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Originally published In Press as doi:10.1074/jbc.M404104200 on May 25, 2004

J. Biol. Chem., Vol. 279, Issue 31, 32716-32727, July 30, 2004
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Nucleolar Nek11 Is a Novel Target of Nek2A in G1/S-arrested Cells*

Kohji Noguchi, Hidesuke Fukazawa, Yuko Murakami, and Yoshimasa Uehara{ddagger}

From the Department of Bioactive Molecules, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan

Received for publication, April 13, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We previously reported that Nek11, a member of the NIMA (never-in-mitosis A) family of kinases, is activated in G1/S-arrested cells. We provide herein several lines of evidence for a novel interaction between Nek11 and Nek2A. Both Nek11 and Nek2A, but not Nek2B, were detected at nucleoli, and the Nek2A-specific C-terminal end (amino acids 399-445) was responsible for nucleolar localization. Endogenous Nek11 coimmunoprecipitated with endogenous Nek2A, and non-catalytic regions of each kinase were involved in the complex formation. Nek11L interacted with phosphorylated Nek2A but barely with the kinase-inactive Nek2A (K37R) mutant. In addition, both Nek2A autophosphorylation activity and the Nek11L-Nek2A complex formation increased in G1/S-arrested cells. These results indicate that autophosphorylation of Nek2A could stimulate its interaction with Nek11L at the nucleolus. Moreover, Nek2 directly phosphorylated Nek11 in the C-terminal non-catalytic region and elevated Nek11 kinase activity. The non-catalytic region of Nek11 showed autoinhibitory activity through intramolecular interaction with its N-terminal catalytic domain. Nek2 dissociated this autoinhibitory interaction. Altogether, our studies demonstrate a unique mechanism of Nek11 activation by Nek2A in G1/S-arrested cells and suggest a novel possibility for nucleolar function of the NIMA family.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The NIMA1 (never-in mitosis A) kinase was first identified in the filamentous fungus Aspergillus nidulans by genetic complementation of the nimA mutation and is essential for nuclear division cycle at G2/M transition (1). Protein kinases structurally related to fungal NIMA have been identified in various organisms (2). In the human genome, eleven NIMA-related kinases (Nek1-11) have been reported (3).

In human, Nek2, the most fungal NIMA-related kinase, is the best characterized (4). Two splice variants of Nek2, Nek2A and Nek2B that encode different C termini have been identified (5, 6). Non-catalytic C-terminal region of Nek2A but not that of Nek2B has the PP1 binding domain (6). PP1 represses Nek2A autophosphorylation and activation by dephosphorylation (7). Both Nek2A and Nek2B are cell cycle-regulated protein kinases detected at the centrosome (6), and Nek2A overexpression causes premature centrosome splitting (8). Although the role of Nek2B is unclear in somatic cells, Nek2B has an important function in zygotic centrosome assembly and maintenance in Xenopus oocytes (9-11). The studies on NIMA-related kinases in lower organisms also support a model that the regulatory function in the spindle pole body/microtubules organization center/centrosome is a conserved activity of Nek2-like kinases (12, 13). Additionally, Nek2 could localize at nucleus in somatic cell lines albeit its physiological significance remains (8, 14), and the functional significance of Nek2A specific extra coiled-coil domain at the C-terminal end has not been addressed. Concerning other member of human Neks, most of their functions have been largely unclear; however, recent studies are beginning to reveal their functions. Interesting findings have been reported that Nercc1/Nek9 activates Nek6/Nek7 during mitosis, representing a novel cascade of human NIMA-related kinases (15, 16). These findings raise a possibility that diversity of human NIMA-related kinases may compose an unknown NIMA family cascade.

We previously identified new members of the mammalian NIMA family of kinases, termed Nek11L and Nek11S (NIMA-related kinase 11 long and short isoform) and showed activation of Nek11 kinase activity by various DNA-damaging agents and DNA replication inhibitors (17). The transient expression of wild-type Nek11L enhanced the aphidicolin-induced S-phase arrest. Conversely, this S-phase arrest was reduced in the U2OS cell lines expressing kinase-inactive Nek11L (K61R) and these cells were more sensitive to aphidicolin-induced cell lethality (17). Therefore, Nek11 appears to have a role in the G1/S-arrested cells, probably in the DNA replication checkpoint, although the molecular mechanism for Nek11 activation was undetermined.

In this study, we investigated the molecular mechanism of Nek11 activation in G1/S-arrested cells. Unexpected colocalization of Nek11 and Nek2A was observed at nucleoli, and association of Nek11 with Nek2A was enhanced especially in G1/S-arrested cells. Biochemical analysis suggested that Nek11 was regulated by Nek2A. Overall, our observations strongly suggest that Nek11 is a novel target of Nek2A at nucleolus in G1/S-arrested cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Immunofluorescence Microscopic Analysis—For immunofluorescence microscopic studies, cells were seeded on multichamber slides (1-4 x 104 per well on 4-well multichamber slides, Lab-Tek II-CC2 chamber slide system, Nalgen Nunc International, Rochester, NY). The next day, cells were washed once with PBS, fixed with 4% formaldehyde/PBS buffer for 10 min, and treated with Triton buffer (0.5% Triton X-100, TBS, 10% glycerol, 1 mM EDTA) for 10 min at room temperature. The permeabilized cells were washed twice with PBS, and primary antibodies (1 µg/ml) in dilution buffer (1% bovine serum albumin, PBS, 0.1% Tween-20) were placed in a multichamber under indicated conditions. The slides were then washed with PBS (10 min x 3) and covered with secondary antibodies in dilution buffer (goat anti-rabbit IgG Alexa Fluor 594-conjugated (x2000), goat anti-mouse IgG Alexa Fluor 488-conjugated (x2000) (Molecular Probes, Inc., Eugene, OR)) for 1 h in a dark box at room temperature. Alternatively, the anti-HA antibody FITC-conjugated (clone F-7, Santa Cruz Biotechnology Inc., Santa Cruz, CA) was used as a primary antibody. Slides were again washed three times with PBS and mounted with DAPI-containing antifade (Vectashield, Vector Laboratories, Inc., Burlingame, CA). Immunostained cells were analyzed by a confocal laser scanning microscope using a Carl Zeiss LSM 510 system or by a conventional fluorescence microscope (OLYMPUS IX70) equipped with fluorescence digital CCD camera (KEYENCE, VB-6010).

Plasmid Construction—The complete open reading frame (ORF) containing cDNAs for Nek2A and Nek2B was isolated from HeLaS3 cells cDNA by RT-PCR method using KODplus DNA polymerase (TOYOBO, Osaka, Japan). The following synthetic primers were used: 5'-GGATCCATGCCTTCCCGGGCTGAGGACTATG-3' for the 5'-end of Nek2A/B, 5'-GGATCCGCGCATGCCCAGGATCTGTCTGC-3' for the 3'-end of Nek2A, 5'-GGATCCTTTGTAGCACCAGCTTCTGTTGAC-3' for the 3'-end of Nek2B, and 5'-GGGATCCCTCTGCTAGTCTCTCACGAACAC-3' for the3'-end of Nek2 (1-342). PCR products were subcloned into pCR®-Blunt II-TOPO plasmid (Invitrogen, CH Groningen, The Netherlands). DNA sequences of PCR products were verified, and these plasmids were termed pCR/nek2A, pCR/nek2B, and pCR/nek2-(1-342), respectively. To generate kinase-inactive Nek2, lysine residue at position 37 in Nek2 was replaced by arginine as described (18) with site-directed mutagenesis using a PCR-based QuikChangeTM site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the instruction manual. Synthetic primers (5'-CAAGATATTAGTTTGGAGAGAACTTGACTATGGC-3' and 5'-GCCATAGTCAAGTTCTCTCCAAACTAATATCTTG-3') were used with template pCR/nek2 plasmids. All mutated Nek2A and Nek2B were fully sequenced and termed pCR/nek2 (K37R). These Nek2A/B ORF containing cDNAs were subcloned into pD3HA plasmid (a pcDNA3-derived plasmid with an HA tag at the C terminus) at the BamHI site to construct a C-terminal HA-tagged Nek2. These plasmids were termed pD3Nek2A-HA, pD3Nek2A (K37R)-HA, pD3Nek2B-HA, pD3Nek2B (K37R)-HA, and pD3Nek2-(1-342)-HA. To generate leucine zipper-deleted Nek2B (deletion of amino acids 306-334) and PP1 non-binding mutant Nek2A (phenylalanine 386 was replaced by alanine) as described (19, 7), site-directed mutagenesis was carried out with template plasmid pD3Nek2B-HA and pD3Nek2A-HA. Synthetic primers (5'-CCAGCCCTGTATTGAGTGAGTGTGTTCGTGAGAGACTAGC-3' and 5'-GCTAGTCTCTCACGAACACACTCACTCAATACAGGGCTGG-3' for deletion of leucine zipper, and 5'-GTAATTAAGAAGAAAGTTCATGCCAGTGGGGAAAGTAAAG-3' and 5'-CTTTACTTTCCCCACTGGCATGAACTTTCTTCTTAATTAC-3' for PP1 non-binding mutant) were used, and sequence-verified plasmids were termed pD3Nek2BdLZ-HA and pD3Nek2A (F386A)-HA. The Nek2B ORF fragment was subcloned into the pEGFP-C1 plasmid (Clontech Laboratories, Inc., Palo Alto, CA) at the BamHI site to obtain the GFP-tagged Nek2B-expressing plasmid pGFP-Nek2B. pGFP-Nek2B plasmid was digested by SacI to remove the Nek2B N-terminal region and subjected to self-ligation to obtain the GFP-tagged Nek2B C-terminal region (amino acids 319-384)-expressing plasmid termed pGFP-Nek2B (319-384). The Nek2A C-terminal region (amino acids 399-445)-containing DNA fragment was obtained by double digestion of the Nek2A ORF by EcoRI and BamHI, and its 3'-end DNA fragment was subcloned into the pEGFP-C1 plasmid at the EcoRI-BamHI site to construct the GFPNek2A-(399-445)-expressing plasmid termed pGFP-Nek2A-(399-445).

N-terminal FLAG-tagged Nek11-expressing plasmids, pFLAG Nek11L and pFLAG-Nek11L (K61R), and EGFP-fused Nek11S fragment (379-470)-expressing plasmid pGFP-Nek11S-(379-470) were constructed previously (17). To obtain C-terminal deletion mutant Nek11s, PCR was carried out with synthetic primers (5'-CGAATTCCATGCTGAAATTCCAAGAGGCAGC-3' for the 5'-end, 5'-CGAATTCTTAATGGAGTACATCAACACTCAGCAG-3' for the 3'-end of Nek11-(1-392), and 5'-CGAATTCTTACTGTACTTCTGACAGTGCCCTCAG-3' for the 3'-end of Nek11-(1-337)) and template plasmid pFLAG-Nek11L. To construct C-terminal FLAG-tagged Nek11L, the stop codon-mutated Nek11L ORF was obtained by PCR with synthetic primers (5'-CGAATTCCATGCTGAAATTCCAAGAGGCAGC-3' for the 5'-end and 5'-GAATTCGGAGATGGTTCTGGAGAGTAGG-3' for the 3'-end) and template plasmid pFLAG-Nek11L. All PCR products were subcloned into pCR®-Blunt II-TOPO plasmid (Invitrogen), and DNA sequences were verified. These plasmids were termed pCR/nek11-(1-392), pCR/nek11-(1-337), and pCR/nek11C. ORFs of Nek11-(1-392) and Nek11-(1-337) were subcloned into pFLAG-CMV2 (Eastman Kodak, Rochester, NY) and the ORF of Nek11C into pFLAG-CMV5c (Eastman Kodak) at each EcoRI site. These expression plasmids were termed pFLAG-Nek11-(1-392), pFLAG-Nek11-(1-337), and pNek11L-FLAG. To obtain FLAG-tagged Nek11L C-terminal fragment (289-645)-expressing plasmid, pGFP-Nek11L plasmid and PCR product generated from pFLAG-Nek11L plasmid using synthetic primers (5'-GGAATTCCGAGCAGCTACAGAACC-3' and 5'-GGAATTCCATTCCTTTTAAATGTG-3') were digested by EcoRI and PstI. These EcoRI-PstI fragments were subcloned into pFLAG-CMV2 plasmid at EcoRI site to generate pFLAG-Nek11L-(289-645). Nek11 cDNAs were subcloned into pD3Myc plasmid (a pcDNA3-derived plasmid with a Myc tag at the N terminus) to obtain N-terminal Myc-tagged Nek11-expressing plasmids termed pMyc-Nek11L and pMyc-Nek11-(1-337). Nek11L C terminus-containing DNA fragments (amino acids 287-337, 385-476, and 412-573) were generated by PCR with synthetic primers (5'-CGAATTCCCTTGATGAGCAGCTACAG-3' and 5'-CGAATTCCTGTACTTCTGACAGTGCCCTCAG-3' for Nek11-(287-337), 5'-GGTCGACGCAGCTGAGTGTTGATGTAC-3' and 5'-GGTCGACGCTACTCATGGTATCCAAGG-3' for Nek11-(385-467), 5'-CGAATTCCTGTTCACCCCAGGACGAGGATGAAGAG-3' and 5'-CGAATTCCCTCATGCGTTTCATCTTGGTCCTGGA-3' for Nek11-(412-573)). These PCR products were subcloned into pCR®-Blunt II-TOPO plasmid, and their sequences were verified. EcoRI-digested insert fragments were subcloned into pGEX6P-3 or pGEX6P-2 (Amersham Biosciences) at an EcoRI site. Then GST fusion protein-expressing plasmids, termed pGEX-Nek11-(287-337), pGEX-Nek11-(385-467), and pGEX-Nek11-(412-573) were obtained.

Transfection, Cell Extract Preparation, Immunoprecipitation, and Immunocomplex Kinase Assay—For immunoprecipitation in the transient transfection, HEK293T cells were transfected with expression plasmid (total 2 µg of plasmid DNA/0.5 x 106 cells/60-mm dish) using FuGENETM 6 reagents (Roche Applied Science) according to the instruction manual. When U2OS cells were used, expression plasmid (total 1 µg of plasmid DNA/4 x 105 cells/60-mm dish) was transfected for a 4-5 h incubation by EffecteneTM reagents (Qiagen GmbH) according to the instruction manual. After 15-44 h, cells were harvested and suspended in Nek11 lysis buffer (20 mM Hepes-NaOH pH 7.5, 420 mM NaCl, 2 mM MgCl2, 0.1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 2 µg/ml pepstatin A, 2 µg/ml heparin, 1 µM okadaic acid, 1 µM cyclosporin A) on ice for 10 min. Soluble cell extracts were obtained by centrifugation (12,000 x g) at 4 °C for 10 min. HeLaS3 and U2OS cell extracts were also prepared as above. To prepare cytosolic and nuclear extracts, HeLaS3 cells were primarily extracted with Nek2 lysis buffer (50 mM Hepes-NaOH pH 7.5, 100 mM NaCl, 5 mM KCl, 10 mM MgCl2, 5 mM MnCl2, 0.1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 2 µg/ml pepstatin A, 2 µg/ml heparin, 1 µM okadaic acid), and centrifuged (10,000 x g, at 4 °C for 10 min) to obtain the cytosolic extract. The resulting cell pellets were re-extracted by Nek11 lysis buffer. FLAG-tagged and HA-tagged proteins were immunoprecipitated by anti-FLAG M2 agarose and anti-HA agarose (Sigma), respectively, for 2-3 h at 4 °C on a rotating wheel. Endogenous Nek11 protein was immunoprecipitated by anti-Nek11 antibody (5 µg/107 cells in extract) for 4 h at 4 °C on a rotating wheel, followed by addition of protein A 4FF Sepharose (Amersham Biosciences) for 1 h. Immunocomplexes were washed with HS buffer (0.1% Nonidet P-40, 50 mM Hepes-NaOH pH 7.5, 1 M NaCl) (1 ml x 5) and 50 mM Hepes-NaOH pH 7.5 (1 ml x 1). For the in vitro immunocomplex kinase assay, reactions were carried out at 30 °C for 15 min in 20 µl of kinase buffer (50 mM Hepes-NaOH pH 7.5, 10 µg/ml heparin, 5 mM MnCl2, 10 µM ATP, 100 µCi/ml of [{gamma}32-P]ATP (Amersham Biosciences), 0.2 mg/ml histone/GST proteins or 0.04 mg/ml FLAG-substrate). Reactions were stopped by the addition of 4x Laemmli SDS sample buffer (7 µl/sample). Samples were resolved by SDS-PAGE, and analyzed using a BAS 1800 bio-image analyzer (Fujifilm).

Western Blot Analysis—For preparation of whole cell lysates, cells were lysed in 1x Laemmli SDS sample buffer and sonicated. Other soluble cell extracts were prepared using Nek11 lysis buffer as described above. Sample proteins from an equal number of cells were mixed with 4x Laemmli SDS sample buffer, heat-denatured for 2 min, resolved by SDS-PAGE, and electrophoretically transferred onto ImmobilonTM polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA). The membranes were blocked in 5% low fat milk or 3% bovine serum albumin/TBS-T (20 mM Tris-HCl, pH 7.5, 137 mM NaCl, 0.1% Tween-20), and incubated with primary antibodies diluted with blocking buffer at 4 °C for 15-20 h. After incubation at room temperature for 1 h with secondary antibodies conjugated with horseradish peroxidase, signals were detected by enhanced chemiluminescence using an ECL detection reagent (Amersham Biosciences) or a Western Lightning chemiluminescence reagent Plus (PerkinElmer Life Sciences, Inc., Boston, MA).

In Vitro Dephosphorylation Assay—For Nek2A dephosphorylation, Nek2A (F386A)-HA was coimmunoprecipitated with FLAG-Nek11L using anti-FLAG M2 agarose from 293T cells. Aliquots of immunocomplex beads were incubated at 25 °C for 30 min in the presence or absence of phosphatases (PP2A (0.5 units/25 µl), PP1 (0.5 units/25 µl), and calf intestine alkaline phosphatase (50 units/25 µl)). For Nek11L de-phosphorylation, Nek11L-FLAG was coexpressed with Nek2B and immunoprecipitated by anti-FLAG M2 agarose from 293T cells. Aliquots of immunocomplex beads were incubated at 37 °C for 30 min in the presence or absence of phosphatases (PP2A and PP1, 0.2 units/20 µl each). Dephosphorylation reaction was carried out using an attached phosphatase buffer (Upstate, Charlottesville, VA). Samples were subjected to SDS-PAGE, and electrophoretic mobility changes were examined by Western blotting.

Recombinant Protein Preparation—GST fusion proteins were expressed in Escherichia coli JM109 by IPTG (isopropyl-{beta}-D-(-)-thiogalactopyranoside) induction (0.2 mM for 2-3 h at 37 °C or 30 °C), collected on GSH-Sepharose 4B (Amersham Biosciences), and eluted by 10 mM GSH in 50 mM Tris-Cl (pH 8.5). FLAG-tagged Nek11 and HA-tagged Nek2 proteins were transiently expressed in 293T cells, immunoprecipitated by anti-FLAG M2 agarose or anti-HA agarose, and eluted by FLAG or HA peptides (200 µg/ml in 50 mM Hepes-NaOH pH 7.5).

Phosphoamino Acid Analysis—FLAG-Nek11L (K61R) protein was phosphorylated by HA-tagged Nek2 (F386A) or Nek2B in vitro as described above, separated by SDS-PAGE, and transferred to the polyvinylidene difluoride membrane. Corresponding bands were cut out and subjected to acid hydrolysis in 6 N HCl at 110 °C for 1 h. Hydrolyzed phosphoamino acids were dried using a Speedvac concentrator, and resolved by pH 1.9 buffer (2.2% formic acid, 7.8% acetic acid). Sample was separated by two-dimensional electrophoresis (pH 1.9 and pH 3.5) on thin layer chromatography plate (MERCK 1.05716.) by the standard method, and results were recorded on x-ray film by autoradiography.

Biochemical Studies of Nek11 C-terminal Non-catalytic Region—For detection of Nek11 homo-oligomerization and intramolecular interaction, FLAG-Nek11-(289-645) and Myc-Nek11-(1-337)-expressing cells were lysed in Nek11 lysis buffer. Immunoprecipitated complexes by anti-Myc agarose were washed with Nek11 lysis buffer (1 ml x 5) and 50 mM Hepes-NaOH pH 7.5 (1 ml x 1) as described above. To examine the effect of in vitro post-phosphorylation by Nek2 on Nek11 intramolecular interaction, Nek2A (F386A)-HA protein was transiently expressed in 293T cells, immunoprecipitated by anti-HA agarose, and eluted by HA peptide (200 µg/ml in 50 mM Hepes-NaOH, pH 7.5). Then aliquots of Myc-agarose immunocomplex beads were subjected to in vitro kinase assay in the presence or absence of Nek2A (F386A)-HA at 37 °C for 30 min. Following a wash by Nek11 lysis buffer (1 ml x 3) and by 50 mM Hepes-NaOH pH 7.5 (1 ml x 1), immunocomplex samples were subjected to Western blot analysis. To examine the effect of the C-terminal non-catalytic region of Nek11 on its kinase activity, equal amounts of FLAG-Nek11-(1-337)-beads were preincubated with GST-Nek11-(412-573) or control GST proteins (1-12 µg/10 µl) for 5 min at room temperature. Subsequently 2 x kinase buffer (10 µl) containing histone H2A substrate (4 µg) was added to perform kinase reaction at 30 °C for 15 min followed by SDS-PAGE. Quantitative analysis was performed as described above.

Cell Culture and Miscellaneous Materials—Human embryonic kidney transformed fibroblast HEK293T cells, cervical epithelioid carcinoma HeLaS3 cells, and osteosarcoma U2OS cells were grown in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% heat-inactivated fetal bovine serum (Sigma) and 5 µg/ml gentamicin (Invitrogen Life Technologies). For cell cycle block, cells were treated with aphidicolin (2 µg/ml) and hydroxyurea (2 mM) or by nocodazole (1 µM) and taxol (1 µg/ml) for 18 h to arrest at the G1/S phase or at the G2/M phase. Anti-FLAG M2 agarose, anti-HA agarose, and anti-FLAG M2 antibodies were purchased from Sigma. Anti-Nucelophosmin/B23 antibody was from Zymed Laboratory Inc. (South San Francisco, CA), and anti-Myc antibody (9E10) from Oncogene Research Products (San Diego, CA). Anti-Nek2 antibody was from Transduction Laboratories (Lexington, KY). Anti-GFP, anti-HA polyclonal (Y-11), and anti-Myc agarose antibodies were purchased from Santa Cruz Biotechnology, Inc., and anti-GST antibody was from Amersham Biosciences. Affinity-purified anti-Nek11 polyclonal antibody was prepared as described (17). PP2A and PP1 were obtained from Upstate (Charlottesville, VA), and calf intestine alkaline phosphatase was from TaKaRa. Nocodazole, aphidicolin, and hydroxyurea were purchased from Sigma and okadaic acid was from Wako Chemicals. Histone H2A was from Roche Applied Science.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Association and Colocalization of Endogenous Nek11 with Nek2A at Nucleolus—We observed enrichment of Nek11 at the nuclear granular structure in the indirect immunofluorescent analysis using anti-Nek11 antibody and U2OS cells. These nuclear small round granular structures appeared to be nucleoli, and we thus compared subnuclear localization of Nek11 with a typical nucleolar protein Nucleophosmin (NPM)/B23 (20). Most Nek11 colocalized with NPM at interphase, telo-phase, and M/G1 transition phase, whereas mitotic Nek11 was detected at perichromosome, and colocalization was partial during metaphase (Fig. 1A). These dynamics of Nek11 resembled nucleolar component disassembly/reassembly during cell cycle (21), and thus we concluded that Nek11 is a novel nucleolar protein.



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  		FIG. 1.
Nucleolar association of Nek11 with Nek2. A, subcellular localization of Nek11 during cell cycle. U2OS cells were fixed with 4% formalin/PBS and treated with 0.5% Triton X-100, TBS, 10% glycerol, and primary antibodies (anti-Nek11 polyclonal and anti-NPM monoclonal antibodies, 1 µg/ml each) were reacted for 18 h at 4 °C. Nek11 was visualized with anti-rabbit-IgG Alexa Fluor 594-conjugated (red), and Nucleophosmin (NPM) with anti-mouse IgG Alexa Fluor 488-conjugated (green) for 1 h of incubation at room temperature. DNA was costained by DAPI (1 µg/ml). Indirect immunofluorescent analysis was performed with a confocal laser scanning microscope LSM510 system. B, detection of endogenous Nek2 at nucleoli. Asynchronous U2OS and HeLaS3 cells were fixed and permeabilized as in A. Endogenous Nek11 (red) and Nek2 (green) were probed with anti-Nek11 polyclonal and anti-Nek2 antibodies (1 µg/ml each) for 4-18 h at 4 °C. DNA was stained with DAPI, and analyzed using a confocal laser scanning microscope LSM510 system. White arrows indicate nucleolar staining, and arrowheads indicate centrosomal staining. C, endogenous Nek11 coimmunoprecipitated with Nek2A. Endogenous Nek11 was immunoprecipitated (IP) with anti-Nek11 polyclonal antibody (anti-Nek11) from U2OS and HeLaS3 cells. Coimmunoprecipitated Nek2A was detected by Western blot analysis using anti-Nek2 monoclonal antibody. As a negative control, normal rabbit IgG (Control Ig) was used for immunoprecipitation. D, Nek11-Nek2A complex recovered from nuclear fraction. HeLaS3 cells were primarily extracted by low salt lysis buffer (0.1 M NaCl, 0.1% Nonidet P-40) (cytoplasmic fraction, L), and cell pellets were secondarily extracted by high salt lysis buffer (0.42 M NaCl, 0.1% Nonidet P-40) (nuclear fraction, H). Nek11 protein was immunoprecipitated by anti-Nek11 polyclonal antibody from each extract, and coimmunoprecipitated Nek2A protein was detected by Western blot analysis using anti-Nek2 antibody. Left panels show the immunoprecipitated Nek11 and Nek2, and right panels show the expression level of Nek11 and Nek2 proteins in each extract.

 
Another well studied NIMA family kinase Nek2 is shown to localize at the centrosome (8). In our preliminary experiments, however, overexpressed exogenous Nek11 and Nek2 were abundant at cytoplasm (probably due to the leptomycin B-sensitive nuclear export system) and some of them colocalized at the pericentrosomal aggresome-like structure in 293T cells (data not shown). Since a recent study reported a NIMA family cascade consisting of Nercc1/Nek9 and Nek6/7 (16), we analogously hypothesized interaction/colocalization of endogenous Nek11 with Nek2. We surveyed subnuclear localization of endogenous Nek2 protein using a confocal laser scanning microscope. Consistent with the previous report, Nek2 protein was detected at the centrosome in both U2OS and HeLaS3 cells (Fig. 1B, arrowheads). Moreover, Nek2 signals also showed colocalization with nucleolar Nek11 in both cell lines (Fig. 1B, arrows). These results indicated that some of the endogenous Nek2 protein could colocalize with nucleolar Nek11. We next tested association of Nek11 with Nek2 by coimmunoprecipitation assay. Endogenous Nek11 was immunoprecipitated from U2OS and HeLaS3 cells, and we detected coimmunoprecipitation of Nek2A but not of Nek2B in both cell lines (Fig. 1C). Coimmunoprecipitation of Nek2A with Nek11 was only observed in the nuclear fraction (H), but not in the cytoplasmic fraction (L) (Fig. 1D). These results suggested that endogenous Nek11 could associate with some Nek2A at the nucleolus.

Specific Nucleolar Targeting of Nek2A Mediated by its C-terminal End Region—We next addressed why endogenous Nek11 coimmunoprecipitated with endogenous Nek2A, but not with Nek2B. As the anti-Nek2 antibody we used here could not distinguish Nek2A and Nek2B, we introduced HA-tagged Nek2A- and Nek2B-expressing plasmids into U2OS cells, and subnuclear localization of each kinase was compared by a confocal laser scanning microscope. Consistent with the results above, nuclear Nek2B did not colocalize with nucleolar Nek11 in interphase cells whereas some Nek2A merged with nucleolar Nek11 (Fig. 2A, left panels). Since Nek2A has an additional coiled-coil domain at the C-terminal end, we introduced a plasmid expressing GFP-fused Nek2A specific coiled-coil domain (amino acids 399-445) into U2OS cells (Fig. 2B). Using a conventional fluorescence microscope, GFP-Nek2A-(399-445) protein showed nuclear (94%, n = 122) and nucleolus-like (89%, n = 122) enrichment in living U2OS cells, but control GFP protein showed diffused distribution (Fig. 2C, left panels). Furthermore, analysis of fixed and permeabilized cells using a confocal laser scanning microscope confirmed that GFP-Nek2A-(399-445) protein indeed colocalized with nucleolar Nek11 whereas GFP-Nek2B-(319-384) did not (Fig. 2C, right panels, arrows). Overall, these experiments demonstrated for the first time that Nek2A, but not Nek2B, has a unique nucleolar targeting/retention activity through its C-terminal end coiled-coil domain (amino acids 399-445). In contrast, mitotic Nek2A and Nek2B proteins were detected on the centrosome and disengaged from perichromosomal Nek11 at metaphase (Fig. 2A, right panels), suggesting that the Nek11-Nek2A complex dissociates during mitosis.



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  		FIG. 2.
Nek2A selective nucleolar targeting. A, Nek2A-HA, Nek2A (F386A)-HA, and Nek2B-HA were transiently expressed in U2OS cells. Fifteen hours after transfection, cells were fixed with 4% formalin/PBS and permeabilized with 0.5% Triton X-100, TBS, 10% glycerol. Endogenous Nek11 protein (red) was probed with anti-Nek11 polyclonal antibody, and Nek2-HA proteins (green) with anti-HA monoclonal antibody FITC conjugate. Primary antibodies were reacted at room temperature for 4 h, and Nek11 was visualized with anti-rabbit IgG Alexa Fluor 594. Left and right panels show images of interphase and mitotic cells, respectively, and white arrows indicate nucleoli. B, scheme for GFP-Nek2 (right scheme) and Nek2-HA (left scheme). C, localization of GFP fusion protein. GFP and GFP-Nek2A-(399-445) proteins were transiently expressed for 15 h and examined by a conventional fluorescence microscope in living U2OS cells (left panels). Colocalization of GFP fusion proteins with Nek11 was analyzed with a confocal laser scanning microscope LSM 510 (right panels) as in A. GFP fusion proteins are shown in green, Nek11 in red, and DAPI-stained DNA in blue. White arrows indicate nucleoli.

 
Nek11 Association With and Activation by Nek2—Endogenous Nek11 and Nek2A formed a complex in vivo, so we next addressed the interaction domain of each kinase. Transient overexpression assay is convenient to estimate the interaction domain of each kinase in vivo, although artificial nonphysiological interaction between Nek2B and Nek11 could be detected (probably due to their cytoplasmic abundance). FLAG-tagged Nek11L protein was coexpressed with various HA-tagged Nek2 proteins (wild-type Nek2A/B, kinase-inactive Nek2A/B (K37R), PP1 binding motif lacking Nek2 (F386A) mutant, leucine zipper-deleted Nek2BdLZ (lacking amino acids 306-334), and C-terminal coiled-coil-deleted Nek2-(1-342)) in 293T cells. Immunoprecipitation-Western blot analysis was carried out to determine the region of Nek2 involved in interaction with Nek11. The results showed that PP1 binding motif lacking Nek2A (F386A) and wild-type Nek2B formed a tight complex with Nek11L, but others did not (Fig. 3A). In addition, in vitro dephosphorylation assay showed that PP2A, PP1, and calf intestine alkaline phosphatase treatments caused rapid electrophoretic migration of Nek11-associated Nek2A (F386A)-HA (Fig. 3B), indicating that Nek11-bound Nek2A (F386A) was phosphorylated. Previous studies have shown that binding of PP1, deletion of the leucine zipper region, and inactivation of mutation (K37R) suppress kinase activity and autophosphorylation of Nek2A (7, 18, 19). As kinase-inactive Nek2 (K37R) mutants hardly interacted with Nek11 (Fig. 3A), association of Nek2 with Nek11 probably depended on autophosphorylation of Nek2. In addition, Nek2-(1-342) mutant lacking the C-terminal coiled-coil region, which has comparable kinase activity with Nek2B (data not shown), did not form a stable complex with Nek11L, indicating that the coiled-coil region common in Nek2A and Nek2B (amino acids 343-370) also has an important role in the functional interaction with Nek11. In parallel with these data, Nek11L immunocomplex kinase activity was remarkably elevated by the coexpressions of Nek2A (F386A) and wild-type Nek2B (Fig. 3A, bottom panel). Because the Nek11L (K61R)-Nek2A (F386A) immunocomplex showed significant kinase activity, Nek2 kinase activity should contribute to total Nek11-Nek2 immunocomplex kinase activity (Fig. 3C). Wild-type Nek11L-Nek2A (F386A) immunocomplex, however, showed higher kinase activity than the expected additive activity (Fig. 3C, upper quantitative graph), suggesting that Nek11 is activated by Nek2.



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  		FIG. 3.
Association between Nek11 and Nek2 mediated by their non-catalytic regions. A, wild-type Nek2A, PP1 binding motif-disrupted Nek2A (F386A), Nek2B, kinase-inactive Nek2A/B (K37R), leucine zipper-deleted Nek2BdLZ (lacking amino acids 306-334), and C-terminal-deleted Nek2-(1-342), were coexpressed with Nek11L-FLAG in 293T cells for 40 h (FLAG-plasmid 2 µg/HA-plasmid 0.3 µg). Nek11L-FLAG was immunoprecipitated (IP) by anti-FLAG agarose followed by Western blot analysis (WB) to detect coimmunoprecipitated Nek2 proteins using anti-HA antibody. Kinase activity of the immunoprecipitated Nek11L-FLAG was measured by in vitro kinase assay using histone H2A as a substrate (bottom panel). B, Nek11-interactive Nek2A is phosphorylated. Nek2A (F386A)-HA was coimmunoprecipitated with FLAG-Nek11L by anti-FLAG M2 agarose, and immunocomplexes were subjected to in vitro dephosphorylation assay using PP2A, PP1, and calf intestine alkaline phosphatase (CIAP). Electrophoretic mobility of Nek2A (F386A) on SDS-PAGE was examined by Western blot analysis using anti-HA antibody. C, Nek11 immunocomplex activation. Wild-type (WT) and kinase-inactive (K61R) Nek11L-FLAG were coexpressed with Nek2A (F386A)-HA in 293T cells as in A. Immunoprecipitation (IP), Western blot (WB), and immunocomplex kinase assay (IVK) of FLAG immunocomplex were performed as in A. Upper graph shows quantitative data obtained from the results of four independent experiments. Relative activities compared with data from cells expressing wild-type Nek11L only are shown. D, various FLAG-tagged Nek11 proteins, wild-type FLAG-Nek11L, FLAG-Nek11S, FLAG-Nek11-(1-392) lacking PEST-like region, and FLAG-Nek11-(1-337) lacking both coiled-coil and PEST-like motifs were coexpressed with Nek2B-HA in 293T cells. Nek11 proteins were immunoprecipitated (IP) and the coprecipitated Nek2B-HA was detected by Western blot analysis (WB) using anti-HA antibody. E, Nek11S-(379-470) region competed with Nek11-Nek2 association in 293T cells. GFP and GFP-Nek11S-(379-470) were coexpressed with FLAG-Nek11L and Nek2B-HA in 293T cells. FLAG-Nek11L was immunoprecipitated (IP) and the coimmunoprecipitated Nek2B-HA was detected by Western blot analysis (WB) using the anti-HA antibody. Expressions of GFP proteins in cell lysates were also confirmed by Western blot analysis using the anti-GFP antibody. F, C-terminal region of Nek11 interacts with Nek2. Nek2A (F386A)-HA and Nek2B-HA expressed in 293T cells were precipitated by GST or GST-Nek11-(385-467) proteins and detected by Western blot analysis (WB) using the anti-HA antibody. GST proteins used for pull-down assay were also detected on the same membrane by anti-GST antibody.

 
To determine an essential domain in Nek11 protein for Nek2 interaction, Nek2B-HA was coexpressed with various FLAG-tagged Nek11 proteins (wild-type Nek11L and Nek11S, C-terminal deleted mutants Nek11-(1-392) and Nek11-(1-337), and kinase-inactive Nek11L (K61R)) in 293T cells. The coimmunoprecipitation assay showed that C-terminal-deleted Nek11-(1-392) and Nek11-(1-337) proteins did not coimmunoprecipitate with Nek2B, but Nek11S (amino acids 1-470) did (Fig. 3D). Amino acids at 393-470 contain PEST-like elements in Nek11L and Nek11S (17), and the above data suggested that this domain in Nek11 was required for complex formation with Nek2 in cells. Furthermore, we tested the effect of GFP-tagged Nek11S fragment (379-470) on Nek11-Nek2 complex formation in 293T cells (Fig. 3E). This experiment showed that coexpression of GFP-Nek11-(379-470) significantly reduced coimmunoprecipitation of Nek2B with Nek11L, suggesting that the Nek11S fragment (379-470) could suppress Nek11-Nek2 association in cells. To confirm involvement of this PEST-like element in Nek11-Nek2 interaction, we carried out a pull-down experiment using bacterially expressed recombinant GST-Nek11-(385-467) with Nek2-HA-expressing cell lysates. As expected, Nek2 was precipitated only by GST-Nek11-(385-467) but not by control GST (Fig. 3F). Collectively, we concluded that Nek11-Nek2 complex formation in vivo depends on phosphorylation of Nek2 and requires both Nek2 coiled-coil region (amino acids 306-384) and Nek11 PEST-like region (amino acids 393-467).

Association of Nek11 with Nek2 Increased in G1/S-arrested Cells—Nek11 is activated in G1/S-arrested cells (17), and Nek2A protein is increased in these cells (18). Therefore, we examined Nek11-Nek2 interaction during cell cycle by coimmunoprecipitation analysis. Endogenous Nek11 protein was immunoprecipitated from HeLaS3 cells chemically synchronized at G1/S or G2/M phase, and we found that coimmunoprecipitation of endogenous Nek2A increased especially in G1/S-arrested cells (Fig. 4A, left panels). In addition, autophosphorylation activity of Nek2A-HA was stimulated in aphidicolin-treated 293T cells (Fig. 4B). Consistent with these data, association of exogenous Nek2A-HA with Nek11L-FLAG increased in G1/S-arrested 293T cells (Fig. 4C, middle panels). As association of Nek2A (F386A)-HA mutant with Nek11L-FLAG also increased in G1/S-arrested cells (Fig. 4C, middle panels), G1/S-arrest-induced increase of Nek2A-Nek11 interaction appeared to be independent of PP1 binding with Nek2A. These results suggested that Nek2A was activated through a PP1-independent mechanism to increase its association with Nek11 in G1/S-arrested cells.



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  		FIG. 4.
Complex formation between Nek11 and Nek2A in G1/S-arrested cells. A, HeLaS3 cells were synchronized at G1/S phase by aphidicolin (APH, 2 µg/ml) and hydroxyurea (HU, 2 mM) or at M phase by nocodazole (Noc, 1 µM) and taxol (1 µg/ml) for 18 h. Endogenous Nek11 was immunoprecipitated (IP) and coimmunoprecipitated endogenous Nek2 was detected by Western blot analysis (WB) using anti-Nek2 antibody (left panels). The amounts of immunoprecipitated endogenous Nek11 were also confirmed with anti-Nek11 antibody (lower panel). The expression levels of endogenous Nek11 and Nek2 proteins were confirmed by Western blot analysis using cell lysates (right panels). B, autophosphorylation activity of Nek2A in G1/S-arrested cells. Nek2A-HA transfected 293T cells were arrested at G1/S phase by aphidicolin (APH, 2 µg/ml) or hydroxyurea (HU, 2 mM) for 16 h, and Nek2A-HA was immunoprecipitated (IP) by anti-HA agarose. The amounts of immunoprecipitated Nek2A-HA protein were examined by Western blot analysis (WB) using anti-HA antibody (upper panel) and autophosphorylation activity of Nek2A-HA was analyzed by in vitro kinase assay (middle panel). Lower graph shows the result from in vitro kinase assay performed in triplicate. C, association between Nek11L-FLAG and Nek2A-HA increased in G1/S-arrested cells. Nek2A-HA and Nek2A (F386A)-HA were coexpressed with Nek11L-FLAG in 293T cells, and transfected 293T cells were treated with aphidicolin (2 µg/ml, APH) for 16 h. Nek2A-HA, Nek2A (F386A)-HA and Nek11L-FLAG were immunoprecipitated (IP) by anti-HA or anti-FLAG agarose from each cell lysate aliquot. The amounts of Nek2As immunoprecipitated by anti-HA-agarose, and the expression level of Nek2As in cell lysates were confirmed by Western blot analysis (WB). Autophosphorylation activities of anti-HA agarose-immunoprecipitated Nek2A-HA and Nek2A (F386A)-HA were examined by in vitro kinase assay. The amounts of Nek11L-FLAG immunoprecipitated by anti-FLAG agarose and coimmunoprecipitated Nek2A-HA were confirmed by Western blot analysis (WB), and kinase activity of immunoprecipitated Nek11L-FLAG was examined by in vitro kinase assay using histone H2A as a substrate. The amounts of coimmunoprecipitated Nek2A-HA were recorded on x-ray films with short and long exposure times.

 
Modulation of Nek11 C-terminal Non-catalytic Function by Nek2—In our studies above, Nek2-HA coexpression caused slower migration of some Nek11L-FLAG on SDS-PAGE (as seen in Fig. 3, A and C), suggesting post-translational modification of Nek11 by Nek2. Thus, we examined whether Nek11 could be a phosphorylation substrate for Nek2. In vitro incubation of Nek11L-FLAG with Nek2B-HA in a kinase reaction buffer induced electrophoretic mobility shift of Nek11L (Fig. 5A). The band shift of Nek11L was reduced by in vitro phosphatase treatment, especially by PP2A but not by PP1 (Fig. 5B), indicating that Nek11L could be phosphorylated by Nek2 in vitro and in vivo. In vitro immunocomplex kinase assay using [{gamma}-32P]ATP showed that Nek2 phosphorylated both FLAG-Nek11L (K61R) and a non-catalytic region containing FLAG-Nek11-(285-645), but FLAG-Nek11 (K61R, 1-337) to a lesser extent (Fig. 5C). GST-Nek11-(287-337) and GST-Nek11-(412-573) but not GST also served as a good substrate for Nek2, while phosphorylation of GST-Nek11-(385-467) was low (Fig. 5D). Phosphoamino acid analysis of the FLAG-Nek11L (K61R) phosphorylated by Nek2A (F386A)-HA in vitro detected predominantly phosphoserine with traces of phosphothreonine, but no phosphotyrosine (Fig. 5E). These data indicated that the non-catalytic region of Nek11L was phosphorylated by Nek2 on multiple serine residues, at least within amino acid positions 287-337 and 468-573 in vitro.



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  		FIG. 5.
Nek2-mediated phosphorylation on Nek11 non-catalytic region. A, Nek2-mediated in vitro phosphorylation caused Nek11L electrophoretic mobility shift. Nek11L (K61R)-FLAG protein was immunoprecipitated and recovered by competitive peptide elution. Nek2B-HA was immunoprecipitated by anti-HA agarose from 293T cells as enzyme source. Nek11L (K61R)-FLAG protein was subjected to in vitro kinase assay in the presence or absence of Nek2B-HA-beads, and Western blotting using anti-Nek11 antibody confirmed electrophoretic mobility of Nek11L (K61R)-FLAG. Nek2B-HA used in this assay was also confirmed by Western blotting using anti-HA antibody. B, electrophoretic mobility change of Nek11 by PP2A treatment. Nek11L-FLAG was coexpressed with Nek2B-HA and immunoprecipitated (IP) by anti-FLAG agarose. Aliquots of immunocomplex were treated by either control buffer, PP2A or PP1. Subsequent Western blotting (WB) showed electrophoretic mobility of Nek11L-FLAG using anti-FLAG antibody. Coimmunoprecipitated Nek2B-HA was also confirmed with anti-HA antibody. C, Nek2 phosphorylated Nek11L in vitro. Nek2A (F386A)-HA immunocomplex beads recovered from 293T cells were used as enzyme source. FLAG-Nek11 proteins recovered by immunoprecipitation from 293T cells were used as substrates for in vitro kinase assay. Left and right panels show the result of autoradiography and Coomassie Blue staining of the gel. D, Nek2-mediated phosphorylation on Nek11 C-terminal region. GST-Nek11-(287-337), GST-Nek11-(385-467), GST-Nek11-(412-573), and GST (4 µg/20 µl each) were subjected to in vitro kinase assay using Nek2A (F386A)-HA immunocomplex. Upper and lower panels show the result of autoradiography and Coomassie Blue staining of the gel. E, phosphoamino acid analysis of phosphorylated Nek11L. FLAG-Nek11L (K61R) was phosphorylated by Nek2A (F386A)-HA in vitro as described above, separated by SDS-PAGE, blotted onto polyvinylidene difluoride membrane, and subjected to acid hydrolysis followed by two-dimensional separation on thin layer chromatography plate. The positions of phosphoamino acids are illustrated at left.

 
Non-catalytic regions of many kinases have autoregulatory functions. Actually, in vitro kinase assay indicated that FLAG-Nek11-(1-337) kinase activity was strongly suppressed by an addition of GST-Nek11-(412-573) but not by GST (Fig. 6A). Further, we found that Myc-Nek11-(1-337) coimmunoprecipitated with FLAG-Nek11-(289-645) from asynchronous cells (Fig. 6B). Coimmunoprecipitation assay, however, showed that FLAG-Nek11L and Myc-Nek11L oligomer was not detected in asynchronous cells despite our being able to detect it in nocodazole-arrested M phase cells (Fig. 6C). These results indicated that C-terminal non-catalytic region of Nek11 would directly interact with its N-terminal catalytic domain during interphase probably in an intramolecular manner as an autorepressor domain. Importantly, Nek2-mediated phosphorylation in vitro caused a dissociation of FLAG-Nek11-(289-645) from Myc-Nek11-(1-337) (Fig. 6D), indicating that Nek2 could disrupt autoinhibitory intramolecular interaction within Nek11. These experiments provided evidence that the C-terminal non-catalytic region of Nek11 has an autorepressive function, and that Nek2-association and/or Nek2-mediated phosphorylation would antagonize this autoinhibitory activity. Overall, these results strongly suggested that Nek2A could activate nucleolar Nek11 by modulating the non-catalytic region of Nek11 (summarized in Fig. 7).



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  		FIG. 6.
Autoregulatory function of Nek11 non-catalytic region. A, autoinhibitory activity of Nek11 C-terminal non-catalytic region. FLAG-Nek11-(1-337) was immunoprecipitated from 293T cells and subjected to in vitro immunocomplex kinase assay using histone H2A as substrate in the presence of GST-Nek11-(412-573) or control GST (1-12 µg/20 µl). Upper and lower panels show the result of autoradiography and Coomassie Blue staining of the gel. B, interaction between Nek11 N-terminal catalytic and C-terminal non-catalytic region. Myc-Nek11-(1-337) was coexpressed with or without FLAG-Nek11-(289-645) and immunoprecipitated (IP) by anti-Myc agarose. Coimmunoprecipitated FLAG-Nek11-(289-645) was detected by Western blotting (WB) using anti-Nek11 antibody. The amounts of immunoprecipitated Myc-Nek11-(1-337) and the expression levels of FLAG-Nek11-(289-645) in cell lysates were also confirmed by Western blotting. C, absence of Nek11L homo-oligomer during interphase. Myc-tagged and FLAG-tagged Nek11L were coexpressed in 293T cells, and these transfected cells were either treated or not by nocodazole (1 µM) for 18 h. The expression levels of Myc-tagged and FLAG-tagged Nek11s were confirmed by Western blotting using cell lysates. FLAG-Nek11L was immunoprecipitated (IP) by anti-FLAG agarose and coimmunoprecipitated Myc-Nek11L was detected by Western blotting (WB) using anti-Myc antibody (bottom panel). D, Nek2-induced disruption of intramolecular interaction between Nek11 N-terminal catalytic and C-terminal non-catalytic region. FLAG-Nek11-(289-645) was coexpressed with Myc-Nek11-(1-337) and coimmunoprecipitated (IP) by anti-Myc antibody. Then immunocomplex beads were subjected to in vitro kinase assay in the presence or absence of Nek2A (F386A)-HA. After washing immunocomplex beads, FLAG-Nek11-(289-645) bound with Myc-Nek11-(1-337) was analyzed by Western blotting (WB) using anti-Nek11 antibody. Anti-Nek11 antibody did not recognize Myc-Nek11-(1-337), and the amounts of immunoprecipitated Myc-Nek11-(1-337) were confirmed by Western blotting using the anti-Myc antibody.

 



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  		FIG. 7.
Possible model for Nek11 activation by Nek2. A, model for interaction between Nek11 and Nek2A. B, schematic hypothesis for Nek11L activation mechanism by Nek2A.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we demonstrated for the first time several lines of evidence indicating a novel interaction between Nek11 and Nek2 in G1/S-arrested cells. Both Nek11 and Nek2 could localize at nucleolus, and Nek11-Nek2 complex formation increased in G1/S-arrested cells. In addition, Nek2 activated Nek11 through modulation of the autorepressive function of the Nek11 C-terminal non-catalytic region. These observations strongly pointed to the likelihood that nucleolar Nek11 is a novel target of Nek2A in G1/S-arrested cells.

In the indirect immunofluorescent analysis, cell preparation protocol and primary antibody are important factors affecting antibody accessibility and reactivity, and experimental protocols also should be adjusted to each cell type. Nek2A and Nek2B localize at centrosome through the cell cycle (6), and our indirect immunofluorescent analysis additionally showed unexpected nucleolar colocalization of endogenous Nek11 and Nek2. Importantly, we here discovered a novel difference between Nek2A and Nek2B. Nek2A has a nucleolar targeting/retention activity via Nek2A specific coiled-coil domain at the C-terminal end, whereas Nek2B does not localize at nucleolus. Nek2A specific C-terminal end coiled-coil domain (amino acids 399-445) contains a lysine/arginine stretch that may contribute to nuclear/nucleolar targeting. Consistent with data from our indirect immunofluorescent analysis, endogenous nucleolar Nek11 associated only with Nek2A, and endogenous Nek11-Nek2A complex was detected only in the nuclear fraction but not the cytosolic fraction. Although overexpressed ectopic Nek11 and Nek2B could interact with each other, probably due to their abundance at the cytoplasm, our observations suggest that nucleolar localization is an important factor for endogenous Nek11-Nek2A complex formation.

In addition, Nek11 formed a complex preferentially with PP1 binding motif-disrupted Nek2A (F386A) but hardly with wild-type Nek2A, suggesting that PP1 might repress the Nek11-Nek2A interaction in cells. Alternatively, as PP1-mediated dephosphorylation inactivates Nek2A (7, 22) and kinase-inactive Nek2B (K37R) hardly formed a complex with Nek11 in cells, activation of Nek2A by autophosphorylation (or upstream regulator) might be required before its interaction with Nek11. The Mos-MAPK-p90RSK pathway activates Nek2 during meiosis in mouse pachytene spermatocytes (23). However, little is known concerning the endogenous Nek2A activation mechanism in human somatic cells. Because endogenous Nek2A protein levels and its autophosphorylation activity increased in G1/S-arrested cells, post-translational regulation of Nek2A would be upstream of the Nek11 activation in these cells. Further exploration of the Nek2A activation mechanism might provide a clue to understand nucleolar regulation of the Nek2A-Nek11 complex in G1/S-arrested cells.

We showed here that the C-terminal non-catalytic region of Nek11L could associate with its N-terminal catalytic domain most probably in an intramolecular manner. Nek2 could phosphorylate the Nek11 C-terminal non-catalytic region and antagonize its autoinhibitory function, which would cause Nek11 activation. Nek2A also autophosphorylates its C-terminal non-catalytic region, which appears to be important for its kinase activity on exogenous substrate (19), suggesting that the Nek11 C-terminal non-catalytic region might contribute to the Nek11 substrate recognition mechanism. Similar autoregulatory mechanisms by non-catalytic domain have been shown in several protein kinases such as Plk, Hsl1, and Chk1 (24-26). Considering these previous studies and our results, we presume that Nek11 kinase is kept latent by a non-catalytic regulatory region in interphase and activated by Nek2A through modulation of this region (as in Fig. 7). Although we did not identify phosphoacceptor serine residues on Nek11, additional experiments indicated that single alanine substitutions at Ser-97, -161, -185, -200, -299-334, -372, and -380 did not affect Nek2-mediated Nek11 activation (data not shown). Since Nek11 seemed to be phosphorylated by Nek2 at multiple sites, we speculate that multiple phosphorylation on the Nek11 non-catalytic regulatory region might be required to neutralize its autoinhibitory activity or would change the total electric charge, which might affect its substrate recognition process.

Our data here suggest a novel possibility that Nek2A and Nek11 have unknown roles at nucleolus. Interestingly, two hybrid-based large scale comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae showed that the yeast NIMA-related kinase Kin3 could interact with some proteins including nucleolar GTP-binding protein Nog1 and mitotic GTP-binding protein Tem1 (27) (also shown in the yeast protein-protein interaction data bases provided by The GRID and Yeast Resource Center). Nucleolar Nog1 has a role in ribosome biogenesis (28, 29), and the protein interaction data base above also suggest that Nog1 could interact with MCM and Yph1/Nop7, both of which have abilities to interact with DNA replication origin recognition complex protein (30). In addition, that two-hybrid study (27) also showed that both Nog1 and Tem1 could interact with Fob1, a DNA replication fork blocking nucleolar protein (31), and that Fob1 could interact with Rad53 (27), a DNA damage checkpoint kinase associated with the DNA replication mechanism (32-34). These collective data suggest to us a possibility that the Kin3-Nog1/Tem1-Fob1-Rad53 complex might be involved in unknown signaling between the DNA replication checkpoint mechanism and ribosome biogenesis at nucleolus. As mammalian nucleolar NIMA-related kinases Nek11 and Nek2A are activated by DNA replication inhibitor-induced G1/S-arrest, nucleolar function of NIMA-related kinase might be conserved between yeast and human. We have a working hypothesis that the Nek11-Nek2A complex might be associated with an unknown signaling pathway for synchronization between ribosome biogenesis and the DNA replication checkpoint mechanism.

Alternatively, yeast Tem1 activates Cdc14 release from nucleolus to trigger mitotic exit network (35), and Fob1 could interact with Spo12, a component of the Cdc14 early anaphase release (FEAR) network (36). If Kin3 actually interacts with Tem1, Kin3 might be involved in the Tem1-Fob1-Spo12 complex-mediated Cdc14 regulation during mitosis. Attractively, in human, both Nek2A and hCdc14A are centrosomal components and important for centrosome separation and chromosome segregation (8, 37-39). The possibility of interplay between Nek2A and hCdc14A was also discussed earlier (39). Another human Cdc14 isoform, hCdc14B is a nucleolar protein phosphatase and negatively regulates a mitotic phosphoprotein SIRT2 in an indirect manner (38-40). Both hCdc14B and SIRT2 show perichromosomal localization during mitosis similar to Nek11, and it may be interesting to explore the functional interaction between Nek11 and hCdc14B/SIRT2.

Collectively, we demonstrated that Nek11 is a nucleolar NIMA-related kinase regulated by Nek2A. We are currently searching for a target of Nek11 among nucleolar components involved in both cell cycle progression and ribosome biogenesis. Future studies could provide significant information concerning unknown cell cycle-related functions of human NIMA-related kinases.


   FOOTNOTES
 
* This work was supported by a grant-in-aid for Young Scientists (B) (to K. N.) and grants for Cancer Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to Y. U.) and by the Japan Health Sciences Foundation for Research on Health Sciences Focusing on Drug Innovation (to K. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 81-3-5285-1111 (ext. 2301); Fax: 81-3-5285-1175; E-mail: yuehara{at}nih.go.jp.

1 The abbreviations used are: NIMA, never in mitosis A; PP1, protein phosphatase 1; PEST, proline-glutamine-serine-threonine rich domain; RT-PCR, reverse transcription polymerase chain reaction; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; DAPI, 4,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate; EGFP, enhanced green fluorescence protein; GST, glutathione S-transferase; HA, hemagglutinin; ORF, open reading frame; Nek11L, NIMA-related kinase 11-long form; Nek11S, NIMA-related kinase 11-short form. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Melvin L. DePamphilis for critical reading of this manuscript and valuable comments on this work. We thank the members of our laboratory for useful discussions.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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