Nek2 localizes to multiple sites in mitotic cells, suggesting its involvement in multiple cellular functions during the cell cycle.

Nek2 is a mammalian protein kinase that is structurally homologous to NIMA, a mitotic regulator in Aspergillus nidulans. To understand the possible cellular processes in which Nek2 participates during the cell cycle, we investigated the expression and subcellular localization of Nek2 in mitotic cells. The Nek2 protein levels were observed to be regulated in a cell cycle stage-specific manner in cultured cells. The cell cycle stage specificity of Nek2 expression was also confirmed in cells undergoing mitosis in vivo. Nek2 proteins were localized in both the nucleus and cytoplasm throughout the cell cycle, but exhibited dynamic changes in distribution, depending on the cell cycle stage. Nek2 was associated with chromosomes from prophase to metaphase and then was dissociated upon entering into anaphase. Nek2 then appeared at the midbody of the cytoplasmic bridge at telophase. Nek2 was also associated with the centrosome throughout the cell cycle as observed previously by others. Additionally, the nuclear localization of Nek2 was increased during S phase. Such dynamic behavior of Nek2 suggests that Nek2 may be a mitotic regulator that is involved in diverse cell cycle events.     

Dynamic recruitment of Nek2 kinase to the centrosome involves microtubules, PCM-1, and localized proteasomal degradation

Centrosomes undergo dramatic changes in composition and activity during cell cycle progression. Yet mechanisms involved in recruiting centrosomal proteins are poorly understood. Nek2 is a cell cycle-regulated protein kinase required for regulation of centrosome structure at the G2/M transition. Here, we have addressed the processes involved in trafficking of Nek2 to the centrosome of human adult cells. We find that Nek2 exists in small, highly dynamic cytoplasmic particles that move to and from the centrosome. Many of these particles align along microtubules and a motif was identified in the Nek2 C-terminal noncatalytic domain that allows both microtubule binding and centrosome localization. FRAP experiments reveal that 70% of centrosomal Nek2 is rapidly turned over (t(1/2) approximately 3 s). Microtubules facilitate Nek2 trafficking to the centrosome but only over long distances. Cytoplasmic Nek2 particles colocalize in part with PCM-1 containing centriolar satellites and depletion of PCM-1 interferes with centrosomal recruitment of Nek2 and its substrate C-Nap1. Finally, we show that proteasomal degradation is necessary to allow rapid recruitment of new Nek2 molecules to the centrosome. Together, these data highlight multiple processes involved in regulating the abundance of Nek2 kinase at the centrosome including microtubule binding, the centriolar satellite component PCM-1, and localized protein degradation.     

Nek10 mediates G2/M cell cycle arrest and MEK autoactivation in response to UV irradiation

Appropriate cell cycle checkpoint control is essential for the maintenance of cell and organismal homeostasis. Members of the Nek (NIMA-related kinase) family of serine/threonine protein kinases have been implicated in the regulation of various aspects of the cell cycle. We explored the cellular functions of Nek10, a novel member of the Nek family, and demonstrate a role for Nek10 in the cellular UV response. Nek10 was required for the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) signaling upon UV irradiation but not in response to mitogens, such as epidermal growth factor stimulation. Nek10 physically associated with Raf-1 and MEK1 in a Raf-1-dependent manner, and the formation of this complex was necessary for Nek10-mediated MEK1 activation. Nek10 did not affect the kinase activity of Raf-1 but instead promoted the autophosphorylation-dependent activation of MEK1. The appropriate maintenance of the G(2)/M checkpoint following UV irradiation required Nek10 expression and ERK1/2 activation. Taken together, our results uncover a role for Nek10 in the cellular response to UV irradiation.     

Mitotic perturbations induced by Nek2 overexpression require interaction with TRF1 in breast cancer cells

NIMA-related kinase 2 (Nek2), a serine–threonine protein kinase, plays a major role in mitotic progression, including timing of mitotic entry, chromatin condensation, spindle organization, and cytokinesis. Nek2 overexpression results in premature centrosome separation, while kinase death Nek2 mutant expression or Nek2-depleted cells lead to centrosome separation failure. In addition, it has been revealed that telomeric repeat binding factor 1 (TRF1) interacts directly with Nek2. TRF1 not only regulates telomere length, but is also associated with cell cycle regulation. However, the interactions and correlations between Nek2 and TRF1 are far from clear. Here, we show that mitotic aberrations through Nek2 overexpression are likely to require TRF1. Our results demonstrate that Nek2 directly binds and phosphorylates TRF1 through multiple sites on TRF1. Nek2 overexpression in breast cancer cells, MDA-MB-231 and MCF7, results in increased numbers of centrosomes and multinucleated cells, which leads to cytokinetic failure and aneuploidization. Additionally, TRF1 depletion by siRNA prevents the phenomenon of unaligned chromosomes by Nek2 overexpression during metaphase. Concurrent Nek2 overexpression and TRF1-depleted cells demonstrated ≤ 2 centrosomes per cell, similar to mock plasmid and negative control siRNA-transfected cells. Interestingly, when exogenous TRF1 was added back in Nek2-overexpressed cells with endogenous TRF1 depletion, cells had re-induced cytokinetic failure. Therefore, we propose that TRF1 is required for overexpressed Nek2 to trigger abnormal mitosis and chromosomal instability.     

Inhibition of centrosome separation after DNA damage: a role for Nek2

DNA damage results in cell cycle arrest in G2. Centrosomes also separate in G2, raising the question of whether separation occurs during the DNA damage-induced G2 arrest. Nek2, the mammalian homologue of NIMA, is a cell cycle-regulated serine/threonine protein kinase that regulates centrosome separation during G2. Here we show that damaged cells fail to activate Nek2. Both Nek2 levels and activity are reduced after DNA damage. Radiation inhibits the premature centrosome splitting induced by overexpression of Nek2, indicating that Nek2 is involved in activation of the G2 checkpoint and is not secondary to cell cycle arrest. We confirm using siRNA that centrosome separation and cell growth are impaired in the absence of Nek2. These studies define a previously unreported DNA damage response of inhibition of centrosome separation mechanistically linked to Nek2.     

Tcp10 promoter-directed expression of the Nek2 gene in mouse meiotic spermatocytes

Nek2 is a cell cycle regulator that is involved in diverse cell cycle events. The expression pattern and biochemical properties of Nek2 in mammalian male germ cells suggested its involvement on regulation of the meiotic cell cycle. To further investigate specific roles of Nek2 in meiosis, we generated transgenic mice in which the Nek2 transgenes were expressed specifically in spermatocytes using the Tcp10 promoter. The Nek2 transgenic mice did not reveal any significant defect in gross testicular anatomy as well as in fertility. However, we observed significant increases in defective spermatogenic cells, such as apoptotic cells and giant degenerating cells, in the Tcp10/Nek2 transgenic mice. These results revealed that even only slightly elevated production of the Nek2 protein disturbed the normal development of male germ cells, possibly in meiosis.     

DYNLL/LC8 protein controls signal transduction through the Nek9/Nek6 signaling module by regulating Nek6 binding to Nek9

The NIMA family protein kinases Nek9/Nercc1 and the highly similar Nek6 and Nek7 form a signaling module activated in mitosis, when they are involved in the control of spindle organization and function. Here we report that Nek9, the module upstream kinase, binds to DYNLL/LC8, a highly conserved protein originally described as a component of the dynein complex. LC8 is a dimer that interacts with different proteins and has been suggested to act as a dimerization hub promoting the organization and oligomerization of partially disorganized partners. We find that the interaction of LC8 with Nek9 depends on a (K/R)XTQT motif adjacent to the Nek9 C-terminal coiled coil motif, results in Nek9 multimerization, and increases the rate of Nek9 autoactivation. LC8 binding to Nek9 is regulated by Nek9 activity through the autophosphorylation of Ser(944), a residue immediately N-terminal to the (K/R)XTQT motif. Remarkably, LC8 binding interferes with the interaction of Nek9 with its downstream partner Nek6 as well as with Nek6 activation, thus controlling both processes. Our work sheds light into the control of signal transduction through the module formed by Nek9 and Nek6/7 and uncovers a novel manner in which LC8 can regulate partner physiology by interfering with protein complex formation. We suggest that this and other LC8 functions can be specifically regulated by partner phosphorylation.     

Live cell imaging reveals distinct roles in cell cycle regulation for Nek2A and Nek2B

Two splice variants of Nek2 kinase, a member of the NIMA-related family, have been identified as Nek2A and Nek2B. Nek2A regulates centrosome disjunction, spindle formation checkpoint signaling, and faithful chromosome segregation. A specific role for Nek2B has not yet been identified. Here, we have examined the distinct roles of Nek2A and Nek2B using timelapse video microscopy to follow the fate of cells progressing through the cell cycle in the absence of either Nek2A or Nek2B. We show that the down-regulation of Nek2B leads to a mitotic delay in the majority of cells. Upon exiting mitosis, cells exhibit mitotic defects such as the formation of multinucleated cells. Such phenotypes are not observed in cells that exit mitosis in the absence of Nek2A. These observations suggest that Nek2B may be required for the execution of mitotic exit.     

Nek2A specifies the centrosomal localization of Erk2

Nek2A is a cell-cycle-regulated protein kinase that localizes to the centrosome and kinetochore. Our recent studies provide a link between Nek2A and spindle checkpoint signaling [J. Biol. Chem. 279 (2004) 20049]. Extracellular signal-regulated kinase 2 (Erk2) is an important kinase, which belongs to mitogen activating protein (MAP) kinase family. Here we demonstrated that Nek2A binds specifically to Erk2. Erk2 interacts with Nek2A via a conserved Erk2 docking site located to the C-terminus of Nek2A. Our studies indicate this docking site is essential and sufficient for a direct Nek2A-Erk2 interaction. In addition, our immunocytochemical studies show that Nek2A and Erk2 are co-localized to centrosome. Significantly, elimination of Nek2A by RNA interference delocalized Erk2 from its centrosomal location, while inhibition of Erk2 kinase activity did not affect the localization of Nek2A in centrosome. We propose that Erk2 links extracellular signaling to centrosome dynamics by Nek2A.     

Giardia lamblia Nek1 and Nek2 kinases affect mitosis and excystation

The NIMA-related serine/threonine kinases (Neks) function in the cell cycle and regulate ciliary and flagellar length. The Giardia lamblia genome encodes 198 Neks, of which 56 are predicted to be active. Here we believe that we report the first functional analysis of two G. lamblia Neks. The GlNek1 and GlNek2 kinase domains share 57% and 43% identity to the kinase domains of human Nek1 and Nek2, respectively. Both GlNeks are active in vitro, have dynamic relocalisation during the cell cycle, and are expressed throughout the life cycle, with GlNek1 being upregulated in cysts. Over-expression of inactive GlNek1 delays disassembly of the parental attachment disc and cytokinesis, whilst over-expression of either wild type GlNek1 or inactive mutant GlNek2 inhibits excystation.     

Purification, cloning, and characterization of Nek8, a novel NIMA-related kinase, and its candidate substrate Bicd2

We describe the isolation, cloning, and characterization of human Nek8, a new mammalian NIMA-related kinase, and its candidate substrate Bicd2. Nek8 was isolated as a beta-casein kinase activity in rabbit lung and has an N-terminal catalytic domain homologous to the Nek family of protein kinases. Nek8 also contains a central domain with homology to RCC1, a guanine nucleotide exchange factor for the GTPase Ran, and a C-terminal coiled-coil domain. Like Nek2, Nek8 prefers beta-casein over other exogenous substrates, has shared biochemical requirements for kinase activity, and is capable of autophosphorylation and oligomerization. Nek8 activity is not cell cycle regulated, but like Nek3, levels are consistently higher in G(0)-arrested cells. During the purification of Nek8 a second protein co-chromatographed with Nek8 activity. This protein, Bicd2, is a human homolog of the Drosophila protein Bicaudal D, a coiled-coil protein. Bicd2 is phosphorylated by Nek8 in vitro, and the endogenous proteins associate in vivo. Bicd2 localizes to cytoskeletal structures, and its subcellular localization is dependent on microtubule morphology. Treatment of cells with nocodazole leads to dramatic reorganization of Bicd2, and correlates with Nek8 phosphorylation. This may be indicative of a role for Nek8 and Bicd2 associated with cell cycle independent microtubule dynamics.     

Human Nek7-interactor RGS2 is required for mitotic spindle organization

The mitotic spindle apparatus is composed of microtubule (MT) networks attached to kinetochores organized from 2 centrosomes (a.k.a. spindle poles). In addition to this central spindle apparatus, astral MTs assemble at the mitotic spindle pole and attach to the cell cortex to ensure appropriate spindle orientation. We propose that cell cycle-related kinase, Nek7, and its novel interacting protein RGS2, are involved in mitosis regulation and spindle formation. We found that RGS2 localizes to the mitotic spindle in a Nek7-dependent manner, and along with Nek7 contributes to spindle morphology and mitotic spindle pole integrity. RGS2-depletion leads to a mitotic-delay and severe defects in the chromosomes alignment and congression. Importantly, RGS2 or Nek7 depletion or even overexpression of wild-type or kinase-dead Nek7, reduced γ-tubulin from the mitotic spindle poles. In addition to causing a mitotic delay, RGS2 depletion induced mitotic spindle misorientation coinciding with astral MT-reduction. We propose that these phenotypes directly contribute to a failure in mitotic spindle alignment to the substratum. In conclusion, we suggest a molecular mechanism whereupon Nek7 and RGS2 may act cooperatively to ensure proper mitotic spindle organization.     

Human Nek7-interactor RGS2 is required for mitotic spindle organization

The mitotic spindle apparatus is composed of microtubule (MT) networks attached to kinetochores organized from 2 centrosomes (a.k.a. spindle poles). In addition to this central spindle apparatus, astral MTs assemble at the mitotic spindle pole and attach to the cell cortex to ensure appropriate spindle orientation. We propose that cell cycle-related kinase, Nek7, and its novel interacting protein RGS2, are involved in mitosis regulation and spindle formation. We found that RGS2 localizes to the mitotic spindle in a Nek7-dependent manner, and along with Nek7 contributes to spindle morphology and mitotic spindle pole integrity. RGS2-depletion leads to a mitotic-delay and severe defects in the chromosomes alignment and congression. Importantly, RGS2 or Nek7 depletion or even overexpression of wild-type or kinase-dead Nek7, reduced γ-tubulin from the mitotic spindle poles. In addition to causing a mitotic delay, RGS2 depletion induced mitotic spindle misorientation coinciding with astral MT-reduction. We propose that these phenotypes directly contribute to a failure in mitotic spindle alignment to the substratum. In conclusion, we suggest a molecular mechanism whereupon Nek7 and RGS2 may act cooperatively to ensure proper mitotic spindle organization.     

The serine/threonine kinase Nek6 is required for cell cycle progression through mitosis

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.     

Alternative splicing controls nuclear translocation of the cell cycle-regulated Nek2 kinase

Nek2 is a cell cycle-regulated serine/threonine protein kinase that is up-regulated in human cancers. Functionally, it is implicated in control of centrosome separation and bipolar spindle formation in mitotic cells and chromatin condensation in meiotic cells. Two major splice variants have been described in vertebrates, Nek2A and Nek2B, that differ in their non-catalytic C termini. Recently, a third splice variant, Nek2C, was identified that lacks an eight-amino acid internal sequence within the C-terminal domain of Nek2A. This excision occurs at the same position as the Nek2A/Nek2B splice point. As predicted from their high degree of similarity, we show here that Nek2C shares many properties with Nek2A including kinase activity, dimerization, protein phosphatase 1 interaction, mitotic degradation, microtubule binding, and centrosome localization. Unexpectedly, though, the non-centrosomal pool of protein exhibits a marked difference in distribution for the three splice variants. Nek2C is mainly nuclear, Nek2B is mainly cytoplasmic, and Nek2A is evenly distributed within nuclei and cytoplasm. Mutagenesis experiments revealed a functional bipartite nuclear localization sequence (NLS) that spans the splice site leading to Nek2C having a strong NLS, Nek2A having a weak NLS, and Nek2B having no NLS. Finally, we identified a 28-kDa protein in nuclear extracts as a potential novel substrate of Nek2. Thus, alternative splicing provides an unusual mechanism for modulating Nek2 localization, enabling it to have both nuclear and cytoplasmic functions.     

Nek2 localises to the distal portion of the mother centriole/basal body and is required for timely cilium disassembly at the G2/M transition

The NIMA-related kinase Nek2 promotes centrosome separation at the G2/M transition and, consistent with this role, is known to be concentrated at the proximal ends of centrioles. Here, we show by immunofluorescence microscopy that Nek2 also localises to the distal portion of the mother centriole. Its accumulation at this site is cell cycle-dependent and appears to peak in late G2. These findings are consistent with previous data implicating Nek2 in promoting reorganisation of centrosome-anchored microtubules at the G2/M transition, given that microtubules are anchored at the subdistal appendages of the mother centriole in interphase. In addition, we report that siRNA-mediated depletion of Nek2 compromises the ability of cells to resorb primary cilia before the onset of mitosis, while overexpression of catalytically active Nek2A reduces ciliation and cilium length in serum-starved cells. Based on these findings, we propose that Nek2 has a role in promoting cilium disassembly at the onset of mitosis. We also present evidence that recruitment of Nek2 to the proximal ends of centrioles is dependent on one of its substrates, the centrosome cohesion protein C-Nap1.     

APC/C-mediated destruction of the centrosomal kinase Nek2A occurs in early mitosis and depends upon a cyclin A-type D-box.

Nek2 is a NIMA-related kinase implicated in regulating centrosome structure at the G(2)/M transition. Two splice variants have been identified that exhibit distinct patterns of expression during cell cycle progression and development. Here we show that Nek2A, but not Nek2B, is destroyed upon entry into mitosis coincident with cyclin A destruction and in the presence of an active spindle assembly checkpoint. Destruction of Nek2A is mediated by the proteasome and is dependent upon the APC/C-Cdc20 ubiquitin ligase. Nek2 activity is not required for APC/C activation. Nek2A destruction in early mitosis is regulated by a motif in its extreme C-terminus which bears a striking resemblance to the extended destruction box (D-box) of cyclin A. Complete stabilization of Nek2A requires deletion of this motif and mutation of a KEN-box. Destruction of Nek2A is not inhibited by the cyclin B-type D-box, but the C-terminal domain of Nek2A inhibits destruction of both cyclins A and B. We propose that recognition of substrates by the APC/C-Cdc20 in early mitosis depends upon possession of an extended D-box motif.     

A mitotic cascade of NIMA family kinases. Nercc1/Nek9 activates the Nek6 and Nek7 kinases

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.     

Nek2A contributes to tumorigenic growth and possibly functions as potential therapeutic target for human breast cancer

Nek2A (NIMA-related kinases 2A) has been known as an important centrosome regulatory factor. The aim of this study was to investigate the expression of Nek2A and the role it played in different stages of breast cancer. We detected the expression of Nek2A in both mRNA and protein levels in MCF10 cell lines including MCF-10A, MCF-10DCIS.com, MCF-10CA1a and in human breast samples which contained normal breast tissue (NBT), breast ductal carcinoma in situ (DCIS), and invasive ductal carcinoma (IDC). Our study revealed that the mRNA and protein expression of Nek2A were significantly up-regulated in MCF-10DCIS.com and MCF-10CA1a cell lines as well as in human primary breast cancer tissue (DCIS and IDC). Our study also presented a correlation between Nek2A mRNA expression and some clinic pathological factors. We found that Nek2A mRNA expression was associated with molecular subtypes, ER, PR and Ki-67 immunoreactivity (P<0.05) in DCIS and associated with histological grade, lymph node metastasis, molecular subtypes, c-erbB-2, and Ki-67 expression (P<0.05) in IDC. In addition, we observed that ectopic expression of Nek2A in "normal" immortalized MCF-10A breast epithelial cell resulted in increased Nek2A which lead to abnormal centrosomes. Furthermore, knockdown of Nek2A in MCF-10DCIS.com could remarkably inhibit cell proliferation and induce cell cycle arrest in MCF-10DCIS.com cell line. These data suggested that Nek2A might bear a close relationship with development and progression of breast carcinoma, and highlighted its role as a novel potential biomarker for diagnosis and a possible therapeutic target for human breast cancer especially for DCIS.