, when endosomal vacuolization was induced by the expression of a dominant-negative mutant of another class E vps protein, human VPS4, a portion of both TSG101 and hVPS28 translocated to the surface of these vacuoles. We conclude that TSG101 and its interacting components are directly involved in endosomal sorting.