Molecular and Cellular Biochemistry

, Volume 329, Issue 1, pp 17–33

The NM23 family in development

Authors

  • Aikaterini Bilitou
    • Department of Oncology, Hutchison/MRC Research CentreUniversity of Cambridge
  • Julie Watson
    • Department of Oncology, Hutchison/MRC Research CentreUniversity of Cambridge
  • Anton Gartner
    • Wellcome Trust Centre for Gene Regulation and ExpressionUniversity of Dundee
    • Department of Oncology, Hutchison/MRC Research CentreUniversity of Cambridge
    • UCL Institute of OphthalmologyUniversity College London
Article

DOI: 10.1007/s11010-009-0121-6

Cite this article as:
Bilitou, A., Watson, J., Gartner, A. et al. Mol Cell Biochem (2009) 329: 17. doi:10.1007/s11010-009-0121-6

Abstract

The NM23 (non-metastatic 23) family is almost universally conserved across all three domains of life: eubacteria, archaea and eucaryotes. Unicellular organisms possess one NM23 ortholog, whilst vertebrates possess several. Gene multiplication through evolution has been accompanied by structural and functional diversification. Many NM23 orthologs are nucleoside diphosphate kinases (NDP kinases), but some more recently evolved members lack NDP kinase activity and/or display other functions, for instance, acting as protein kinases or transcription factors. These members display overlapping but distinct expression patterns during vertebrate development. In this review, we describe the functional differences and similarities among various NM23 family members. Moreover, we establish orthologous relationships through a phylogenetic analysis of NM23 members across vertebrate species, including Xenopus laevis and zebrafish, primitive chordates and several phyla of invertebrates. Finally, we summarize the involvement of NM23 proteins in development, in particular neural development. Carcinogenesis is a process of misregulated development, and NM23 was initially implicated as a metastasis suppressor. A more detailed understanding of the evolution of the family and its role in vertebrate development will facilitate elucidation of the mechanism of NM23 involvement in human cancer.

Keywords

NM23DevelopmentXenopusNeurogenesisNDP kinase

Introduction

Carcinogenesis is a process of misregulated development. Therefore, almost all developmentally important signals are involved in carcinogenesis, while oncogenes and tumour suppressor genes often have developmental roles. The non-metastatic 23 (NM23) family’s role in carcinogenesis is well established, but its role in normal development has not been studied extensively. Nonetheless, the NM23 proteins are attractive candidates as potential regulators of development due to their many fundamental roles in the cell. They interact with, and regulate the expression and activity of, numerous molecules already known to be involved in development. Evidently, a greater understanding of NM23 function in normal homoeostasis and development may well give us a greater appreciation of what exactly is going wrong when NM23 is misexpressed in cancer.

NM23 was the first metastasis suppressor gene identified [1], and its expression has been correlated with histopathological indicators of metastatic potential, lymph node infiltration and poor prognosis in several tumour types (reviewed in [2, 3]). Its ability to suppress metastasis was functionally confirmed by the observation that its transfection into cancer cell lines reduced their in vivo metastatic potential [47]. Much work has focused on identifying the mechanism of NM23-mediated metastasis suppression and this work has attributed an astonishing array of biochemical activities to the NM23 family. NM23 proteins are nucleoside transphosphorylases, catalyzing the transfer of γ-phosphate from a nucleoside triphosphate (NTP) to a nucleoside diphosphate (NDP) [8, 9]. They can also act as histidine-dependent protein kinases and phosphorylate proteins such as the kinase suppressor of Ras (KSR), a scaffold protein for the extracellular signal-regulated kinase (ERK) pathway [10], metabolic products of geranyl and farnesyl diphosphates [11], the β-subunit of G proteins and the potassium channel KCa3.1 [12, 13]. In addition to binding nucleotides, NM23 family members can also bind polymeric DNA and act as transcription factors. They regulate the expression of c-myc [14], platelet-derived growth factor-A promoter [15] and gelatinase A [16]. NM23-H2 can also bind single-stranded DNA and polypyrimidine-rich RNAs [17] and cleave DNA after formation of a protein–DNA complex [18], while NM23-H1 possesses 3′–5′ exonuclease activity [19] and can mediate apoptosis by nicking DNA [20]. NM23 proteins interact with the components of cytoskeleton, including the centrosomal kinase Aurora A/STK15 [21, 22] and integrin cytoplasmic-associated protein 1α [23]. They regulate endocytosis [2426]. The relative importance of each of these activities for metastasis suppression is disputed, but they are so fundamental and various that they are likely important in normal homoeostasis and development, as well as in cancer.

In this review, we provide a phylogenetic analysis of NM23 across the kingdoms of life. We describe what is known about the expression patterns and functions of NM23 proteins in various developing animals and include novel data regarding the expression patterns of Xenopus laevis orthologs in the developing embryo.

Structures and evolution of the NM23 family

The first member of the NM23 family, NM23-M1, was identified as a candidate suppressor of cancer metastasis by searching for genes suppressed in highly metastatic melanoma cell lines [1]. At almost same time, Shearn’s group identified, through a genetic screen for defects in imaginal disc development, a mutant fly gene, the abnormal wing discs (awd), that was highly homologous to NM23-M1. In the awd mutant, wing, leg, eye-antenna and ovary did not develop normally [27, 28]. Subsequently, the cloning of NDP kinases, which convert NDP to NTP, from Myxococcus xanthus [29], Dictyostelium discoideum [30] and rat [30] revealed that NM23-M1 and awd are orthologs of NDP kinase.

Members of the NM23 family

After the isolation of NM23-M1 and awd, further screening for homologous genes and searching of genomic databases revealed that almost all organisms, including eubacteria, archaea and eucaryotes, have homologous genes. The only exception is the Mycoplasma taxon. In silico search of the completed Mycoplasma genome databases such as that of Mycoplasma genitalium did not identify any NM23 genes [31]. The widely conserved existence of genes indicates that NM23 family members must have a basic role in cellular activity. Studies in unicellular organisms like Escherichia coli, Mycobacterium tuberculosis and Saccharomyces cerevisiae have shown that they contain only one member of the NM23 family. The most primitive multicellular organism, D. discoideum, has three ndk genes: ndkC, ndkM and DDBDRAFT_0191701 [32]. The fruit fly, Drosophila melanogaster, has four NM23 members: awd, nmdyn-D7/CG8362, nmdyn-D6/CG5310 and CG15547. The nematode worm Caenorhabditis elegans also has NM23 genes: F25H2.5, F55A3.6 and Y48G8AL.15. The NM23 family has been identified in several plant species (Oryza sativa, Arabidopsis thaliana, Triticum aestivum, Nicotiana tabacum) that all seem to possess several members.

Mammalian organisms have 8–10 members of the NM23 family, among which NM23-9 and NM23-10/RP2 that have been identified most recently (see also reviews in this issue). The existence of at least eight NM23 genes in all vertebrate species examined suggests that some of the multiplication of genes happened prior to the evolutionary divergence of vertebrate species. Historically, there has been little consideration of the phylogenetic relationship of genes between species when naming new NM23 members. This led to a confusing nomenclature. In this review, we use a novel system of nomenclature under which vertebrate non-human NM23 orthologs are named to correspond to their most closely related human counterparts. Under this system, mouse, rat, Xenopus and zebrafish NM23 members are called, for example, NM23-M1, R1, X1 and Z1, respectively. The human NM23 family members had been subdivided into two groups according to their genomic architecture and phosphotransferase activity and we provide strong correlation with the phylogenetic data produced in this review to support this (Fig. 1). Members of Group I (NM23-H1, H2, H3 and H4) possess well-conserved NDP kinase active site motifs and are catalytically active, while Group II members (NM23-H5, H6, H7 and H8) show high diversion from their counterparts in sequence and motifs. The two novel members, NM23-H9 and H10/RP2, appear to belong in the Group II as well, based on motif prediction and the phylogenetic tree.
https://static-content.springer.com/image/art%3A10.1007%2Fs11010-009-0121-6/MediaObjects/11010_2009_121_Fig1_HTML.gif
Fig. 1

Orthologous relationships of NM23 members. The phylogenetic tree includes all the known and newly identified NM23 members of the human (Homo sapiens, Hs.NM23-H1 to -H9 and Hs.RP2), mouse (Mus musculus, Mm.NM23-M1 to -M9 and Mm.RP2), rat (Rattus norvegicus, Rn.NM23-R1 to -R8), chick (Gallus gallus, Gg. NM23-G1 to -G8), frog (Xenopus laevis, Xl.NM23-X1 to -X8 and Xl.RP2) and zebrafish (Danio rerio, Dr.NM23-Z1 to -Z9) families. Also shown members of Drosophila melanogaster (Dm), Caenorhabditis elegans (Ce), Dictyostelium discoideum (Dd), Arabidopsis thaliana (At), Escherichia coli (Ec), Mycobacterium tuberculosis (Mt) and Saccharomyces cerevisiae (Sc) denoted with their gene identity (gi) numbers. The alignment of protein sequences generated on Jalview 2.4 software [99] with Clustal W algorithm was used to construct the tree using Splitstree software with NJ (Neighbour Joining) and equal angle method (filter set max dimension 4, use weights, run convex hull, daylight iterations 0) [100]. All sequences used were retrieved from the NCBI database, and homologues were identified through BLAST searches. The gene ID numbers for each NM23 member is given below. Human members (Homo sapiens, Hs): NM23-H1 (non-metastatic cells 1, NME1, NDPKA, NM23A) gi:4830, NM23-H2 (non-metastatic cells 2, NM23B) gi:4831, NM23-H3 (non-metastatic cells 3, nme3, NDPKC, DR-NM23, KIAA0516) gi:4832, NM23-H4 (non-metastatic cells 4) gi:4833, NM23-H5 (NME5, non-metastatic cells 5) gi:8382, NM23-H6 (non-metastatic cells 6, NDP kinase 6, inhibitor of p53-induced apoptosis-alpha, IPIA-alpha) gi:10201, NM23-H7 (non-metastatic cells 7, nucleoside diphosphate kinase 7) gi:29922, NM23-H8 (thioredoxin domain-containing protein 3, spermatid-specific thioredoxin-2, Sptrx-2) gi:51314, NM23-H9 (thioredoxin domain-containing protein 6, thioredoxin-like protein 2, Txl-2) gi:347736, NM23-H10 (retinitis pigmentosa RP2) gi:6102. Mouse members (Mus musculus, Mm): NM23-M1 (NM23A, NDPK-A, AL024257) gi:18102, NM23-M2 (Nme2) gi:18103, NM23-M3 (Ndk3, DR-NM23, AI413736) gi:79059, NM23-M4 (non-metastatic cells 4) gi:56520, NM23-M5 gi:75533, NM23-M6 (non-metastatic cells 6) gi:54369, NM23-M7 gi:171567, NM23-M8 (thioredoxin-containing protein 3, Txndc3) gi:73412, NM23-M9 (hypothetical protein LOC623534) gi:623534, NM23-M10 (retinitis pigmentosa 2 homologue) gi:19889. Rat members (Rattus norvegicus, Rn): NM23-R1 gi:191575, NM23-R2 (NM23B) gi:83782, NM23-R3 (Nme3) gi:85269, NM23-R4 (non-metastatic cells 4) gi:685679, NM23-R5 (similar to NDK-H5, NDP kinase homologue 5) gi:682386, NM23-R6 non-metastatic cells 6) gi:58964, NM23-R7 gi:171566, NM23-R8 (Txndc3) gi:364729. Chick (Gallus gallus, Gg): NM23-G1 (NM23A) gi:422094, NM23-G2 (NM23B) gi:395916, NM23-G3 gi:416399, NM23-G4 (RAB11 family interacting protein 3) gi:395915, NM23-G5 (NME5) gi:416373, NM23-G6 (non-metastatic cells 6) gi:426866, NM23-G8 (Txndc3) gi:428461. Xenopus (Xenopus laevis or tropicalis, Xl or Xt): NM23-X1 (NM23ndk-a NM23/nucleoside diphosphate kinase) gi:394350, NM23-X2 (NM23/nucleoside diphosphate kinase) gi:399333, NM23-X3 (hypothetical protein LOC100037175) gi:100037175, NM23-X4 (hypothetical protein MGC81083) gi:414658, NM23-X5 (MGC85510 protein) gi:447618, NM23-X6 (hypothetical protein LOC734821) gi:734821, NM23-X7 (hypothetical protein MGC75677) gi:394498, NM23-X8 (hypothetical protein LOC432114) gi:432114, NM23-X9 (hypothetical protein LOC432114) gi:432114, NM23-X10 (retinitis pigmentosa 2) gi:379779. Zebrafish (Danio rerio, Dr): NM23-Z1 (non-metastatic cells 2, protein NM23B, nucleoside diphosphate kinase Z1) gi:30083, NM23-Z2 (ndpkz2) gi:30084, non-metastatic cells 2-like, protein (NM23B) expressed in (nme2l) gi:335716, NM23-Z3 (NDPK-Z3, MGC92672) gi:30085, NM23-Z4 (Nme4, non-metastatic cells 4) gi:394170, NM23-Z5 (hypothetical protein LOC436789 homologue of NM23-H5) gi:436789, NM23-Z6 (ndpk6) gi:58120, NM23-Z7 (nucleoside diphosphate kinase Z4, NDPK-Z4, NDPK-Z7) gi:30086, NM23-Z8 (hypothetical protein LOC100037319) gi:147898395. Other organisms. Drosophila melanogaster (Dm) awd: (CG2210 gene product, Killer-of-prune; NDP kinase; nucleoside diphosphate kinase; abnormal wing discs) gi:43739, Dm.3287 (nmdyn-D6) gi:32396, Dm.1965 (nmdyn-D7) gi:41174; Dm.31359 (CG15547) gi:43661; Caenorhabditis elegans (Ce): F25H2.5 (Unigene Cel.8678) gi:172939, F55A3.6 (Cel.26984) gi:186271, Y48G8AL.15 (Cel.8464) gi:259363; Dictyostelium discoideum (Dd): ndkC (nucleoside diphosphate kinase-ndkC2) gi:3395654, ndkM gi:3392555, DDBDRAFT_0191701 gi:3386090; Arabidopsis thaliana (At): At.5402, NDPK1 (nucleoside diphosphate kinase 1) ATP binding/nucleoside diphosphate kinase, gi:826515; At.32397, NDK4 (AT4G23900) gi:828490; At.47514 (NDPK3) gi:826702; At.24504 (NDPK2) gi:836451; At.41837 (nucleoside diphosphate kinase family protein) gi:838313; At. F1L3.7 Accession number: AC022492.5; At. AC007843.6 Protein id: AAF97316; Escherichia coli Ec.ndk gi:1038720; Mycobacterium tuberculosis Mt.ndk gi:885905; Saccharomyces cerevisiae Sc.YNK1 gi:853798; Human members (Homo sapiens, Hs) NM23-H1 (non-metastatic cells 1, NME1, NDPKA, NM23A) gi:4830, NM23-H2 (non-metastatic cells 2, NM23B) gi:4831, NM23-H3 (non-metastatic cells 3, nme3, NDPKC, DR-NM23, KIAA0516) gi:4832, NM23-H4 (non-metastatic cells 4) gi:4833, NM23-H5 (NME5, non-metastatic cells 5) gi:8382, NM23-H6 (non-metastatic cells 6, NDP kinase 6, inhibitor of p53-induced apoptosis-alpha, IPIA-alpha) gi:10201, NM23-H7 (non-metastatic cells 7, nucleoside diphosphate kinase 7) gi:29922, NM23-H8 (thioredoxin domain-containing protein 3, spermatid-specific thioredoxin-2, Sptrx-2) gi:51314, NM23-H9 (thioredoxin domain-containing protein 6, thioredoxin-like protein 2, Txl-2) gi:347736, NM23-H10 (retinitis pigmentosa RP2) gi:6102. Mouse members (Mus musculus, Mm) NM23-M1 (NM23A, NDPK-A, AL024257) gi:18102, NM23-M2 (Nme2) gi:18103, NM23-M3 (Ndk3, DR-NM23, AI413736) gi:79059, NM23-M4 (non-metastatic cells 4) gi:56520, NM23-M5 gi:75533, NM23-M6 (non-metastatic cells 6) gi:54369, NM23-M7 gi:171567, NM23-M8 (thioredoxin-containing protein 3, Txndc3) gi:73412, NM23-M9 (hypothetical protein LOC623534) gi:623534, NM23-M10 (retinitis pigmentosa 2 homologue) gi:19889. Rat members (Rattus norvegicus, Rn) NM23-R1 gi:191575, NM23-R2 (NM23B) gi:83782, NM23-R3 (Nme3) gi:85269, NM23-R4 (non-metastatic cells 4) gi:685679, NM23-R5 (similar to NDK-H5, NDP kinase homologue 5) gi:682386, NM23-R6 non-metastatic cells 6) gi:58964, NM23-R7 gi:171566, NM23-R8 (Txndc3) gi:364729. Chick (Gallus gallus, Gg) NM23-G1 (NM23A) gi:422094, NM23-G2 (NM23B) gi:395916, NM23-G3 gi:416399, NM23-G4 (RAB11 family interacting protein 3) gi:395915, NM23-G5 (NME5) gi:416373, NM23-G6 (non-metastatic cells 6) gi:426866, NM23-G8 (Txndc3) gi:428461. Xenopus (Xenopus laevis or tropicalis, Xl or Xt) NM23-X1 (NM23ndk-a NM23/nucleoside diphosphate kinase) gi:394350, NM23-X2 (NM23/nucleoside diphosphate kinase) gi:399333, NM23-X3 (hypothetical protein LOC100037175) gi:100037175, NM23-X4 (hypothetical protein MGC81083) gi:414658, NM23-X5 (MGC85510 protein) gi:447618, NM23-X6 (hypothetical protein LOC734821) gi:734821, NM23-X7 (hypothetical protein MGC75677) gi:394498, NM23-X8 (hypothetical protein LOC432114) gi:432114, NM23-X9 (hypothetical protein LOC432114) gi:432114, NM23-X10 (retinitis pigmentosa 2) gi:379779. Zebrafish (Danio rerio, Dr) NM23-Z1 (non-metastatic cells 2, protein NM23B, nucleoside diphosphate kinase Z1) gi:30083, NM23-Z2 (ndpkz2) gi:30084, non-metastatic cells 2-like, protein (NM23B) expressed in (nme2l) gi:335716, NM23-Z3 (NDPK-Z3, MGC92672) gi:30085, NM23-Z4 (Nme4, non-metastatic cells 4) gi:394170, NM23-Z5 (hypothetical protein LOC436789 homologue of NM23-H5) gi:436789, NM23-Z6 (ndpk6) gi:58120, NM23-Z7 (nucleoside diphosphate kinase Z4, NDPK-Z4, NDPK-Z7) gi:30086, NM23-Z8 (hypothetical protein LOC100037319) gi:147898395. Other organisms.Drosophila melanogaster (Dm) awd (CG2210 gene product, Killer-of-prune; NDP kinase; nucleoside diphosphate kinase; abnormal wing discs) gi:43739, Dm.3287 (nmdyn-D6) gi:32396, Dm.1965 (nmdyn-D7) gi:41174; Dm.31359 (CG15547) gi:43661; Caenorhabditis elegans (Ce) F25H2.5 (Unigene Cel.8678) gi:172939, F55A3.6 (Cel.26984) gi:186271, Y48G8AL.15 (Cel.8464) gi:259363; Dictyostelium discoideum (Dd) ndkC (nucleoside diphosphate kinase-ndkC2) gi:3395654, ndkM gi:3392555, DDBDRAFT_0191701 gi:3386090; Arabidopsis thaliana (At) At.5402, NDPK1 (nucleoside diphosphate kinase 1) ATP binding/nucleoside diphosphate kinase, gi:826515; At.32397, NDK4 (AT4G23900) gi:828490; At.47514 (NDPK3) gi:826702; At.24504 (NDPK2) gi:836451; At.41837 (nucleoside diphosphate kinase family protein) gi:838313; At. F1L3.7 Accession number: AC022492.5; At. AC007843.6 Protein id: AAF97316; Escherichia coli Ec.ndk gi:1038720; Mycobacterium tuberculosis Mt.ndk gi:885905; Saccharomyces cerevisiae Sc.YNK1 gi:853798

Recently, our studies of the African clawed frog, Xenopus laevis, have led us to the identification of novel members of the NM23 family [33]. Previously, Ouatas et al. [34, 35] had reported three Xenopus homologues of the NM23 family. Our phylogenetic analysis based on multiple sequence alignments indicates that these two previously reported genes, namely NM23-X1 and X2 [34, 35] as well as zebrafish NM23 Z1 and Z2 arose before human, mouse and rat NM23 split into NM23-1 and -2 groups. These observations indicate that the NM23-X1 and X2 are derived from a single ancestral gene (see Fig. 2) consistent with the pseudo-tetraploidy of Xenopus laevis. This phylogenetic relationship is also consistent with biochemical data of Ouatas et al. that NM23-X1, but not NM23-X3, is able to bind to the nuclease hypersensitive element (NHE) of the human c-myc promoter, in vitro [34], like NM23-H2 [14]. Although it is not perfectly clear, which NM23-X1 and X2 are true orthologs of either NM23-H1 or H2 based on their sequence similarity to human orthologs, we use the revised names in this review (the previously named NM23-X1, X2 and X3 are re-named as NM23-X1a, X1b and X2, respectively). The other Xenopus members of the NM23 family have been named NM23-X3, X4, X5, X6, X7, X8 and X10/RP2 according to their homology to human and mouse members. When there is a homologous allele, then the two are classified as a and b. We could not identify an ortholog of NM23-H9 in the Xenopus laevis or tropicalis databases. The closest similarity was given for the Xenopus NM23-X8 sequence, but we are convinced it is a true NM23-8 ortholog because it contains three NDPK domains like the human NM23-H8 and mouse NM23-M8.
https://static-content.springer.com/image/art%3A10.1007%2Fs11010-009-0121-6/MediaObjects/11010_2009_121_Fig2_HTML.gif
Fig. 2

Phylogenetic tree of vertebrate and invertebrate NM23 members. This phylogenetic tree considered all known NM23s shown in Fig. 1 as well as the indicated invertebrate species. In order to persevere clarity Dictyostelium discoideum, Arabidopsis thaliana, Escherichia coli, Mycobacterium tuberculosi,Saccharomyces cerevisiae, Rattus norvegicus and Gallus gallus, sequences are not shown. Nematode species analyzed were Caenorhabditis elegans (Ce), Caenorhabditis briggsae (Cb), Brugia malayi (Bm) and Pristionchus pacificus (Pp). Protein sequences were aligned using Jalview 2.4 software and employing the CLUSTAL W algorithm (the MAFT algorithm essentially gave similar results). Phylogenetic trees were generated as described in Fig. 1. Sequences not described in Fig. 1 were retrieved from the NCBI database and gene ID numbers are indicated in the tree. All sequences are available upon request from the authors

Three NM23 members have been previously reported for zebrafish [36] and two for the Atlantic salmon [37]. A search in zebrafish protein and open reading frame databases has revealed eight NM23 members in total. Based on their phylogenetic relationships, we termed these members as NM23-Z1 (previously named ndpk-z1, also referred as nme2) with two isoforms, NM23-Z2 (ndpk-z2), NM23-Z3 (ndpk-z3), NM23-Z4 (known as nme), NM23-Z5, NM23-Z6 (ndkp-z6), NM23-Z7 (previously referred as ndpk-z4 but is structurally similar to NM23-H7) and NM23-Z8.

Evolution

As mentioned, unicellular organisms have only one member of the NM23 family, while lower multicellular organisms such as D. discoideum, Drosophila and C. elegans have a few members. All vertebrates analyzed have 8–10 members. How were these members duplicated in evolution? An insightful study of NM23 evolution was reported by Kimura and co-workers [38].

We have updated their phylogenetic analysis, by addition of the novel human, mouse, rat, chick, Xenopus and zebrafish members and also include sequences from Drosophila, C. elegans, A. thaliana, D. discoideum and M. tuberculosis (Fig. 1). In Fig. 2, we describe phylogenetic relationships of vertebrate NM23 family members to primitive chordates and representative members of several phyla of invertebrates. As shown in the phylogenetic tree (Fig. 1), all vertebrate orthologs of NM23-H3, H4, H5, H6, H7, H8, H9 and H10/RP2 form clear homologous groups, indicating that these members were duplicated before emergence of these animals. As mentioned, the branch including NM23-X1, X2, Z1 and Z2 is separated from the corresponding mammalian branch. Drosophila members, awd and nmdyn-D7/CG8362, being most related to the NM23-1 and NM23-3 groups provides evidence for a single ancestral gene duplicated several times in vertebrate evolution (see above). This phylogenetic relationship that places awd within the NM23H-1/2 group (Figs. 1, 2) is consistent with the partial rescue of awd mutations by human NM23-H2 [39]. Including non-vertebrate chordate, NM23 sequences and multiple invertebrate NM23 sequences allows for a comprehensive dissection of NM23 evolution (Fig. 2). The most surprising result of this analysis is that all group II NM23 family members (NM23-10/RP2, NM23-6, NM23-8/9 and NM23-7) evolved very early in animal evolution and already occur in animals that are thought to form sister groups of bilaterian animals. These “early” metazoans (indicated in green) include the cnidarian (corals, sea anemones and jellyfish) starlet sea anemone Nematostella vectensis (Nv) [40] and the placozoan Trichoplax adherens (Ta) [41]. Trichoplax is a representative of a basal eumetazoan lineage (all animal clades except sponges) that diverged before the separation of cnidarians and bilaterians [41]. At present, we cannot know if the absence of a NM23-8/9 family member in Trichoplax or the absence of a NM23-10/RP2 family member is genuine or whether this is related to the non-complete genome assembly of these species. Trichoplax has one group I NM23 family member while Nematostella has two family members, potentially related to the chordate (red and orange) NM23-1 and NM23-2 groups. Thus, at least one group I NM23 family member and all group II family members evolved during early metazoan evolution. NM23-3 and NM23-4 members appear as vertebrate-specific inventions, since no such invertebrate sequences occur. NM23-1 and NM23-2 groups first appeared to split up during the evolution of invertebrate chordates (orange) as evidenced by NM23-1 and NM23-2 sequences of each group from the cephalochordate Brachistoma floridae (florida lancelet) and the urochordate Ciona intestinalis (sea squirt). The presence of two fairly distant NM23-1/2 Nematostella sequences indicates that this split might have occurred much earlier in evolution. Most likely nematodes (indicated in dark blue) encode a single group I NM23 member that is highly diverged from chordate and cnidarian group I NM23 family members as evidenced by a highly related group of sequences from all four examined nematodes. Similar to nematodes, phylogenetic distant Trematodes represented by Schistosoma mansoni (Sm, indicated in blue) and Echinoderms (sea urchins) represented by Strongylocentrotus purpuratus (Stp, indicated in violet) diverge from other group I NM23 enzymes. Analysis of the phylogeny of group II enzymes provides clear evidence for widespread loss of these enzymes in various subgroups especially in nematodes and insects while most other above described animal species have at least one family member in each subgroup. More careful analysis provides evidence that this loss occurred independently within insects and nematodes. For instance, there is a clear nematode NM23-6 representative encoded by Brugia malayi, while no other group II NM23 is encoded in the four fully sequenced nematode species examined. Within insects, Apis melifera (Am, honey bee) and Tribolium castaneum (Tc, flour beetle) encode a single NM23-5 family member while there is no such protein found in the nearly fully sequenced D. melanogaster genome. Intriguingly, only Tribolium encodes a NM23-7 family member amongst the examined insects while a single NM23-6 member occurs in D. melanogaster and T. castaneum but is absent in A. melifera.

Structural differences

The identification of multiple NM23 members and their alignment suggests that the multiplication of genes may have significantly contributed to their evolution from unicellular to higher multicellular organisms and indicates that each NM23 member in higher organisms may serve unique functions. What is the functional difference between the members? As mentioned in the introduction, so far, several unique activities have been reported in the NM23 family, summarized in Table 1. Here, we will describe the correlation between their sequences and function.
Table 1

The Xenopus NM23 proteins share conserved motifs but also have unique domains

Predicted sites and domains

Vertebrate NM23 members

NM23-1

NM23-2

NM23-3

NM23-4

NM23-5

NM23-6

NM23-7

NM23-8

NDP kinase motif

+

+

+ (H3, M3, Z3)

+

+

+(X7, Z7)

CKII phosphorylation site

+(Z2)

+(H4, Z4)

+

+(M6, Z6)

+

+

Tyr kinase phosphorylation site

+

+(Z5)

+(X6)

+(H7, M7, X7)

RGD cell adhesion tripeptide

+

+

+

+

+(X6)

DM10 domain

+

Thioredoxin family active site

+

Prokaryotic lipoprotein attachment site

+(X4)

cAMP, cGMP-dependent protein kinase phosphorylation site

+

Protein domain analysis was made using Prosite website after submitting the deduced amino acid sequences of human, mouse, zebrafish and Xenopus orthologs as indicated above for Fig. 1. The symbols represent presence (+) or absence (−) of a motif or site among all orthologs, unless otherwise stated in the parenthesis

Activity

Multiple activities have been attributed to NM23 family members. In this section, we present a sequence alignment of NM23 members from yeast to humans including the newly identified Xenopus orthologs (Fig. 3) and link structure–function relationships based on current knowledge and amino acid conservation. As mentioned, NM23 members found in unicellular organisms are confirmed NDP kinases. This suggests that the NM23 family originated from the bacterial form, that NDPK kinase activity is the primary enzymatic activity of the NM23 proteins and any additional activities were introduced later in evolution. So far, NDP kinase activity has been confirmed in NM23-H1, H2, H3, H4 and H6, while NM23-H5, H7 and H8 are reported to be enzymatically inactive [42]. Sequence alignment and structure–function analysis revealed the consensus active centre (NXXHG/ASD) of NDP kinase activity and the essential amino acid residues of proline-96, histidine-118 and serine-120 (based on NM23-H1) [43]. The consensus sequence is highly conserved between vertebrate group I members (NM23-1, 2, 3, 4) and all orthologs of NM23-H6 (Fig. 3). However, it is not highly conserved between mammalian orthologs of NM23-H5, H7 and H8, although Xenopus and zebrafish NM23-H7 orthologs contain the motif. Structure–function analysis has shown that the residues that form the NDP kinase active site are lysine-12, tyrosine-52, arginine-88, threonine-94, arginine-105, asparagine-115 and glutamic acid-129 with histidine-118 and phenylalanine-60 forming the catalytic cleft (numbering for NM23-H1) [4345]. Histidine-118 is the essential amino acid for the catalytic activity, and its phosphorylation is an obligatory step in the catalytic reaction. All of these residues are well conserved in the members that possess the NDP motif across species (see alignment with highlighted amino acids).
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Fig. 3

Amino acid conservation among NM23 family members. Protein sequence alignment of the NDP kinase motif and the Kpn loop of the NM23 family from human (NM23-H1 to H8), Xenopus laevis (NM23-X1 to X8), zebrafish (NM23-Z1-to Z8) and representatives of chick (NM23-G1 to G6) and Drosophila is shown. The conserved residues are highlighted in black. The residues that form the NDP active site are shown in green fonts and the conserved serine residues in yellow. The insertion of three amino acids before the Kpn loop (domain shown) of the group II members is boxed. The NDPK motif (NXIHGSDSV) is indicated by a red box. Proline-96 is highlighted in red fonts. The alignment was made using Clustal W software at default settings (matrix Gonnet 250, gap open 10, exclude endgaps, gap extension penalty 0.2, gap distance 4, no iteration). The amino acids of the protein sequence used for the alignment are indicated by the numbers flanking each fragment

NM23-H1 and NM23-H2 have been shown to possess histidine-dependent protein kinase activity, by which the catalytic histidine residue is auto-phosphorylated by ATP (His118 for NM23-H1, H2) and then the intermediate phosphate is transferred to other target residues. So far, ATP citrate lyase, aldolase C and KSR are known as substrates [10, 46, 47]. Interestingly, an aspartate residue of aldolase C works as an acceptor of phosphate as reported in the bacterial two-component system [48]. Also, heterotrimeric G proteins and the potassium channel KCa3.1 have been reported to be regulated by complex formation with NM23-H2 and subsequent phosphorylation on a conserved histidine residue [12, 13]. Proline-96, histidine-118 and serine-120 are essential for the histidine protein kinase activity [47] and these residues are conserved in group I. So far, the number of substrates identified is limited probably because of unstable nature of the phosphoaspartate residue. Also, the biological significance of known substrates remains to be elucidated and very little is known about protein kinase activity of NM23 family members other than NM23-H1. It would be interesting to analyze phosphorylation of NM23-binding proteins that have been reported on many occasions.

The DNA repair functions and 3′–5′ exonuclease activities of NM23-H1 and H2 have been attributed to the residue lysine-12 that lies in the enzymatic pocket [19, 49]. Lysine-12 is the amino acid that mediates the covalent complex formation with DNA [18], and the alignment shows that it is phylogenetically conserved in all the members of invertebrate and vertebrate families except only for the eighth member (NM23-8). Human NM23-H1 and H2 have been shown to bind nucleotides and histidine-118 and phenylalanine-60 have a major contribution for the interaction [50].

Structure

NM23 members are known to be functional as oligomers. NM23 members of Dictyostelium [51], Drosophila [8] and vertebrates make hexamers, while bacterial proteins seem to make tetramers [29]. The Kpn loop of NM23 members forms part of the active centre of the NDP kinase and protein kinase activities and also contributes to the oligomer formation. The Kpn loop, named after the Killer of prune mutation in Drosophila that leads to lethality of the prune mutant (see Hsu’s review this issue), includes the highly conserved residue proline-97. Overall, the Kpn loop is highly conserved across NM23 members, although group II is characterized by an insertion of three additional amino acids (shown in Fig. 3). In Dictyostelium, the corresponding Kpn mutation makes the protein unstable due to dissociation of the hexamer [52, 53]. Interestingly, all NM23-H4 orthologs naturally possess the equivalent of the killer of prune mutation (a serine instead of proline at position 129 of NM23-H4). Mutation of this serine residue in Xenopus NM23-X4 did not alter the activity of NM23-X4 on retinal cell fate determination [33], suggesting that the structure of loop in these orthologs might be different.

Although the residues responsible for the NDP kinase and protein kinase activities appear largely conserved between members, there are some differences in the domain structure. NM23-H1 to H6 orthologs only have a single NDP kinase domain, while NM23-H7 orthologs have duplicated NDP kinase modules and NM23-H8 have triplicate. Also, NM23-H5 has an additional Dpy-30 superfamily domain at the C-terminus, while NM23-H7 and H8 have DUF1126/DM10 and thioredoxin superfamily motif at the N-terminus, respectively (Table 1). The DUF1126/DM10 superfamily domain has only been identified in two types of proteins: the NM23-H7 and the Rib72/Efhc1 type of proteins in mammals. It has been proposed that these domains act as flagellar NDK regulatory modules or units involved in axonemal targeting or assembly. The thioredoxin superfamily module has a redox-active disulphide bond, an amidation site and a cAMP/cGMP-dependent protein kinase phosphorylation site.

In the last 20 years, many binding partners of the NM23 family members have been identified (see reviews [54] and special issue of J Bioenerg Biomembr 2006). Recently, we have identified cyclin-dependent kinase inhibitors (CDKIs) as novel binding partners of the NM23 family [33]. Our analysis has shown that NM23-X3 and X4 have the highest affinity although other Xenopus members also bind weakly. A well-known binding partner is Prune, a DHH phosphodiesterase that binds to NM23-H1 through its C-terminal region [28, 55]. Point mutations in conserved serine residues of NM23 (serines-120, 122 and 125 in NM23-H1) impaired this interaction. Interestingly, the serine residues between 120 and 125 are phosphorylated by casein kinase I and II, and this phosphorylation is important for the modulation of the interaction with Prune. Although identification of binding partners suggests involvement of the NM23 family in many cellular events, we need to know much more about structural and functional differences among the NM23 family members.

Cellular localization

Localized cellular distribution of NM23 family members has been reported. A majority of papers show that NM23-H1 is largely localized in the cytosol, while NM23-H2 localizes in the nucleus. This localization pattern correlates with the NDP kinase and protein kinase activities of NM23-H1 and the transcriptional role of NM23-H2, respectively [14]. Similar localization data have been reported for the rodent orthologs [56]. However, other papers provide evidence of NM23 members H1, H2 and H3 being localized to the plasma membrane and/or nucleus [57, 58]. The reported NM23-H1 protein functions of exonuclease or DNase in the nucleus [19, 20] may suggest the possibility of protein translocation. Furthermore, NM23-H1 is also reported as a secreted protein [59]. NM23-H3 is cytoplasmic and NM23-M3 was reported to be localized in Golgi and endoplasmic reticulum membranes [56]. NM23-H4 has a signal sequence for mitochondria localization and indeed distributes in the mitochondria [60]. However, we have found that the gliogenic activity of NM23-X4 in Xenopus retinal cell fate determination does not require its mitochondrial localization [33]. NM23-H6 is known to localize in both cytosol and mitochondria [42].

Expression pattern of NM23 family members in vertebrates

The genes of the mouse NM23 family show overlapping and distinct spatio-temporal patterns of expression during embryonic development and adult life. Gene expression analysis of NM23-M1, M2, M3 and M4 by in situ hybridization shows that they are highly expressed in the central nervous system [61]. NM23-M1, M2 and M3 are expressed preferentially in the postmitotic neuronal differentiation layer, while NM23-M4 is expressed in the proliferating layer. In the case of the peripheral nervous system, expression of NM23-M1 is detected immediately at the end of migration of the dorsal root ganglia from neural crest precursors and before their start of differentiation in ganglia [56]. Immunohistochemical analysis of NM23-M1 showed that an increase in NM23-M1 level coincides with the onset of organogenesis and persists in tissues of epithelial origin, neural tissue and the heart [62]. Transient expression of NM23-M1 in intestinal epithelia or mammary glands suggests a role in cell differentiation and maintenance of stably differentiated epithelia [62].

In the case of human, NM23-H1 and H2 are ubiquitous with higher expression in brain, kidney, liver, pancreas and heart. NM23-H3 expression is ubiquitous but enriched in specific regions of the central nervous system such as cerebellum and pituitary. NM23-H4 has higher expression in heart, prostate and liver. NM23-H5 is specifically expressed during spermatogenesis like its mouse homologue NM23-M5 although the expression timing seems to be different from that of NM23-M5 [63, 64]. NM23-H5 is also expressed in brain, parathyroid, testis and ovary [65]. NM23-H6 is found in most human tissues tested, but most abundant in skeletal muscle, kidney, prostate, ovary, intestine and spleen [66]. NM23-H7 and NM23-H8 are found in testis, liver, heart, brain, ovary, small intestine and spleen.

In the case of Xenopus, Ouatas et al. reported that NM23-X1a, X1b and X2 are differentially distributed throughout the Xenopus oocyte and early embryos with a high level of transcription in somites, brain, optic vesicle and branchial arches. We have performed in situ hybridization (Fig. 4). All of the Xenopus NM23 members show high expression in the head at tadpole stages, especially in brain regions and differentiating tissues of the CNS, the otic vesicle, the branchial arches and the eye (Fig. 4). Looking more closely at retinal sections throughout development, NM23-X3 and X4 show prominent staining in the ciliary marginal zone (CMZ) [33]. The CMZ is a proliferating region in the retina that stays mitotically active throughout the life of the frog. The CMZ has retinal stem cells at the most peripheral region, then proliferating retinoblasts and finally differentiating retinoblasts. NM23-X3 and X4 are expressed in proliferating retinoblasts and beginning of differentiating retinoblasts. On the other hand, other members of the Xenopus NM23 family are expressed mainly in the postmitotic differentiated retina (unpublished data). This expression pattern among proliferating neural cells is consistent with expression pattern of mouse NM23 members, in which NM23-M1 and M2 are expressed in postmitotic cells, while NM23-M3 is expressed in mitotically active cells [61]. In addition to their expression in the nervous system, Xenopus NM23 members show additional patterns, distinct for each member (Fig. 4). For example, NM23-X8 has a dotted pattern in epidermis, revealing staining of ciliated cells. NM23-X7 has three positive spots in the pancreas precursor region. NM23-X2 and X5 are strongly expressed in somites.
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Fig. 4

Expression patterns of NM23 family members in Xenopus laevis embryos at tadpole stages. Whole mount in situ hybridisation was undertaken on fixed embryos using DIG-labelled probes against Xenopus NM23-X2, X3, X5, X6, X7 and X8. Higher magnification images are included to show areas of interest. All NM23 members have stronger expression in the central nervous system including brain, eye and otic vesicle. Note: NM23-X2 and X5 are expressed in the somites. NM23-X7 has specific expression in three spots of pancreas as indicated by white arrows. NM23-X8 is expressed in some cells in the epidermis. The developmental stage is shown in each case. Sense probe was used as a negative control

The roles of the NM23 family in development

The NM23 genes were multiplied during evolution but then remain conserved after their appearance in vertebrates. We referred to the expression patterns of the NM23 family during the embryonic and adult stages of organisms. Taken together, all data suggest that they have important roles in vertebrate development. In the second part of this review, we will summarize the current understanding of the roles of the NM23 family in development, in particular, neural development and gamete development.

The role of the NM23 family in development of lower organisms

The M. xanthus homologue of NM23, known as ndk, seems to be essential for bacterial growth [29]. The expression of the D. discoideum homologues, gip17 and guk7.2, is modulated during the development phases of cell growth or aggregation due to starvation [67]. In Drosophila, awd is required for proper differentiation of many tissues including brain and eye. Null or hypomorphic mutations allow normal embryonic development but cause widespread abnormalities in larvae including altered morphology and cell necrosis in the wing discs, vacuolated larval brain, poor differentiation of the eye/antenna and ovaries [27, 68, 69]. NDP kinase activity is necessary but not sufficient for the biological rescue [39]. It is also required for proper cell migration of tracheal cells by regulating FGF receptor levels and synergizing with shibire/dynamin in vesicular recycling pathway during tracheal development [24]. A more detailed review concerning Drosophila awd is available in this issue by Hsu’s group.

The roles of NM23 family members in neural development

As mentioned, all members of the NM23 family are expressed in the developing and mature nervous system and some members show very unique expression patterns. Their functions in distinct aspects of neural development and neural function have been documented in several papers. Neural development starts by neural induction in late gastrulation and is then achieved by consecutive multiple steps of neural patterning, cell fate determination, differentiation and cell migration. NM23 members seem to work in all of these developmental processes. Also, NM23 members have roles in mature neurons and glial cells through regulation of synaptic function. Here, we summarize the roles of NM23 family members in distinct aspects of neural development.

Neural induction and patterning

It was known that some NM23 members are associated with cell membranes [50, 51]. Recently, Mitchell et al. [57] showed that NM23-M1, M2 and possibly their Xenopus orthologs are localized in the developing primary cilia of murine NIH3T fibroblasts and Xenopus A6 kidney epithelial cell. Also they are localized in sperm flagella [64]. These observations indicate that NM23-M1 and M2 are universal components of primary cilia. As mentioned, these NM23 members are expressed in neuroectoderm-derived tissues during neural induction and patterning. In Xenopus, NM23-X2 (previously known as NM23-X3) is expressed in the organizer, which induces neural cell fate in overlaying tissues [35]. Also, all members of the Xenopus NM23 family are enriched in neuroectodermal regions. Cilia play very important roles in early embryogenesis. In particular, many reports indicate that cilia provide functional sites for shh signalling, Wnt signalling and TGF-β superfamily signalling [70]. These pathways have fundamental roles in neural patterning. The function of cilia is mediated by regulation of microtubules. The regulators of microtubules are largely managed by the intraflagellar transport proteins, which are required for shh-signalling machinery [70], and are indeed co-localized with NM23 proteins [57]. NM23-R1/H1 and NM23-H2 are known to interact with tubulins [21, 71, 72]. NM23-H1 and awd interact with dynamin, which regulates microtubule polymerization [24, 25]. Also, NM23 members regulate microtubule function through interaction with and activation of the small monomeric GTPases like Tiam1 [73] and ICAP-1 [23]. These observations suggest a possible role of NM23 members in neural patterning. Although we still don’t have direct functional evidence of NM23 involvement in neural patterning through regulation of cilia function, these circumstantial observations seem to propose a novel function of NM23 in neural development.

Cell fate determination

In vertebrates, neural cell fates are determined before or around the final cell cycle. Interestingly, NM23-M4 is expressed in proliferating cells, while NM23-M1 is mainly expressed in postmitotic cells in E18.5 brain cortex [61]. Recently, our studies of retinal cell fate determination have identified NM23-X4 as a binding partner of the Xenopus CDKI, p27Xic1 [33]. In Xenopus retinogenesis, p27Xic1 functions as a determinant of Müller glial cells in addition to its role as a cell cycle inhibitor. Expression of NM23-X4 and NM23-X3 in the retina starts slightly earlier than p27Xic1 and then overlaps with p27Xic1. This expression timing is coincident with the timing of retinal cell fate determination. On the other hand, NM23 members other than these two are mainly expressed after cell fate determination (unpublished data). Our in vivo functional analysis using Xenopus retina has revealed that introduction of shRNAs against NM23-X4 into retinal stem cells/progenitor cells activates gliogenesis and that co-overexpression of NM23-X4 with p27Xic1 inhibits p27Xic1-mediated gliogenesis [33]. Further analysis has revealed that the NM23-X4 shRNA-mediated activation of gliogenesis does not happen in the presence of p27Xic1 shRNA. In addition, the inhibition of p27Xic1-mediated gliogenesis requires direct interaction of NM23-X4 with the N-terminal side of p27Xic1. These observations clearly demonstrate that NM23-X4 functions as an inhibitor of gliogenesis through interaction with p27Xic1. This activity requires serine-150 and histidine-148, which correspond to the important residues for the kinase activities of NM23 family members. Our previous analysis showed that in addition to the activation of gliogenesis, p27Xic1 has the ability to potentiate neurogenesis. In Xenopus retinal development, gliogenesis always proceeds after the completion of neurogenesis. Therefore, it was a question how p27Xic1-mediated gliogenic activity is suppressed during neurogenesis. NM23-X4 expression commences earlier than p27Xic1 and inhibits p27Xic1-mediated gliogenic activity. These observations propose a model of NM23-X4 and p27Xic1 function in retinogenesis, in which NM23-X4 contributes to delaying gliogenesis and inhibits precocious gliogenesis in the sequential production of retinal neurons and glial cells.

The role of the NM23 family in cell fate determination was also reported in other systems such as hematopoietic cells (NM23-H1) [74, 75] and bone marrow cells (NM23-H3) [76]. Although we need to wait for further detailed studies of their mechanisms, the NM23 family seems to have a fundamental role in cell fate determination.

Differentiation and migration of neural cells

In the presence of nerve growth factor, NM23-M1 activation in PC12 cells induces neurite outgrowth, while its down-regulation inhibits neuronal differentiation [77]. Also, inactive mutants of NM23-R1 and R2 suppressed neurite outgrowth induced by nerve growth factor or by cyclic AMP in PC12D cells [78], demonstrating the role of NM23 family members on neuronal differentiation. Also, NM23 is well known to regulate cancer cell migration [3].

NM23s associate with microtubules [21, 71, 72]. NM23-M1 co-precipitates with Tiam1 from mouse brain extract [73]. Tiam1 is the nucleotide exchange factor for Rac1. Also, NM23-H2 binds to ICAP-1 [23]. ICAP-1 interacts with Rac1 and regulates integrin-mediated cell adhesion. Furthermore, we found that NM23 members directly bind to the Kip/Cip CDKI family. Interestingly, CDKIs have been reported to regulate neural cell migration [79] through interaction of RhoA, ROCK, LIMK and stathmin [8083]. RhoA, ROCK and LIMK are components of the Rho signalling pathway and stathmin is a regulator of actin fibres. The small GTPases of Rho, Rac and cdc42 are major regulators of cytoskeleton which is important for both neurite outgrowth and migration [84] and their activities are regulated by GTP levels. Although little is yet known about the role of NDP kinase activity of NM23 family members on the small GTPases, control of GTP level by NM23s might have a contribution. All these observations indicate that, through interaction with Rac-, Rho-, cdc42-related signalling pathways, NM23s seem to regulate neural differentiation and neural cell migration.

Neural activity

NM23 members are essential for normal presynaptic function by regulating synaptic vesicle internalization [25]. We have previously referred to the role of the Drosophila member in synergizing with shibire/dynamin in vesicular recycling and receptor internalization. A rat NM23 member was also found to interact with dynamin at the synapses and with phocein at the dendritic region of rat hippocampal neurons [85]. Phocein is a clathrin adaptor protein homologue that localizes in dendritic spines of neurons in the central and peripheral nervous system [85]. It has been proposed that a complex between dynamin, phocein, NM23 and Eps regulates vesicular trafficking in neurons.

Cell cycle regulation in neural development

Co-ordination of cell cycle with multiple developmental processes is fundamentally important to achieve proper neural development [8688]. The role of the NM23 members on cell cycle regulation is evident based on their expression during neural development and their effect on cell cycle. As mentioned, NM23-M4 is largely expressed in proliferative cells, while NM23-M1 is expressed in postmitotic cells [61]. Also, in Xenopus retina, NM23-X3 and X4 are expressed in proliferating retinoblasts, while NM23-X1 and X2 are mainly expressed in postmitotic cells [33]. The contribution of NM23 family to the regulation of the cell cycle has been studied extensively in the cancer field. Enforced expression of NM23-H1 in the breast cancer cell line MDA-MB435 induces growth arrest [89]. Overexpression of NM23-H6 in SAOS2 cells resulted in growth suppression and generated multinucleated cells [42]. On the one hand, NM23-H2-transfected keratinocytes display enhanced proliferation [90]. In neural development, in association with neuronal differentiation, overexpression of NM23-H3 in neuroblastoma cells [91] and overexpression of NM23-M1 in PC12 cells [77] result in cell cycle arrest. On the other hand, we have found that NM23-X4 works as an activator of cell cycle [33]. These observations indicate that NM23 members have the ability to influence cell cycle, but the direction is context dependent.

How do NM23 members influence cell cycle? NM23-H1 and H2 show cell cycle-dependent nuclear localization [92]. NM23 members may influence the cell cycle through regulation of microtubules during mitosis. The intracellular level of GTP, which has multiple contributions in the cell cycle, might contribute to NM23-mediated cell cycle regulation. Also, NM23-H1 has been reported to work as a component of the centrosome through interaction with centrosomal kinase, aurora A [21, 22]. Furthermore, we have found that NM23 directly binds to CDK inhibitors such as p27Xic1 and that NM23-X4 activates cell cycle through inhibition of activity of p27Xic1. Further analysis will be required to understand the mechanisms of cell cycle regulation.

NM23 in oogenesis and spermatogenesis

Drosophila awd is required for normal oocyte differentiation and female fertility [39, 69]. It was shown that the Drosophila homologue awd acts as a negative regulator of the directional migration of border cells during fly oogenesis by interacting with the pathways of Pvf receptor and JAK/STAT signalling pathways [93]. During Xenopus oogenesis, NM23 transcripts are expressed in previtellogenic oocytes and localize at the germinal vesicle and the Balbiani body [35]. Progesterone is known to induce maturation of Xenopus oocytes. NM23-H1 and H2 were shown to inhibit the process of progesterone-induced maturation of the frog oocyte [94]. The production of a fertilisable egg involves cell-cycle regulation, signal transduction through MEK and p42/MAPK and translational control of Mos. The two NM23s appear able to inhibit this process by acting upstream of the Mos/MEK/p42 cascade and this activity is independent of their NDP kinase activity.

NM23-H5 is expressed in the male gametes during spermiogenesis and at later stages of spermatogenesis [65], and the protein is located in the flagella of spermatids and spermatozoa, adjacent to the central pair and outer doublets of microtubules. Also, NM23-H8, which is found in human sperm flagella, has a potential role in flagellar movement in spermiogenesis [95]. On the other hand, NM23-H1 and H2 are found in postmeiotic germinal cells and spermatids [64]. NM23-M5 is also expressed in late stage spermatids and adult testis [63]. Although their function has not been reported, current information supports their role in flagellar movement in spermiogenesis. Also, after fertilization, transcripts of NM23-H1, H2 and H3 are detected in distinct cell populations in the human placenta, the foetomaternal structure essential for the nourishment of the embryos. In situ hybridization and immunohistochemistry in floating villi and placentae shows that NM23-H1 and H2 are found exclusively in the cytotrophoblasts, the trophoblastic stem cell population, while NM23-H3 is localized in plasma membrane of the differentiated multinucleated syncytiotrophoblast layer [96]. Also, all Xenopus members are expressed during early embryogenesis [35]. These observations show that the NM23 family members have important roles in oogenesis, spermatogenesis and early embryogenesis.

Role of the NM23 family in skin homeostasis

Expression levels of NM23 members are modulated in mouse primary keratinocytes [97] and the epidermal fraction of normal and hyperplastic human skin [98]. Also, NM23-H1 and H2 members have been shown to be targets of keratinocyte and epidermal growth factors that are key regulators of keratinocyte proliferation, differentiation and survival [90]. Also, our expression analysis of Xenopus members shows that NM23-X8 is specifically expressed in a subpopulation of epidermal cells (Fig. 3). These data support involvement of the NM23 family in skin homeostasis and repair.

Conclusions

The NM23 family is almost universally conserved, and highly homologous orthologs exist in everything from bacteria to man. The phylogenetic analysis performed in this review reveals that unicellular life forms have single NM23 orthologs, but vertebrates have as many as 10. Most NM23 family members arose very early in animal evolution and 10 different members could be clearly defined in multiple vertebrate species. The conservation of the NM23 family suggests that they have an important fundamental cellular function. However, the multiplication of NM23 members in animal species indicates that different members are likely to have distinct roles. Their expression and functional analyses have shed some light on their developmental roles. In particular, the current observations emphasize their roles in neural development. The NM23 family members seem to have multiple roles during the processes of neural development including neural patterning, cell fate determination, differentiation and cell migration. Interestingly, some of these activities might be NDP kinase independent. All these observations indicate the importance of the NM23 family in development and normal homeostasis. There is still much work to be done, however, to determine the roles and the detailed mechanisms of the NM23 family. We believe that greater focus on these questions is pertinent and timely, and that it will lead to a greater understanding of NM23’s role in the process of development gone awry that is carcinogenesis.

Acknowledgements

We are grateful to Dr. T. Mochizuki for contribution of the in situ images. This work was supported by Cancer Research, UK, and Fight for Sight.

Copyright information

© Springer Science+Business Media, LLC. 2009