Molecular cloning and characterization of the human NIMA-related protein kinase 3 gene (NEK3)

NEKs (NIMA-related kinases) are a group of protein kinases sharing high amino acid sequence identities with NIMA (never in mitosis gene a) which control mitosis in Aspergillus nidulans. We have cloned a cDNA for human NEK3, a novel human gene structurally related to NIMA, by RT-PCR. Its open reading frame encodes a protein of 489 amino acid residues with the calculated molecular mass of 56.0 kDa and a predicted pI of 6.58. Phylogenetic analysis suggests that mouse and human NEK3s constitute a subfamily within the NIMA family of protein kinases. The expression pattern of NEK3 was studied by RT-PCR and a high level of expression was detected in testis, ovary, and brain, with low-level expression being detected in most of the tissues studied. NEK3 mRNA was detected in all the proliferating cell lines studied, and the amount did not change during the cell cycle. The human NEK3 gene was assigned to human chromosome 13 by somatic cell hybrids and 13q14.2 by radiation hybrid mapping.     

NEK7 is a centrosomal kinase critical for microtubule nucleation

NIMA in Aspergillus nidulans is a mitotic kinase for chromosome condensation and segregation. We characterized NEK7, a human homologue of Aspergillus NIMA. NEK7 was located at the centrosome throughout the cell cycle. Temporal localization of NEK7 at midbody of the cytokinetic cell was also observed. NEK7 knockdown by RNAi caused a prometaphase arrest of the cell cycle with monopolar or disorganized spindle. We propose that such defects in spindle assembly are resulted from reduction in microtubule nucleation activity at the centrosome. In consistent to the proposal, we observed a decrease in the centrosomal gamma-tubulin levels and reduction of the microtubule re-growth activity in the NEK7-suppressed cells. In addition, it was evident that NEK7 was directly involved in cytokinesis.     

Identification of proteins that interact with the central coiled-coil region of the human protein kinase NEK1

NEK protein kinases are evolutionarily conserved kinases structurally related to the Aspergillus nidulans mitotic regulator NIMA. At least nine members of the NEK family in vertebrates have been described to date, but for most of them the interacting protein partners are unknown. The pleiotropic deleterious effects and the formation of kidney cysts caused by NEK1 mutation in mice emphasize its involvement in the regulation of diverse cellular processes and in the etiology of polycystic kidney disease (PKD), respectively. Here we report the identification of proteins that interacted with the human NEK1 protein kinase in a yeast two-hybrid screen of a human fetal brain cDNA library, using the catalytic and regulatory domains of NEK1 separately as baits. These proteins are known to take part either in the development of PKD, in the double-strand DNA break repair at the G2/M transition phase of the cell cycle, or in neural cell development. The proteins involved in PKD include the motor protein KIF3A and the proteins tuberin and alpha-catulin. Mapping studies of the human NEK1 regulatory domain (NRD) indicated a strong interaction of most of the proteins retrieved from the library with putative coiled coils located in the central region of NRD. Our results give further support to the previous observation that NEK1 is of functional importance for the etiology of PKD.     

Isolation and characterization of two evolutionarily conserved murine kinases (Nek6 and nek7) related to the fungal mitotic regulator, NIMA

Entrance and exit from mitosis in Aspergillus nidulans require activation and proteolysis, respectively, of the NIMA (never in mitosis, gene A) serine/threonine kinase. Four different NIMA-related kinases were reported in mammals (Nek1-4), but none of them has been shown to perform mitotic functions related to those demonstrated for NIMA. We describe here the isolation of two novel murine protein kinase genes, designated nek6 and nek7, which are highly similar to each other (87% amino acid identity in the predicted kinase domain). Interestingly, Nek6 and Nek7 are also highly similar to the F19H6.1 protein kinase of Caenorhabditis elegans (76 and 73% amino acid identity in the kinase domain, respectively), and phylogenetic analysis suggests that these three proteins constitute a novel subfamily within the NIMA family of serine/threonine kinases. In contrast to the other documented NIMA-related kinases, Nek6/7 and F19H6.1 harbor their catalytic domain in the C-terminus of the protein. Immunofluorescence suggests that Nek6 and Nek7 are cytoplasmic. Linkage analysis, using the murine BXD recombinant inbred strain panel, localized nek6 to chromosome 2 at 28 cM. Using a mouse/hamster radiation hybrid panel, we assigned the nek7 gene to chromosome 1 at approximately 73 cM.     

NEK2A interacts with MAD1 and possibly functions as a novel integrator of the spindle checkpoint signaling

Chromosome segregation in mitosis is orchestrated by protein kinase signaling cascades. A biochemical cascade named spindle checkpoint ensures the spatial and temporal order of chromosome segregation during mitosis. Here we report that spindle checkpoint protein MAD1 interacts with NEK2A, a human orthologue of the Aspergillus nidulans NIMA kinase. MAD1 interacts with NEK2A in vitro and in vivo via a leucine zipper-containing domain located at the C terminus of MAD1. Like MAD1, NEK2A is localized to HeLa cell kinetochore of mitotic cells. Elimination of NEK2A by small interfering RNA does not arrest cells in mitosis but causes aberrant premature chromosome segregation. NEK2A is required for MAD2 but not MAD1, BUB1, and HEC1 to associate with kinetochores. These NEK2A-eliminated or -suppressed cells display a chromosome bridge phenotype with sister chromatid inter-connected. Moreover, loss of NEK2A impairs mitotic checkpoint signaling in response to spindle damage by nocodazole, which affected mitotic escape and led to generation of cells with multiple nuclei. Our data demonstrate that NEK2A is a kinetochore-associated protein kinase essential for faithful chromosome segregation. We hypothesize that NEK2A links MAD2 molecular dynamics to spindle checkpoint signaling.     

Cloning and characterization a novel human 1-acyl-sn-glycerol-3-phosphate acyltransferase gene AGPAT7.

The 1-Acylglycerolphosphate acyltransferase is crucial enzyme for synthesis of glycerolipids as well as triacylglylcerol biosynthesis in eukaryotes. Six members of 1-acyl-sn-glycerol-3-phosphate acyltransferase family in human have been described, which were AGPAT1, 2, 3, 4, 5 and 6. Here we report the cloning and characterization of another novel human 1-acyl-sn-glycerol-3-phosphate acyltransferase member AGPAT7 (1-acyl-sn-glycerol-3-phosphate acyltransferase 7) gene, which was mapped to human chromosome 15q14. The AGPAT7 cDNA is 1898 bp in length, encoding a putative protein with 524 amino acid residues, which contains an acyltransferase domain in 123-234 aa. RT PCR amplification in 18 human tissues indicated that human AGPAT7 gene was widely expressed in uterus, thymus, pancreas, skeletal muscle, bladder, stomach, lung and testis. AGPAT7 protein was mainly localized to the endoplasmic reticulum (ER) in Hela cells.     

A gemcitabine sensitivity screen identifies a role for NEK9 in the replication stress response

The Replication Stress Response (RSR) is a signaling network that recognizes challenges to DNA replication and coordinates diverse DNA repair and cell-cycle checkpoint pathways. Gemcitabine is a nucleoside analogue that causes cytotoxicity by inducing DNA replication blocks. Using a synthetic lethal screen of a RNAi library of nuclear enzymes to identify genes that when silenced cause gemcitabine sensitization or resistance in human triple-negative breast cancer cells, we identified NIMA (never in mitosis gene A)-related kinase 9 (NEK9) as a key component of the RSR. NEK9 depletion in cells leads to replication stress hypersensitivity, spontaneous accumulation of DNA damage and RPA70 foci, and an impairment in recovery from replication arrest. NEK9 protein levels also increase in response to replication stress. NEK9 complexes with CHK1, and moreover, NEK9 depletion impairs CHK1 autophosphorylation and kinase activity in response to replication stress. Thus, NEK9 is a critical component of the RSR that promotes CHK1 activity, maintaining genome integrity following challenges to DNA replication.     

Differential control of the NIMA-related kinases,Nek6 and Nek7, by serum stimulation

Neks (NIMA-related kinases) are mammalian serine/threonine (Ser/Thr) protein kinases structurally related to Aspergillus NIMA (Never in Mitosis, gene A), which plays essential roles in mitotic signaling. Among these kinases, Nek6 and Nek7 are structurally related and constitute a subfamily in the NIMA/Nek family, although their functions still remain almost elusive. In this report, we studied the enzymatic regulation of Nek6 and Nek7 to gain an insight into their cellular functions. Recombinant Nek7 produced in bacteria was active comparably to Nek6; however, the Nek7 activity in mammalian cells was found to be significantly lower than Nek6. Since Nek6 previously has been reported to in vitro phosphorylate p70 ribosomal S6 kinase at Thr412, we examined if Nek6 and Nek7 activities were controlled by the amino acid supplement, which is known to affect the phosphorylation at Thr412, and did not observe any significant effect. However, we unexpectedly found that Nek7 kinase activity was rapidly and efficiently increased by serum deprivation, while Nek6 activity was decreased. This is well consistent with the lower activity of Nek7 in cells under normal growth conditions. In addition, it was suggested that Nek7 activity would be regulated in a cell cycle-dependent manner, although Nek6 was not. These clear differences in enzymatic control between the highly similar kinases, Nek6 and Nek7, suggest their distinct signaling functions in mammalian cells.     

The Tumor Suppressor pVHL Down-regulates Never-in-Mitosis A-related Kinase 8 via Hypoxia-inducible Factors to Maintain Cilia in Human Renal Cancer Cells

NEK8 (never in mitosis gene A (NIMA)-related kinase 8) is involved in cytoskeleton, cilia, and DNA damage response/repair. Abnormal expression and/or dysfunction of NEK8 are related to cancer development and progression. However, the mechanisms that regulate NEK8 are not well declared. We demonstrated here that pVHL may be involved in regulating NEK8. We found that CAK-I cells with wild-type vhl expressed a lower level of NEK8 than the cells loss of vhl, such as 786-O, 769-P, and A-498 cells. Moreover, pVHL overexpression down-regulated the NEK8 protein in 786-O cells, whereas pVHL knockdown up-regulated NEK8 in CAK-I cells. In addition, we found that the positive hypoxia response elements (HREs) are located in the promoter of the nek8 sequence and hypoxia could induce nek8 expression in different cell types. Consistent with this, down-regulation of hypoxia-inducible factors α (HIF-1α or HIF-2α) by isoform-specific siRNA reduced the ability of hypoxia inducing nek8 expression. In vivo, NEK8 and HIF-1α expression were increased in kidneys of rats subjected to an experimental hypoxia model of ischemia and reperfusion. Furthermore, NEK8 siRNA transfection significantly blocked pVHL-knockdown-induced cilia disassembling, through impairing the pVHL-knockdown-up-regulated NEK8 expression. These results support that nek8 may be a novel hypoxia-inducible gene. In conclusion, our findings show that nek8 may be a new HIF target gene and pVHL can down-regulate NEK8 via HIFs to maintain the primary cilia structure in human renal cancer cells.     

Identification of the NIMA family kinases NEK6/7 as regulators of the p70 ribosomal S6 kinase

BACKGROUND: The p70 S6 kinase, like several other AGC family kinases, requires for activation the concurrent phosphorylation of a site on its activation loop and a site carboxyterminal to the catalytic domain, situated in a hydrophobic motif site FXXFS/TF/Y, e.g.,Thr412 in p70 S6 kinase (alpha 1). Phosphorylation of the former site is catalyzed by PDK1, whereas the kinase responsible for the phosphorylation of the latter site is not known. RESULTS: The major protein kinase that is active on the p70 S6 kinase hydrophobic regulatory site, Thr412, was purified from rat liver and identified as the NIMA-related kinases NEK6 and NEK7. Recombinant NEK6 phosphorylates p70 S6 kinase at Thr412 and other sites and activates the p70 S6 kinase in vitro and in vivo, in a manner synergistic with PDK1. Kinase-inactive NEK6 interferes with insulin activation of p70 S6 kinase. The activity of recombinant NEK6 is dependent on its phosphorylation, but NEK6 activity is not regulated by PDK1 and is only modestly responsive to insulin and PI-3 kinase inhibitors. CONCLUSION: NEK6 and probably NEK7 are novel candidate physiologic regulators of the p70 S6 kinase.     

NIMA-related kinase 7 amplifies NLRP3 inflammasome pro-inflammatory signaling in microglia/macrophages and mice models of spinal cord injury

BackgroundNIMA-related kinase-7 (NEK7) is a serine/threonine kinase that drives cell-cycle dynamics by modulating mitotic spindle formation and cytokinesis. It is also a crucial modulator of the pro-inflammatory effects of NOD-like receptor 3 (NLRP3) inflammasome. However, the role of NEK7 in microglia/macrophages post-spinal cord injury (SCI) is not well defined.MethodsIn this study, we performed both in vivo and in vitro experiments. Using an in vivo mouse SCI model, NEK7 siRNAs were administered intraspinally. For in vitro analysis, BV-2 microglia cells with NEK7-siRNA were stimulated with 1 μg/ml lipopolysaccharide (LPS) and 2 mM Adenosine triphosphate (ATP).ResultsHere, we found that the mRNA and protein levels of NEK7 and NLRP3 inflammasomes were upregulated in spinal cord tissues of injured mice and BV-2 microglia cells exposed to Lipopolysaccharide (LPS) and Adenosine triphosphate (ATP). Further experiments established that NEK7 and NLRP3 interacted in BV-2 microglia cells, an effect that was eliminated following NEK7 ablation. Moreover, NEK7 ablation suppressed the activation of NLRP3 inflammasomes. Although NEK7 inhibition did not significantly improve motor function post-SCI in mice, it was found to attenuate local inflammatory response and inhibit the activation of NLRP3 inflammasome in microglia/macrophages of the injured spinal cord.ConclusionNEK7 amplifies NLRP3 inflammasome pro-inflammatory signaling in BV-2 microglia cells and mice models of SCI. Therefore, agents targeting the NEK7/NLRP3 signaling offers great promise in the treatment of inflammatory response post-SCI.     

Protein kinases predominately expressed in human ES cell lines during differentiation

Capacity of human embryonic stem cells (ESC) for unlimited proliferation and differentiation make them an attractive object in fundamental science and medicine. Little is known about the mechanisms that direct cells to particular differentiation or sustain them in an undifferentiated state. Activation of these mechanisms is determined by gene expression mediated by cascades of signal transduction. Protein kinases are essential components of signal pathways. The study of protein kinases expression in ESC and embryoid bodies facilitates a better understanding of the processes underlying the differentiation stages. We isolated cDNA libraries with fragments of catalytic domains of protein kinases expressed in human ESC and embryoid bodies (EB) of hESM01 and hESM02 cell lines. Using Northern hybridization, we revealed a high level of protein kinases MAK-V in human ESC. Expressions of MAK-V, A-RAF-1, MAPK3, IGF1R, NEK3, and NEK7 in ESC and EB in hESM01 and hESM02 cell lines were compared by the semiquantitative method RT-PCR.     

A NIMA-related protein kinase suppresses ectopic outgrowth of epidermal cells through its kinase activity and the association with microtubules

To study cellular morphogenesis genetically, we isolated loss-of-function mutants of Arabidopsis thaliana , designated ibo1 . The ibo1 mutations cause local outgrowth in the middle of epidermal cells of the hypocotyls and petioles, resulting in the formation of a protuberance. In Arabidopsis, the hypocotyl epidermis differentiates into two alternate cell files, the stoma cell file and the non-stoma cell file, by a mechanism involving TRANSPARENT TESTA GLABRA1 ( TTG1 ) and GLABRA2 ( GL2 ). The ectopic protuberances of the ibo1 mutants were preferentially induced in the non-stoma cell files, which express GL2 . TTG1-dependent epidermal patterning is required for protuberance formation in ibo1 , suggesting that IBO1 functions downstream from epidermal cell specification. Pharmacological and genetic analyses demonstrated that ethylene promotes protuberance formation in ibo1 , implying that IBO1 acts antagonistically to ethylene to suppress radial outgrowth. IBO1 is identical to NEK6 , which encodes a Never In Mitosis A (NIMA)-related protein kinase (Nek) with sequence similarity to Neks involved in microtubule organization in fungi, algae, and animals. The ibo1-1 mutation, in which a conserved Glu residue in the activation loop is substituted by Arg, completely abolishes its kinase activity. The intracellular localization of GFP-tagged NEK6 showed that NEK6 mainly accumulates in cytoplasmic spots associated with cortical microtubules and with a putative component of the γ-tubulin complex. The localization of NEK6 is regulated by the C-terminal domain, which is truncated in the ibo1-2 allele. These results suggest that the role of NEK6 in the control of cellular morphogenesis is dependent on its kinase action and association with the cortical microtubules.