Protein kinase which plays an important role in mitotic cell cycle progression. Required for chromosome segregation at metaphase-anaphase transition, robust mitotic spindle formation and cytokinesis. Phosphorylates ATF4, CIR1, PTN, RAD26L, RBBP6, RPS7, RPS6KB1, TRIP4, STAT3 and histones H1 and H3. Phosphorylates KIF11 to promote mitotic spindle formation. Involved in G2/M phase cell cycle arrest induced by DNA damage. Inhibition of activity results in apoptosis. May contribute to tumorigenesis by suppressing p53/TP53-induced cancer cell senescence.
Physical protein-protein interactions are fundamental to all biological processes and are organized in complex networks. One branch of the kinome network is the evolutionarily conserved NIMA-related serine/threonine kinases (Neks). Most of the 11 mammalian Neks studied so far are related to cell cycle regulation, and due to association with diverse human pathologies, Neks are promising chemotherapeutic targets. Human Nek6 was associated to carcinogenesis, but its interacting partners and signaling pathways remain elusive. Here we introduce hNek6 as a highly connected member in the human kinase interactome. In a more global context, we performed a broad data bank comparison based on degree distribution analysis and found that the human kinome is enriched in hubs. Our networks include a broad set of novel hNek6 interactors as identified by our yeast two-hybrid screens classified into 18 functional categories. All of the tested interactions were confirmed, and the majority of tested substrates were phosphorylated in vitro by hNek6. Notably, we found that hNek6 N-terminal is important to mediate the interactions with its partners. Some novel interactors also colocalized with hNek6 and γ-tubulin in human cells, pointing to a possible centrosomal interaction. The interacting proteins link hNek6 to novel pathways, for example, Notch signaling and actin cytoskeleton regulation, or give new insights on how hNek6 may regulate previously proposed pathways such as cell cycle regulation, DNA repair response, and NF-κB signaling. Our findings open new perspectives in the study of hNek6 role in cancer by analyzing its novel interactions in specific pathways in tumor cells, which may provide important implications for drug design and cancer therapy.
Nek6 is a recently identified NIMA-related kinase that is required for mitotic cell cycle progression. In the present study, we examined the role of Nek6 in the DNA damage response. We found that Nek6 is phosphorylated upon IR and UV irradiation through the DNA damage checkpoint in vivo. Nek6 is also directly phosphorylated by the checkpoint kinases Chk1 and Chk2 in vitro. Notably, Nek6 activation during mitosis is completely abolished by IR and UV irradiation. Moreover, the ectopic expression of Nek6 overrides DNA damage-induced G(2)/M arrest. These results suggest that Nek6 is a novel target of the DNA damage checkpoint and that the inhibition of Nek6 activity is required for proper cell cycle arrest in the G(2)/M phase upon DNA damage.
In Aspergillus nidulans, the kinase activity of NIMA (never in mitosis, gene A) is critical for the initiation of mitosis. NIMA regulates mitotic chromatin condensation through phosphorylation of histone H3 at serine 10. In the present study, we identified human Nek6 (hNek6), a member of the mammalian NIMA-related kinases. The predicted hNek6 protein is comprised of 338 amino acids. Northern blot analysis revealed that hNek6 transcripts are ubiquitously expressed with the highest expression found in the heart and skeletal muscle. Lower cell cycle-dependent expression of hNek6 transcripts was observed in the early G1 phase. GFP-fused hNek6 protein showed both nuclear and cytoplasmic localizations in HeLa cells. Fluorescence in situ hybridization using full-length hNek6 cDNA as a probe showed that the hNek6 gene is localized to human chromosome 9q33-34, a region at which the loss of heterozygosity is associated with transitional cell carcinomas. Importantly, recombinant hNek6 protein produced in insect cells effectively phosphorylated histones H1 and H3, but not casein. Thus, these results suggest that, unlike other mammalian NIMA-related kinases, Nek6 is a mitotic histone kinase which regulates chromatin condensation in mammalian cells.
Nek6 and Nercc1 (also known as Nek9) belong to the NIMA family of protein kinases. Nercc1 is activated in mitosis, whereupon it binds, phosphorylates and activates Nek6. Interference with Nek6 or Nercc1 in mammalian cells causes prometaphase-metaphase arrest, and depletion of Nercc1 from Xenopus egg extracts prevents normal spindle assembly. Herein we show that Nek6 is constitutively associated with Eg5 (also known as Kinesin-5 and Kif11), a kinesin that is necessary for spindle bipolarity. Nek6 phosphorylated Eg5 at several sites in vitro and one of these sites, Ser1033, is phosphorylated in vivo during mitosis. Whereas CDK1 phosphorylates nearly all Eg5 at Thr926 during mitosis, Nek6 phosphorylates approximately 3% of Eg5, primarily at the spindle poles. Eg5 depletion caused mitotic arrest, resulting in cells with a monopolar spindle. This arrest could be rescued by wild-type Eg5 but not by Eg5[Thr926Ala]. Despite substantial overexpression, Eg5[Ser1033Ala] rescued 50% of cells compared with wild-type Eg5, whereas an Eg5[Ser1033Asp] mutant was nearly as effective as wild type. Thus, during mitosis Nek6 phosphorylates a subset of Eg5 polypeptides at a conserved site, the phosphorylation of which is crucial for the mitotic function of Eg5.
The Aspergillus nidulans protein NIMA (never in mitosis, gene A) is a protein kinase required for the initiation of mitosis, whereas its inactivation is necessary for mitotic exit. Here, we demonstrate that human NIMA-related kinase 6 (Nek6) is required for mitotic progression of human cells. Nek6 is phosphorylated and activated during M phase. Inhibition of Nek6 function by either overexpression of an inactive Nek6 mutant or elimination of endogenous Nek6 by siRNA arrests cells in M phase and triggers apoptosis. Time-lapse recording of the cell cycle progression of cells expressing kinase-inactive Nek6 reveals mitotic arrest at the metaphase stage prior to cells entering apoptosis. In contrast to NIMA and the closely related mammalian Nek2 kinase, which regulate centrosome function and separation, our data demonstrate an important function for Nek6 during mitosis and suggest that Nek6 kinase is required for metaphase-anaphase transition.
Nek6 and Nek7 are members of the NIMA-related serine/threonine kinase family. Previous work showed that they contribute to mitotic progression downstream of another NIMA-related kinase, Nek9, although the roles of these different kinases remain to be defined. Here, we carried out a comprehensive analysis of the regulation and function of Nek6 and Nek7 in human cells. By generating specific antibodies, we show that both Nek6 and Nek7 are activated in mitosis and that interfering with their activity by either depletion or expression of reduced-activity mutants leads to mitotic arrest and apoptosis. Interestingly, while completely inactive mutants and small interfering RNA-mediated depletion delay cells at metaphase with fragile mitotic spindles, hypomorphic mutants or RNA interference treatment combined with a spindle assembly checkpoint inhibitor delays cells at cytokinesis. Importantly, depletion of either Nek6 or Nek7 leads to defective mitotic progression, indicating that although highly similar, they are not redundant. Indeed, while both kinases localize to spindle poles, only Nek6 obviously localizes to spindle microtubules in metaphase and anaphase and to the midbody during cytokinesis. Together, these data lead us to propose that Nek6 and Nek7 play independent roles not only in robust mitotic spindle formation but also potentially in cytokinesis.
Nek6 is an NIMA-related kinase that plays a critical role in mitotic cell cycle progression. Recent studies have shown that Nek6 is upregulated in various human cancers, but the function of Nek6 in tumorigenesis is largely unknown. Here, we examined the role of Nek6 in cellular senescence. Our data revealed that Nek6 expression is decreased both in both the replicative senescence of human normal fibroblasts and premature senescence induced by p53 expression in EJ human bladder cancer cells and H1299 human lung cancer cells. Interestingly, the enforced expression of Nek6 in EJ and H1299 cells completely suppresses p53-induced senescence, whereas the expression of kinase-dead Nek6 did not affect p53-induced senescence. Mechanistic studies revealed that cell cycle arrest in the G1 and G2/M phases, as well as the reduction of cyclin B and cdc2 protein level upon p53 expression were significantly reduced by Nek6 overexpression. In addition, p53-induced increases in intracellular levels of ROS were also inhibited in cells overexpressing Nek6. These results suggest that the downregulation of Nek6 expression is required for the onset of p53-induced cellular senescence and imply a possible role of Nek6 in tumorigenesis.
We investigated the role of Nek6, a member of the NIMA-related serine/threonine kinase family, in tumorigenesis. Transcript, protein, and kinase activity levels of Nek6 were highly elevated in the malignant tumors and human cancer cell lines compared with normal tissue and fibroblast cells. Expression of exogenous wild-type Nek6 increased anchorage-independent growth of a variety of human cancer cell lines, whereas overexpression of the kinase-dead Nek6 and RNAi knockdown of endogenous Nek6 suppressed cancer cell transformation and induced apoptosis. Additionally, in in vivo xenograft nude mouse model, knockdown of Nek6 in HeLa cells resulted in reduction of tumor size relative to control siRNA tumors. Most importantly, knocking down endogenous Nek6 levels or exogenous expression of the kinase-dead form did not inhibit cell proliferation, nor did it induce apoptosis in normal fibroblast cells. Taken together, our data indicate a pivotal role for Nek6 in tumorigenesis and establish Nek6 as a potential target for treatment of a variety of human cancers.
The Aspergillus nidulans protein NIMA (never in mitosis, gene A) is a protein kinase required for the initiation of mitosis, whereas its inactivation is necessary for mitotic exit. Here, we demonstrate that human NIMA-related kinase 6 (Nek6) is required for mitotic progression of human cells. Nek6 is phosphorylated and activated during M phase. Inhibition of Nek6 function by either overexpression of an inactive Nek6 mutant or elimination of endogenous Nek6 by siRNA arrests cells in M phase and triggers apoptosis. Time-lapse recording of the cell cycle progression of cells expressing kinase-inactive Nek6 reveals mitotic arrest at the metaphase stage prior to cells entering apoptosis. In contrast to NIMA and the closely related mammalian Nek2 kinase, which regulate centrosome function and separation, our data demonstrate an important function for Nek6 during mitosis and suggest that Nek6 kinase is required for metaphase-anaphase transition.
Interacting selectively and non-covalently and stoichiometrically with kinesin, a member of a superfamily of microtubule-based motor proteins that perform force-generating tasks such as organelle transport and chromosome segregation.
Evidence
1:
Inferred from Physical InteractionUniProtKB
Nek6 and Nercc1 (also known as Nek9) belong to the NIMA family of protein kinases. Nercc1 is activated in mitosis, whereupon it binds, phosphorylates and activates Nek6. Interference with Nek6 or Nercc1 in mammalian cells causes prometaphase-metaphase arrest, and depletion of Nercc1 from Xenopus egg extracts prevents normal spindle assembly. Herein we show that Nek6 is constitutively associated with Eg5 (also known as Kinesin-5 and Kif11), a kinesin that is necessary for spindle bipolarity. Nek6 phosphorylated Eg5 at several sites in vitro and one of these sites, Ser1033, is phosphorylated in vivo during mitosis. Whereas CDK1 phosphorylates nearly all Eg5 at Thr926 during mitosis, Nek6 phosphorylates approximately 3% of Eg5, primarily at the spindle poles. Eg5 depletion caused mitotic arrest, resulting in cells with a monopolar spindle. This arrest could be rescued by wild-type Eg5 but not by Eg5[Thr926Ala]. Despite substantial overexpression, Eg5[Ser1033Ala] rescued 50% of cells compared with wild-type Eg5, whereas an Eg5[Ser1033Asp] mutant was nearly as effective as wild type. Thus, during mitosis Nek6 phosphorylates a subset of Eg5 polypeptides at a conserved site, the phosphorylation of which is crucial for the mitotic function of Eg5.
The Aspergillus nidulans protein NIMA (never in mitosis, gene A) is a protein kinase required for the initiation of mitosis, whereas its inactivation is necessary for mitotic exit. Here, we demonstrate that human NIMA-related kinase 6 (Nek6) is required for mitotic progression of human cells. Nek6 is phosphorylated and activated during M phase. Inhibition of Nek6 function by either overexpression of an inactive Nek6 mutant or elimination of endogenous Nek6 by siRNA arrests cells in M phase and triggers apoptosis. Time-lapse recording of the cell cycle progression of cells expressing kinase-inactive Nek6 reveals mitotic arrest at the metaphase stage prior to cells entering apoptosis. In contrast to NIMA and the closely related mammalian Nek2 kinase, which regulate centrosome function and separation, our data demonstrate an important function for Nek6 during mitosis and suggest that Nek6 kinase is required for metaphase-anaphase transition.
Interacting selectively and non-covalently with any protein or protein complex (a complex of two or more proteins that may include other nonprotein molecules).
Evidence
1:
Inferred from Physical InteractionIntAct
The peptidyl-prolyl isomerase Pin1 is prevalently overexpressed in human cancers and is regarded as a new diagnostic and therapeutic target. Pin1 interacts with several proteins involved in cell cycle events in a phosphorylation-dependent manner. Among them, NIMA (never in mitosis, gene A) was first identified to interact with Pin1. In this report, we found that Pin1 could interact with Nek6, one of the human NIMA-related kinases (Neks). This interaction was confirmed by GST pull-down assay, which was further confirmed by immunoprecipitation experiments, as well as immunofluorescence colocalization. We further studied Pin1 and Nek6 mRNA level in 40 pairs of hepatocellular carcinoma cases, finding significant correlations between Nek6 and Pin1 mRNA expression levels in these samples.
Interacting selectively and non-covalently with a protein kinase, any enzyme that catalyzes the transfer of a phosphate group, usually from ATP, to a protein substrate.
Evidence
1:
Inferred from Physical InteractionUniProtKB
Nek6 and Nercc1 (also known as Nek9) belong to the NIMA family of protein kinases. Nercc1 is activated in mitosis, whereupon it binds, phosphorylates and activates Nek6. Interference with Nek6 or Nercc1 in mammalian cells causes prometaphase-metaphase arrest, and depletion of Nercc1 from Xenopus egg extracts prevents normal spindle assembly. Herein we show that Nek6 is constitutively associated with Eg5 (also known as Kinesin-5 and Kif11), a kinesin that is necessary for spindle bipolarity. Nek6 phosphorylated Eg5 at several sites in vitro and one of these sites, Ser1033, is phosphorylated in vivo during mitosis. Whereas CDK1 phosphorylates nearly all Eg5 at Thr926 during mitosis, Nek6 phosphorylates approximately 3% of Eg5, primarily at the spindle poles. Eg5 depletion caused mitotic arrest, resulting in cells with a monopolar spindle. This arrest could be rescued by wild-type Eg5 but not by Eg5[Thr926Ala]. Despite substantial overexpression, Eg5[Ser1033Ala] rescued 50% of cells compared with wild-type Eg5, whereas an Eg5[Ser1033Asp] mutant was nearly as effective as wild type. Thus, during mitosis Nek6 phosphorylates a subset of Eg5 polypeptides at a conserved site, the phosphorylation of which is crucial for the mitotic function of Eg5.
The Aspergillus nidulans protein NIMA (never in mitosis, gene A) is a protein kinase required for the initiation of mitosis, whereas its inactivation is necessary for mitotic exit. Here, we demonstrate that human NIMA-related kinase 6 (Nek6) is required for mitotic progression of human cells. Nek6 is phosphorylated and activated during M phase. Inhibition of Nek6 function by either overexpression of an inactive Nek6 mutant or elimination of endogenous Nek6 by siRNA arrests cells in M phase and triggers apoptosis. Time-lapse recording of the cell cycle progression of cells expressing kinase-inactive Nek6 reveals mitotic arrest at the metaphase stage prior to cells entering apoptosis. In contrast to NIMA and the closely related mammalian Nek2 kinase, which regulate centrosome function and separation, our data demonstrate an important function for Nek6 during mitosis and suggest that Nek6 kinase is required for metaphase-anaphase transition.
Physical protein-protein interactions are fundamental to all biological processes and are organized in complex networks. One branch of the kinome network is the evolutionarily conserved NIMA-related serine/threonine kinases (Neks). Most of the 11 mammalian Neks studied so far are related to cell cycle regulation, and due to association with diverse human pathologies, Neks are promising chemotherapeutic targets. Human Nek6 was associated to carcinogenesis, but its interacting partners and signaling pathways remain elusive. Here we introduce hNek6 as a highly connected member in the human kinase interactome. In a more global context, we performed a broad data bank comparison based on degree distribution analysis and found that the human kinome is enriched in hubs. Our networks include a broad set of novel hNek6 interactors as identified by our yeast two-hybrid screens classified into 18 functional categories. All of the tested interactions were confirmed, and the majority of tested substrates were phosphorylated in vitro by hNek6. Notably, we found that hNek6 N-terminal is important to mediate the interactions with its partners. Some novel interactors also colocalized with hNek6 and γ-tubulin in human cells, pointing to a possible centrosomal interaction. The interacting proteins link hNek6 to novel pathways, for example, Notch signaling and actin cytoskeleton regulation, or give new insights on how hNek6 may regulate previously proposed pathways such as cell cycle regulation, DNA repair response, and NF-κB signaling. Our findings open new perspectives in the study of hNek6 role in cancer by analyzing its novel interactions in specific pathways in tumor cells, which may provide important implications for drug design and cancer therapy.
Nek6 and Nercc1 (also known as Nek9) belong to the NIMA family of protein kinases. Nercc1 is activated in mitosis, whereupon it binds, phosphorylates and activates Nek6. Interference with Nek6 or Nercc1 in mammalian cells causes prometaphase-metaphase arrest, and depletion of Nercc1 from Xenopus egg extracts prevents normal spindle assembly. Herein we show that Nek6 is constitutively associated with Eg5 (also known as Kinesin-5 and Kif11), a kinesin that is necessary for spindle bipolarity. Nek6 phosphorylated Eg5 at several sites in vitro and one of these sites, Ser1033, is phosphorylated in vivo during mitosis. Whereas CDK1 phosphorylates nearly all Eg5 at Thr926 during mitosis, Nek6 phosphorylates approximately 3% of Eg5, primarily at the spindle poles. Eg5 depletion caused mitotic arrest, resulting in cells with a monopolar spindle. This arrest could be rescued by wild-type Eg5 but not by Eg5[Thr926Ala]. Despite substantial overexpression, Eg5[Ser1033Ala] rescued 50% of cells compared with wild-type Eg5, whereas an Eg5[Ser1033Asp] mutant was nearly as effective as wild type. Thus, during mitosis Nek6 phosphorylates a subset of Eg5 polypeptides at a conserved site, the phosphorylation of which is crucial for the mitotic function of Eg5.
Conveys a signal across a cell to trigger a change in cell function or state. A signal is a physical entity or change in state that is used to transfer information in order to trigger a response.
Evidence
1:
Inferred from Mutant PhenotypeUniProtKB
We have carried out a large-scale identification and characterization of human genes that activate the NF-kappaB and MARK signaling pathways. We constructed full-length cDNA libraries using the oligo-capping method and prepared an arrayed cDNA pool consisting of 150 000 cDNAs randomly isolated from the libraries. For analysis of the NF-kappaB signaling pathway, we introduced each of the cDNAs into human embryonic kidney 293 cells and examined whether it activated the transcription of a luciferase reporter gene driven by a promoter containing the consensus NF-kappaB binding sites. In total, we identified 299 cDNAs that activate the NF-kappaB pathway, and we classified them into 83 genes, including 30 characterized activator genes of the NF-kappaB pathway, 28 genes whose involvement in the NF-kappaB pathways have not been characterized and 25 novel genes. We then carried out a similar analysis for the identification of genes that activate the MARK pathway, utilizing the same cDNA resource. We assayed 145 000 cDNAs and identified 57 genes that activate the MARK pathway. Interestingly, 27 genes were overlapping between the NF-kappaB and the MAPK pathways, which may indicate that these genes play cross-talking roles between these two pathways.
A programmed cell death process which begins when a cell receives an internal (e.g. DNA damage) or external signal (e.g. an extracellular death ligand), and proceeds through a series of biochemical events (signaling pathways) which typically lead to rounding-up of the cell, retraction of pseudopodes, reduction of cellular volume (pyknosis), chromatin condensation, nuclear fragmentation (karyorrhexis), plasma membrane blebbing and fragmentation of the cell into apoptotic bodies. The process ends when the cell has died. The process is divided into a signaling pathway phase, and an execution phase, which is triggered by the former.
The process in which genetic material, in the form of chromosomes, is organized into specific structures and then physically separated and apportioned to two or more sets. In eukaryotes, chromosome segregation begins with the alignment of chromosomes at the metaphase plate, includes chromosome separation, and ends when chromosomes have completed movement to the spindle poles.
The Aspergillus nidulans protein NIMA (never in mitosis, gene A) is a protein kinase required for the initiation of mitosis, whereas its inactivation is necessary for mitotic exit. Here, we demonstrate that human NIMA-related kinase 6 (Nek6) is required for mitotic progression of human cells. Nek6 is phosphorylated and activated during M phase. Inhibition of Nek6 function by either overexpression of an inactive Nek6 mutant or elimination of endogenous Nek6 by siRNA arrests cells in M phase and triggers apoptosis. Time-lapse recording of the cell cycle progression of cells expressing kinase-inactive Nek6 reveals mitotic arrest at the metaphase stage prior to cells entering apoptosis. In contrast to NIMA and the closely related mammalian Nek2 kinase, which regulate centrosome function and separation, our data demonstrate an important function for Nek6 during mitosis and suggest that Nek6 kinase is required for metaphase-anaphase transition.
The division of the cytoplasm and the plasma membrane of a cell and its separation into two daughter cells. Cytokinesis usually occurs after growth, replication, and segregation of cellular components, and occurs after division of the nucleus.
Nek6 is a recently identified NIMA-related kinase that is required for mitotic cell cycle progression. In the present study, we examined the role of Nek6 in the DNA damage response. We found that Nek6 is phosphorylated upon IR and UV irradiation through the DNA damage checkpoint in vivo. Nek6 is also directly phosphorylated by the checkpoint kinases Chk1 and Chk2 in vitro. Notably, Nek6 activation during mitosis is completely abolished by IR and UV irradiation. Moreover, the ectopic expression of Nek6 overrides DNA damage-induced G(2)/M arrest. These results suggest that Nek6 is a novel target of the DNA damage checkpoint and that the inhibition of Nek6 activity is required for proper cell cycle arrest in the G(2)/M phase upon DNA damage.
A cell cycle process comprising the steps by which the nucleus of a eukaryotic cell divides; the process involves condensation of chromosomal DNA into a highly compacted form. Canonically, mitosis produces two daughter nuclei whose chromosome complement is identical to that of the mother cell.
Nek6 and Nercc1 (also known as Nek9) belong to the NIMA family of protein kinases. Nercc1 is activated in mitosis, whereupon it binds, phosphorylates and activates Nek6. Interference with Nek6 or Nercc1 in mammalian cells causes prometaphase-metaphase arrest, and depletion of Nercc1 from Xenopus egg extracts prevents normal spindle assembly. Herein we show that Nek6 is constitutively associated with Eg5 (also known as Kinesin-5 and Kif11), a kinesin that is necessary for spindle bipolarity. Nek6 phosphorylated Eg5 at several sites in vitro and one of these sites, Ser1033, is phosphorylated in vivo during mitosis. Whereas CDK1 phosphorylates nearly all Eg5 at Thr926 during mitosis, Nek6 phosphorylates approximately 3% of Eg5, primarily at the spindle poles. Eg5 depletion caused mitotic arrest, resulting in cells with a monopolar spindle. This arrest could be rescued by wild-type Eg5 but not by Eg5[Thr926Ala]. Despite substantial overexpression, Eg5[Ser1033Ala] rescued 50% of cells compared with wild-type Eg5, whereas an Eg5[Ser1033Asp] mutant was nearly as effective as wild type. Thus, during mitosis Nek6 phosphorylates a subset of Eg5 polypeptides at a conserved site, the phosphorylation of which is crucial for the mitotic function of Eg5.
We have carried out a large-scale identification and characterization of human genes that activate the NF-kappaB and MARK signaling pathways. We constructed full-length cDNA libraries using the oligo-capping method and prepared an arrayed cDNA pool consisting of 150 000 cDNAs randomly isolated from the libraries. For analysis of the NF-kappaB signaling pathway, we introduced each of the cDNAs into human embryonic kidney 293 cells and examined whether it activated the transcription of a luciferase reporter gene driven by a promoter containing the consensus NF-kappaB binding sites. In total, we identified 299 cDNAs that activate the NF-kappaB pathway, and we classified them into 83 genes, including 30 characterized activator genes of the NF-kappaB pathway, 28 genes whose involvement in the NF-kappaB pathways have not been characterized and 25 novel genes. We then carried out a similar analysis for the identification of genes that activate the MARK pathway, utilizing the same cDNA resource. We assayed 145 000 cDNAs and identified 57 genes that activate the MARK pathway. Interestingly, 27 genes were overlapping between the NF-kappaB and the MAPK pathways, which may indicate that these genes play cross-talking roles between these two pathways.
The Aspergillus nidulans protein NIMA (never in mitosis, gene A) is a protein kinase required for the initiation of mitosis, whereas its inactivation is necessary for mitotic exit. Here, we demonstrate that human NIMA-related kinase 6 (Nek6) is required for mitotic progression of human cells. Nek6 is phosphorylated and activated during M phase. Inhibition of Nek6 function by either overexpression of an inactive Nek6 mutant or elimination of endogenous Nek6 by siRNA arrests cells in M phase and triggers apoptosis. Time-lapse recording of the cell cycle progression of cells expressing kinase-inactive Nek6 reveals mitotic arrest at the metaphase stage prior to cells entering apoptosis. In contrast to NIMA and the closely related mammalian Nek2 kinase, which regulate centrosome function and separation, our data demonstrate an important function for Nek6 during mitosis and suggest that Nek6 kinase is required for metaphase-anaphase transition.
Physical protein-protein interactions are fundamental to all biological processes and are organized in complex networks. One branch of the kinome network is the evolutionarily conserved NIMA-related serine/threonine kinases (Neks). Most of the 11 mammalian Neks studied so far are related to cell cycle regulation, and due to association with diverse human pathologies, Neks are promising chemotherapeutic targets. Human Nek6 was associated to carcinogenesis, but its interacting partners and signaling pathways remain elusive. Here we introduce hNek6 as a highly connected member in the human kinase interactome. In a more global context, we performed a broad data bank comparison based on degree distribution analysis and found that the human kinome is enriched in hubs. Our networks include a broad set of novel hNek6 interactors as identified by our yeast two-hybrid screens classified into 18 functional categories. All of the tested interactions were confirmed, and the majority of tested substrates were phosphorylated in vitro by hNek6. Notably, we found that hNek6 N-terminal is important to mediate the interactions with its partners. Some novel interactors also colocalized with hNek6 and γ-tubulin in human cells, pointing to a possible centrosomal interaction. The interacting proteins link hNek6 to novel pathways, for example, Notch signaling and actin cytoskeleton regulation, or give new insights on how hNek6 may regulate previously proposed pathways such as cell cycle regulation, DNA repair response, and NF-κB signaling. Our findings open new perspectives in the study of hNek6 role in cancer by analyzing its novel interactions in specific pathways in tumor cells, which may provide important implications for drug design and cancer therapy.
The Nek family of protein kinases in humans is composed of 11 members that share an amino-terminal catalytic domain related to NIMA, an Aspergillus kinase involved in the control of several aspects of mitosis, and divergent carboxyl-terminal tails of varying length. Nek6 (314AA) and Nek7 (303AA), 76% identical, have little noncatalytic sequence but bind to the carboxyl-terminal noncatalytic tail of Nercc1/Nek9, a NIMA family protein kinase that is activated in mitosis. Microinjection of anti-Nercc1 antibodies leads to spindle abnormalities and prometaphase arrest or chromosome missegregation. Herein we show that Nek6 is increased in abundance and activity during mitosis; activation requires the phosphorylation of Ser206 on the Nek6 activation loop. This phosphorylation and the activity of recombinant Nek6 is stimulated by coexpression with an activated mutant of Nercc1. Moreover, Nercc1 catalyzes the direct phosphorylation of prokaryotic recombinant Nek6 at Ser206 in vitro concomitant with 20-25-fold activation of Nek6 activity; Nercc1 activates Nek7 in vitro in a similar manner. Nercc1/Nek9 is likely to be responsible for the activation of Nek6 during mitosis and probably participates in the regulation of Nek7 as well. These findings support the conclusion that Nercc1/Nek9 and Nek6 represent a novel cascade of mitotic NIMA family protein kinases whose combined function is important for mitotic progression.
The Nek family of cell-cycle kinases is widely represented in eukaryotes and includes numerous proteins that were described only recently and remain poorly characterized. Comparing Neks in the context of clades allows us to examine the question of whether microbial eukaryotic Neks, although not strictly orthologs of their vertebrate counterparts, can provide clues to ancestral functions that might be retained in the vertebrate Neks. Relatives of the Nek2/NIMA proteins play important roles at the G2-M transition in nuclear envelope breakdown and centromere separation. Nek6, Nek7 and Nek9 also seem to regulate mitosis. By contrast, Nek1 and Nek8 have been linked with polycystic kidney disease. Results of statistical analysis indicate that the family coevolved with centrioles that function as both microtubule-organizing centers and the basal bodies of cilia. This evolutionary perspective, taken together with functional studies of microbial Neks, provides new insights into the cellular roles of the proteins and disease with which some of them have been linked.
The Aspergillus nidulans protein NIMA (never in mitosis, gene A) is a protein kinase required for the initiation of mitosis, whereas its inactivation is necessary for mitotic exit. Here, we demonstrate that human NIMA-related kinase 6 (Nek6) is required for mitotic progression of human cells. Nek6 is phosphorylated and activated during M phase. Inhibition of Nek6 function by either overexpression of an inactive Nek6 mutant or elimination of endogenous Nek6 by siRNA arrests cells in M phase and triggers apoptosis. Time-lapse recording of the cell cycle progression of cells expressing kinase-inactive Nek6 reveals mitotic arrest at the metaphase stage prior to cells entering apoptosis. In contrast to NIMA and the closely related mammalian Nek2 kinase, which regulate centrosome function and separation, our data demonstrate an important function for Nek6 during mitosis and suggest that Nek6 kinase is required for metaphase-anaphase transition.
The cellular process in which a signal is conveyed to trigger a change in the activity or state of a cell. Signal transduction begins with reception of a signal (e.g. a ligand binding to a receptor or receptor activation by a stimulus such as light), or for signal transduction in the absence of ligand, signal-withdrawal or the activity of a constitutively active receptor. Signal transduction ends with regulation of a downstream cellular process, e.g. regulation of transcription or regulation of a metabolic process. Signal transduction covers signaling from receptors located on the surface of the cell and signaling via molecules located within the cell. For signaling between cells, signal transduction is restricted to events at and within the receiving cell.
We have carried out a large-scale identification and characterization of human genes that activate the NF-kappaB and MARK signaling pathways. We constructed full-length cDNA libraries using the oligo-capping method and prepared an arrayed cDNA pool consisting of 150 000 cDNAs randomly isolated from the libraries. For analysis of the NF-kappaB signaling pathway, we introduced each of the cDNAs into human embryonic kidney 293 cells and examined whether it activated the transcription of a luciferase reporter gene driven by a promoter containing the consensus NF-kappaB binding sites. In total, we identified 299 cDNAs that activate the NF-kappaB pathway, and we classified them into 83 genes, including 30 characterized activator genes of the NF-kappaB pathway, 28 genes whose involvement in the NF-kappaB pathways have not been characterized and 25 novel genes. We then carried out a similar analysis for the identification of genes that activate the MARK pathway, utilizing the same cDNA resource. We assayed 145 000 cDNAs and identified 57 genes that activate the MARK pathway. Interestingly, 27 genes were overlapping between the NF-kappaB and the MAPK pathways, which may indicate that these genes play cross-talking roles between these two pathways.
The aggregation, arrangement and bonding together of a set of components to form the spindle, the array of microtubules and associated molecules that serves to move duplicated chromosomes apart.
Nek6 and Nercc1 (also known as Nek9) belong to the NIMA family of protein kinases. Nercc1 is activated in mitosis, whereupon it binds, phosphorylates and activates Nek6. Interference with Nek6 or Nercc1 in mammalian cells causes prometaphase-metaphase arrest, and depletion of Nercc1 from Xenopus egg extracts prevents normal spindle assembly. Herein we show that Nek6 is constitutively associated with Eg5 (also known as Kinesin-5 and Kif11), a kinesin that is necessary for spindle bipolarity. Nek6 phosphorylated Eg5 at several sites in vitro and one of these sites, Ser1033, is phosphorylated in vivo during mitosis. Whereas CDK1 phosphorylates nearly all Eg5 at Thr926 during mitosis, Nek6 phosphorylates approximately 3% of Eg5, primarily at the spindle poles. Eg5 depletion caused mitotic arrest, resulting in cells with a monopolar spindle. This arrest could be rescued by wild-type Eg5 but not by Eg5[Thr926Ala]. Despite substantial overexpression, Eg5[Ser1033Ala] rescued 50% of cells compared with wild-type Eg5, whereas an Eg5[Ser1033Asp] mutant was nearly as effective as wild type. Thus, during mitosis Nek6 phosphorylates a subset of Eg5 polypeptides at a conserved site, the phosphorylation of which is crucial for the mitotic function of Eg5.
Mitosis is controlled by multiple protein kinases, many of which are abnormally expressed in human cancers. Nek2, Nek6, Nek7, and Nek9 are NIMA-related kinases essential for proper mitotic progression. We determined the atomic structure of Nek7 and discovered an autoinhibited conformation that suggests a regulatory mechanism not previously described in kinases. Additionally, Nek2 adopts the same conformation when bound to a drug-like molecule. In both structures, a tyrosine side chain points into the active site, interacts with the activation loop, and blocks the alphaC helix. Tyrosine mutants of Nek7 and the related kinase Nek6 are constitutively active. The activity of Nek6 and Nek7, but not the tyrosine mutant, is increased by interaction with the Nek9 noncatalytic C-terminal domain, suggesting a mechanism in which the tyrosine is released from its autoinhibitory position. The autoinhibitory conformation is common to three Neks and provides a potential target for selective kinase inhibitors.
Protein involved in apoptotic programmed cell death. Apoptosis is characterized by cell morphological changes, including blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation and chromosomal DNA fragmentation, and eventually death. Unlike necrosis, apoptosis produces cell fragments, called apoptotic bodies, that phagocytic cells are able to engulf and quickly remove before the contents of the cell can spill out onto surrounding cells and cause damage. In general, apoptosis confers advantages during an organism's life cycle.
Protein involved in the complex series of events by which the cell duplicates its contents and divides into two. The eukaryotic cell cycle can be divided in four phases termed G1 (first gap period), S (synthesis, phase during which the DNA is replicated), G2 (second gap period) and M (mitosis). The prokaryotic cell cycle typically involves a period of growth followed by DNA replication, partition of chromosomes, formation of septum and division into two similar or identical daughter cells.
Protein involved in the separation of one cell into two daughter cells. In eukaryotic cells, cell division includes the nuclear division (mitosis) and the subsequent cytoplasmic division (cytokinesis).
Protein involved in chromosome partition, the process by which newly replicated plasmids and chromosomes are actively segregated prior to cell division. E.g., par and soj which contribute to efficient chromosome partitioning by serving functions analogous to centromeres (i.e. pairing or positioning of sister chromosomes).
Protein involved in mitosis, the nuclear division in eukaryotic cells involving the exact duplication and separation of the chromosome threads so that each daughter nucleus carries a chromosome complement identical to that of the parent nucleus. Mitosis is divided into four substages: prophase, metaphase, anaphase and telophase.
Protein which catalyzes the phosphorylation of serine or threonine residues on target proteins by using ATP as phosphate donor. Such phosphorylation may cause changes in the function of the target protein. Protein kinases share a conserved catalytic core common to both serine/ threonine and tyrosine protein kinases.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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