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Nek7 Kinase Targeting Leads to Early Mortality, Cytokinesis Disturbance and Polyploidy

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Nek7 Kinase Targeting Leads to Early Mortality, Cytokinesis Disturbance and Polyploidy Oncogene (2010) 29, 4046–4057 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 www.nature.com/onc ORIGINAL ARTICLE Nek7 kinase targeting leads to early mortality, cytokinesis disturbance and polyploidy H Salem1,5, I Rachmin1,5, N Yissachar1,4,5, S Cohen1, A Amiel1,2, R Haffner3, L Lavi1 and B Motro1 1The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel; 2Genetics Institute, Meir Medical Center, Kfar-Saba, Israel and 3Department of Veterinary Resources, Weizmann Institute of Science, Rehovot, Israel The mammalian NIMA-related kinases (Neks) are eukaryotic evolution. Homologs for the Ser/Thr kinase commonly referred to as mitotic kinases, although a NIMA, an essential mitotic regulator of the filamentous definitive in vivo verification of this definition is largely fungus Aspergillus nidulans (Osmani et al., 1991),canbe missing. Reduction in the activity of Nek7 or its close found in almost all protists, plants and animals (Parker paralog, Nek6, has previously been shown to arrest cells in et al., 2007). As implied by its name (‘never in mitosis mitosis, mainly at metaphase. In this study, we investigate A’), NIMA activity is vital for fungal cells to overstep the developmental and cellular roles of Nek7 kinase the G2/M checkpoint and to enter mitosis. The NIMA through the generation and analysis of Nek7-deficient protein has been shown to be implicated in chromatin mice. We show that absence of Nek7 leads to lethality condensation, cell division cycle 2/cyclin B localization in late embryogenesis or at early post-natal stages and to and microtubule network dynamics (Fry and Nigg, severe growth retardation. Mouse embryonic fibroblasts 1995; De Souza et al., 2003; O’Connell et al., 2003). The (MEFs) derived from Nek7À/À embryos show increase mammalian genome has 11 NIMA-related kinases, tendency for chromosomal lagging, micronuclei formation designated Nek1-11 (O’Connell et al., 2003; O’Regan and cytokinesis failure. Tetraploidy and aneuploidy et al., 2007). Although many of these kinases are poorly were commonly observed and their prevalence arises with characterized, recent studies assign aspects of cell cycle MEFs passages. The frequency of multicentrosomal cells regulation to some of the Neks, which therefore are in the mutant’s MEF cells was higher, and it commonly mostly referred to as a family of ‘mitotic kinases’ occurred concurrently with a binuclear phenotype, sug- (O’Regan et al., 2007). gesting cytokinesis failure etiology. Lastly, the percentage Mammalian Nek7, and its close paralog, Nek6, of mutant MEF cells bearing primary cilia (PC) was low, represent a subgroup of the smallest Neks within whereas a cell population having two cilia appeared in the this family, consisting of a core kinase domain and a mutant MEFs. Taken together, these results confirm very short N-terminal tail (Kandli et al., 2000). They are Nek7 as a regulator of cell division, and reveal it as an remarkably similar to each other (87% identity in the essential component for mammalian growth and survival. kinase domain), and are highly conserved evolutionarily The intimate connection between tetraploidy, aneuploidy (for example, the kinase domain of the mammalian and cancer development suggests that Nek7 deregulation Nek6/7 is 76% identical to the Nek6/7 ortholog in can induce oncogenesis. Caenorhabditis elegans). A comparative analysis of Oncogene (2010) 29, 4046–4057; doi:10.1038/onc.2010.162; murine nek6 and nek7 expression reveals a complemen- published online 17 May 2010 tary tissue-specific expression pattern during embryonic development and in adults (Feige and Motro, 2002). Keywords: NIMA; kinase; gene targeting; early Interestingly, Nek6 and Nek7 bind to and are phos- lethality; cytokinesis; mitosis phorylated by another Nek kinase, designated Nercc1/ Nek9 (Roig et al., 2002; Belham et al., 2003). Nercc1 has been shown to function as a mitotic regulator required Introduction for accurate spindle assembly and cell cycle progression, and is recruited to the centrosome in its phosphorylated The cell cycle is tightly regulated by numerous enzymes, active form (Roig et al., 2005). The enrichment of Nek7 many of which have been conserved throughout in the centrosome has been demonstrated (Yissachar et al., 2006; Kim et al., 2007), strengthening the idea Correspondence: Dr B Motro, The Mina and Everard Goodman that Nercc1 and Nek7 (and presumably also Nek6) are Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel. components of a mitotic spindle-signaling cascade. E-mail: [email protected] We have shown previously that knock down of Nek7 4Current address: Department of Immunology, Weizmann Institute of by small interfering RNA (siRNA) results in a range of Science, Rehovot 76100, Israel. 5These authors contributed equally to this work. mitotic defects, assigning Nek7 as a centrosomal protein Received 12 November 2009; revised 1 April 2010; accepted 5 April 2010; involved in mitotic regulation (Yissachar et al., 2006). published online 17 May 2010 In line with these results, Nek7 involvement in g-tubulin Nek7 kinase targeting leads to polyploidy H Salem et al 4047 recruitment to the centrosome, together with micro- To explore in vivo the impact of Nek7 loss on mam- tubule nucleation activity was recently reported (Kim malian embryonic development and on the cell cycle, et al., 2007). Very recently, O’Regan and Fry (2009) Nek7-null mice were generated. The Nek7 mutants reported that depletion of Nek7 by siRNA or over- demonstrated prenatal and neonatal lethality. Mouse expression of dominant negative constructs leads to embryonic fibroblast (MEF) cells derived from Nek7 metaphase arrest depending on spindle assembly check- mice showed various mitotic defects and polyploidy. point activation. The overexpression of hypomorphic Our analysis indicated that Nek7 is essential for the mutant Nek7 protein increases the tendency for late accurate completion of cytokinesis, and that it is mitotic delay and cytokinesis failure. necessary for correct mammalian growth and survival. Interestingly, in spite of the proposition that Nek kinases are involved in mitotic regulation, the two naturally occurring mouse mutants for Nek kinases, kat (Nek1) and jck (Nek8), do not display obvious cell Results cycle disorders. Instead, in both cases polycystic kidney disease is a major or the sole phenotype, respectively Generation and analysis of Nek7 mutant mice (Upadhya et al., 2000; Liu et al., 2002). Consistent To investigate the in vivo functions of the mammalian with the polycystic kidney disease, localization of these Nek7 gene, we constructed a replacement vector to proteins to the centrosome and to the primary cilium replace most of exons 3 and 4 with b-galactosidase (Mahjoub et al., 2005; Shalom et al., 2008; White (b-gal)–NeoR cassette in mouse R1 embryonic stem cells and Quarmby, 2008), and ciliary phenotypes (Smith (Figures 1a and b). Homologous recombination should et al., 2006; Shalom et al., 2008) have been reported. insert the b-gal sequence at amino-acid 75 and delete EcoRI EcoRI 14 Kb 15.8Kb EcoRV EcoRV 4 5 Nek7 genomic 5’ probe 3’ probe locus 4 N-Lacz Neo 5 Replacement vector SacII NotIEcoRVHindIII SalI Homologous recombination 6.2 Kb 11.6 Kb 10.6 Kb 9 Kb EcoRIEcoRV EcoRI EcoRV EcoRI EcoRV 4 N-Lacz Neo 5 1 Kb NotI HindIII 5’ probe 3’ probe wt het wt het WT KO 14Kb 250 30 19.6Kb 130 95 25 72 20 9.0 Kb 55 15 10 Nek7 36 % Nek7 -/- 5 6.2Kb 28 0 Actin DPC Figure 1 Nek7 targeting results in dwarfism and early mortality. (a–b) Schematic representation of nek7 gene-targeting strategy. (a) nek7 genomic locus and the replacement vector. The replacement vector replaces most of exons 3 and 4 with b-galactosidase (b-gal)–Neo resistance cassette. (b) nek7 locus after expected recombination. Exons and major restriction enzymes used for vector creation and genotyping are indicated. The 50 and 30 external probes and the expected size of the restricted fragments before and after recombination are indicated. (c–d) Southern blot analysis of mice with (c)50 and (d)30 probes. (e) Western blot analysis of protein extracts from wild-type WT and mutant mouse embryonic fibroblasts using polyclonal antibodies against Nek7, and as a control with anti-actin antibodies. (f) Survival curve of homozygote embryos for nek7 mutation at the indicated days post-coitum after a cross between nek7 heterozygotes. (g) Nek7À/À newborns are dwarf. Photographs of WT (left) and mutant (right) siblings at postnatal day 5. Oncogene Nek7 kinase targeting leads to polyploidy H Salem et al 4048 amino-acids 76–112 of Nek7. The b-gal reporter gene is 38.3±9.5% binuclear cells in the Nek7À/À MEFs com- in frame with the initiating methionine, resulting in b-gal pared with about 14.6±5% in the WT (Figure 2d). In expression under the control of the endogenous Nek7 addition, the percentage of cells having more than promoter. The early termination of Nek7 should yield two nuclei (henceforth designated as multinuclear) was a null mutation, and potential splicing event from exon elevated in the mutants’ MEFs, with 4.4% of the late 2 to exon 5 will generate a nonfunctional kinase, lacking passage MEF cells being multinuclear compared with kinase subdomains II–V. Two independently targeted about 0.8% in the WT. embryonic stem (ES) cell clones were aggregated with The examination of nuclear size in mutant MEF cells wild-type (WT) ICR morulae, to generate two lines of revealed a higher proportion of the mononuclear cells mutant mice. Southern blotting was used to genotype which had large nuclei (Figures 2e–h at P3 and P7, WT, Nek7 þ /À and Nek7À/À mice (Figures 1c and d). respectively). To quantify this feature, nuclear size was Western blot analysis confirmed that the mutation measured using ImageJ software (National Institutes of abolishes Nek7 protein expression (Figure 1e).
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