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NEK2A Interacts with MAD1 and Possibly Functions as a Novel Integrator of the Spindle Checkpoint Signaling

2004; Elsevier BV; Volume: 279; Issue: 19 Linguagem: Inglês

10.1074/jbc.m314205200

1083-351X

Yang Lou, Xuebiao Yao, Arzhang Zereshki, Zhen Dou, Kashif Ahmed, Hongmei Wang, Jun-Bin Hu, Yuzhen Wang, Xuebiao Yao,

Genomics and Chromatin Dynamics

Chromosome segregation in mitosis is orchestrated by protein kinase signaling cascades. A biochemical cascade named spindle checkpoint ensures the spatial and temporal order of chromosome segregation during mitosis. Here we report that spindle checkpoint protein MAD1 interacts with NEK2A, a human orthologue of the Aspergillus nidulans NIMA kinase. MAD1 interacts with NEK2A in vitro and in vivo via a leucine zipper-containing domain located at the C terminus of MAD1. Like MAD1, NEK2A is localized to HeLa cell kinetochore of mitotic cells. Elimination of NEK2A by small interfering RNA does not arrest cells in mitosis but causes aberrant premature chromosome segregation. NEK2A is required for MAD2 but not MAD1, BUB1, and HEC1 to associate with kinetochores. These NEK2A-eliminated or -suppressed cells display a chromosome bridge phenotype with sister chromatid inter-connected. Moreover, loss of NEK2A impairs mitotic checkpoint signaling in response to spindle damage by nocodazole, which affected mitotic escape and led to generation of cells with multiple nuclei. Our data demonstrate that NEK2A is a kinetochore-associated protein kinase essential for faithful chromosome segregation. We hypothesize that NEK2A links MAD2 molecular dynamics to spindle checkpoint signaling. Chromosome segregation in mitosis is orchestrated by protein kinase signaling cascades. A biochemical cascade named spindle checkpoint ensures the spatial and temporal order of chromosome segregation during mitosis. Here we report that spindle checkpoint protein MAD1 interacts with NEK2A, a human orthologue of the Aspergillus nidulans NIMA kinase. MAD1 interacts with NEK2A in vitro and in vivo via a leucine zipper-containing domain located at the C terminus of MAD1. Like MAD1, NEK2A is localized to HeLa cell kinetochore of mitotic cells. Elimination of NEK2A by small interfering RNA does not arrest cells in mitosis but causes aberrant premature chromosome segregation. NEK2A is required for MAD2 but not MAD1, BUB1, and HEC1 to associate with kinetochores. These NEK2A-eliminated or -suppressed cells display a chromosome bridge phenotype with sister chromatid inter-connected. Moreover, loss of NEK2A impairs mitotic checkpoint signaling in response to spindle damage by nocodazole, which affected mitotic escape and led to generation of cells with multiple nuclei. Our data demonstrate that NEK2A is a kinetochore-associated protein kinase essential for faithful chromosome segregation. We hypothesize that NEK2A links MAD2 molecular dynamics to spindle checkpoint signaling. Chromosome movements during mitosis are governed by the interaction of spindle microtubules with a specialized chromosome domain located within the centromere. This specialized region, called the kinetochore (1Brinkley B.R. Stubblefield E. Chromosoma. 1966; 19: 28-43Crossref PubMed Scopus (146) Google Scholar, 2Roos U.-P. J. Cell Biol. 1976; 54: 363-385Google Scholar), is the site for spindle microtubule-centromere association. In addition to providing a physical link between chromosomes and spindle microtubules, the kinetochore has an active function in chromosomal segregation through microtubule motors and spindle checkpoint sensors located at or near it (3Nicklas R.B. J. Cell Biol. 1989; 109: 2245-2255Crossref PubMed Scopus (147) Google Scholar, 4Rieder C.L. Alexander S.P. J. Cell Biol. 1990; 110: 81-95Crossref PubMed Scopus (360) Google Scholar, 5Yao X. Abrieu A. Zheng Y. Sullivan K.F. Cleveland D.W. Nat. Cell Biol. 2000; 2: 484-491Crossref PubMed Scopus (307) Google Scholar, 6Li R. Murray A.W. Cell. 1991; 66: 519-531Abstract Full Text PDF PubMed Scopus (933) Google Scholar). Several lines of evidence have implicated the kinetochore in generation of a diffusible checkpoint signal that can block cell cycle progression into anaphase until all kinetochores have successfully attached to spindle microtubules. Delayed attachment of one or more chromosomes to the spindle is correlated with a corresponding delay in the onset of anaphase. For mutants that fail to arrest the cell cycle in mitosis after disassembly of microtubules in budding yeast, genetic screen has identified three MAD (mitotic arrest deficiency) and three BUB (budding uninhibited by benomyl) genes (7Hoyt M.A. Totis L. Roberts B.T.S. Cell. 1990; 66: 507-517Abstract Full Text PDF Scopus (899) Google Scholar). Vertebrate homologues of MAD1 (8Jin D.Y. Spencer F. Jeang K.T. Cell. 1998; 93: 81-91Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar), MAD2 (9Chen R.H. Waters J.C. Salmon E.D. Murray A.W. Science. 1996; 274: 242-246Crossref PubMed Scopus (379) Google Scholar, 10Li Y. Benezra R. Science. 1996; 274: 246-248Crossref PubMed Scopus (521) Google Scholar), BUB3 (11Taylor S.S. Ha E. McKeon F. J. Cell Biol. 1998; 142: 1-11Crossref PubMed Scopus (361) Google Scholar, 12Martinez-Exposito M.J. Kaplan K.B. Copeland J. Sorger P.K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8493-8498Crossref PubMed Scopus (83) Google Scholar), BUB1, and BUBR1 (13Taylor S.S. McKeon F. Cell. 1997; 89: 727-735Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar, 14Cahill D.P. Lengauer C. Yu J. Riggins G.J. Willson J.K. Markowitz S.D. Kinzler K.W. Vogelstein B. Nature. 1998; 392: 300-303Crossref PubMed Scopus (1314) Google Scholar, 15Chan G.K. Jablonski S.A. Sudakin V. Hittle J.C. Yen T.J. J. Cell Biol. 1999; 146: 941-954Crossref PubMed Scopus (312) Google Scholar) are spindle checkpoint components transiently associated with kinetochore. Expression of the kinetochore binding domain of murine BUB1 (13Taylor S.S. McKeon F. Cell. 1997; 89: 727-735Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar) or injection of antibodies against BUBR1 (15Chan G.K. Jablonski S.A. Sudakin V. Hittle J.C. Yen T.J. J. Cell Biol. 1999; 146: 941-954Crossref PubMed Scopus (312) Google Scholar) results in premature onset of anaphase, presumably by replacement of the endogenous proteins at kinetochores. Collectively, these data indicate that binding of these spindle checkpoint components at the kinetochores may generate a signal in response to spindle defects and/or aberrant kinetochore protein-protein interactions. Mitosis is orchestrated by signaling cascades that coordinate mitotic processes and ensure accurate chromosome segregation. The key switch for the onset of mitosis is the archetypal cyclin-dependent kinase Cdc2. Besides the master mitotic kinase Cdc2, there are three protein serine/threonine kinase families as follows: the Polo kinases, Aurora kinases, and the NEK (NIMA-related kinases) (16Nigg E.A. Nat. Rev. Mol. Cell. Biol. 2001; 2: 21-32Crossref PubMed Scopus (1252) Google Scholar, 17O'Connell M.J. Krien M.J. Hunter T. Trends Cell Biol. 2003; 13: 221-228Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). The latter family has proven the most enigmatic in function, although recent advances from several sources are beginning to reveal a common functional theme. NIMA (never in mitosis A) is vital in Aspergillus nidulans for entry into mitosis (17O'Connell M.J. Krien M.J. Hunter T. Trends Cell Biol. 2003; 13: 221-228Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 18Oakley B.R. Morris N.R. J. Cell Biol. 1983; 96: 1155-1158Crossref PubMed Scopus (93) Google Scholar). Mutation of NIMA arrests cells in G2 without interfering with p34cdc2 activation, suggesting that the NIMA protein has a central role in the G2/M transition. Moreover, if the G2 arrest of nimA mutants is bypassed by additional mutations, the resulting mitotic cells show aberrant spindle and nuclear envelope organization (17O'Connell M.J. Krien M.J. Hunter T. Trends Cell Biol. 2003; 13: 221-228Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 18Oakley B.R. Morris N.R. J. Cell Biol. 1983; 96: 1155-1158Crossref PubMed Scopus (93) Google Scholar, 19Lu K.P. Hunter T. Cell. 1995; 81: 413-424Abstract Full Text PDF PubMed Scopus (141) Google Scholar), pointing to functions of NIMA beyond the control of mitotic entry. NEK2A, the homologue in human cells with the greatest structural similarity to NIMA within the catalytic domain, is regulated in yeast in a manner similar to regulation of NIMA in A. nidulans; its expression and serine/threonine kinase activity is highest during late G2 phase, when NEK2A is expected to function critically (20Fry A.M. Meraldi P. Nigg E.A. EMBO J. 1998; 17: 470-481Crossref PubMed Scopus (343) Google Scholar). Furthermore, a portion of NEK2A localizes to centrosomes and in mammalian cells appears to play a role similar to NIMA in controlling entry into mitosis (16Nigg E.A. Nat. Rev. Mol. Cell. Biol. 2001; 2: 21-32Crossref PubMed Scopus (1252) Google Scholar, 17O'Connell M.J. Krien M.J. Hunter T. Trends Cell Biol. 2003; 13: 221-228Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). NEK2A may have more diverse roles during several phases of the cell cycle, from S phase to multiple phases of mitosis, based on its dynamic expression and subcellular localization in cytosol, nucleus, and chromosome portions other than the centrosome (16Nigg E.A. Nat. Rev. Mol. Cell. Biol. 2001; 2: 21-32Crossref PubMed Scopus (1252) Google Scholar, 17O'Connell M.J. Krien M.J. Hunter T. Trends Cell Biol. 2003; 13: 221-228Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 21Chen Y. Riley D.J. Zheng L. Chen P.L. Lee W.H. J. Biol. Chem. 2002; 277: 49408-49416Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Although it has been proposed that NEK2A is associated with centromere (17O'Connell M.J. Krien M.J. Hunter T. Trends Cell Biol. 2003; 13: 221-228Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar), it was not clear whether NEK2A is indeed localized to the kinetochore and whether NEK2A functions during chromosome movements in mitosis. The mitotic spindle checkpoint is a chromosome segregation surveillance mechanism that prevents chromosome from premature segregation in the presence of mis-aligned chromosomes. This surveillance system is highly sensitive as a single unaligned chromosome or perturbation of kinetochore protein interactions is sufficient to block the transit of cells from metaphase to anaphase (22Li X. Nicklas R.B. J. Cell Sci. 1997; 110: 537-545PubMed Google Scholar). The initial description of the mitotic checkpoint in yeast defined a nonessential pathway invoked only in response to spindle damage. But in metazoans, the checkpoint has evolved into an essential feature of normal mitoses and meioses (23Shah J.V. Cleveland D.W. Cell. 2000; 103: 997-1000Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). Microinjection of antibodies to MAD2 into cultured animal cells first revealed premature anaphase onset and chromosome mis-segregation (24Gorbsky G.J. Chen R.H. Murray A.W. J. Cell Biol. 1998; 141: 1193-1205Crossref PubMed Scopus (199) Google Scholar). In budding yeast, the spindle checkpoint depends on a tight complex between the MAD1 and MAD2 proteins (25Chen R.H. Brady D.M. Smith D. Murray A.W. Hardwick K.G. Mol. Biol. Cell. 1999; 10: 2607-2618Crossref PubMed Scopus (150) Google Scholar). Although MAD1 is required for MAD2 localization to the kinetochore (25Chen R.H. Brady D.M. Smith D. Murray A.W. Hardwick K.G. Mol. Biol. Cell. 1999; 10: 2607-2618Crossref PubMed Scopus (150) Google Scholar), it is unknown how MAD1 function is integrated into the mitotic regulation. To explore the nature of MAD1 function in mitotic spindle checkpoint signaling, we carried out yeast genetic screening and identified the novel MAD1-interacting partner NEK2A. Our studies show that NEK2A interacts with MAD1 via the leucine zipper motif on the MAD1 molecule. In addition, we show that both NEK2A and MAD1 are co-localized to the kinetochore of mitotic cells. Given the involvement of MAD1 in spindle checkpoint surveillance and novel function of NEK2A revealed in this study, we proposed that NEK2A links mitotic checkpoint signaling to chromosome segregation dynamics. Yeast Two-hybrid Assay—Full-length human MAD1 cDNA, a gift from Dr. Dong-Yan Jin (University of Hong Kong), was employed to screen a HeLa cDNA library made in fusion with the GAL4 activation domain in the vector pGBD-T7 (Clontech, Palo Alto, CA) by using yeast strain AH109 containing the reporter genes his3 and lacZ. About 1 million co-transformants were screened for the two reporter genes on the plates containing media with amino acid selection. To map the region(s) of MAD1, which interact with NEK2A, four deletion mutants of MAD1 were selected based on the secondary structure prediction (cubic.bioc.columbia.edu/predictprotein/). MAD1 bait deletion constructs were PCR-amplified and obtained by digestion of the bait with BamHI and EcoRI followed by gel purification of respective bands and sub-cloning into pGBD-T7 vector. In addition, six deletion mutants of NEK2A were selected to map the interface(s) between NEK2A and MAD1. NEK2A deletion constructs were generated by digesting the prey with BamHI and XhoI followed by the gel purification of respective bands and then sub-cloning into pGADT7 vector. cDNA Construction—Total RNAs, isolated from HeLa cells by using standard protocol, were treated with RNase-free DNase I (Takara Biotechnology, Dalian, China) for 15 min at room temperature before addition of 25 mm EDTA and were heated to 65 °C for 10 min to stop the reaction. DNase I-treated RNAs were reverse-transcribed at 42 °C for 50 min using oligo(dT) and Moloney murine leukemia virus-reverse transcriptase RNase H (Promega, Madison, WI). By using this cDNA as template, PCR was performed with related primer. The resulting fragment was cloned into TA cloning vector (Takara Biotechnology, Dalian, China) and completely sequenced. For mammalian expression of the full-length NEK2A, NEK2A cDNA was digested with SalI and BamHI and cloned into pEGFP C1 vector (Clontech, Palo Alto, CA), whereas the full length of MAD1 cDNA was digested with EcoRI and BamHI and cloned into pEGFP C2 vector (Clontech, Palo Alto, CA). Recombinant Protein Production—NEK2A, MAD1, and their fragments were expressed in bacteria as fusion proteins. Briefly, the full length of MAD1 cDNA was cloned into pMal C2 vector (New England Biolabs, Beverly, MA), whereas a BamHI/EcoRI fragment (amino acids 480–680) was cloned into pGEX-2T vector (Amersham Biosciences). The partial cDNA sequence of NEK2A (amino acids 305–446) was obtained from yeast screen. After digestion with NotI and SalI, it was fused in-frame with His6 at the C terminus (pET22b; Novagen) and GST 1The abbreviations used are: GST, glutathione S-transferase; GFP, green fluorescent protein; RNAi, RNA interference; PBS, phosphate-buffered saline; siRNA, small interfering RNA; PIPES, 1,4-piperazinediethanesulfonic acid; BSA, bovine serum albumin; DAPI, 4,6-diamidino-2-phenylindole; ACA, anti-centromere antibody. at the N terminus (pGEX-5X-3), respectively. Purification of recombinant proteins was carried out as described previously (26Zhou R. Cao X. Watson C. Miao Y. Guo Z. Forte J.G. Yao X. J. Biol. Chem. 2003; 278: 35651-35659Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Briefly, 1 liter of LB media was inoculated with bacteria transformed either with NEK2A or MAD1. The expression of protein was induced by addition of 0.5 mm isopropyl-β-d-thiogalactopyranoside. Bacteria were harvested by centrifugation 3 h after the induction, re-suspended in phosphate-buffered saline (PBS) containing proteinase inhibitors (leupeptin, pepstatin, and chymostatin; 5 μg/ml), and sonicated for four bursts of 10 s each by using a probe-tip sonicator. The lysis solution was clarified by centrifugation for 20 min at 10,000 × g. The soluble fraction was applied to a column packed with glutathione-agarose beads, followed by extensive washes with PBS. Affinity Precipitation of MAD1 and NEK2A—His-tagged NEK2A-transformed bacterial cell lysate was used as a source of NEK2A protein. The purified soluble GST-fused MAD1-(380–680) protein was pre-bound to glutathione-agarose (Sigma). GST-MAD1-(380–680) bound beads were washed with 5× column volume using wash buffer (10 mm phosphate buffer, pH 7.4, 150 mm NaCl, 1% Triton X-100, 0.01% phenylmethylsulfonyl fluoride) and equilibrated with 10× column volume of incubation buffer (50 mm Tris-HCl, pH 7.5, 100 mm NaCl, 0.1% Triton X-100, 0.01% phenylmethylsulfonyl fluoride). The purified His-fused NEK2A-(305–446), re-suspended in incubation buffer, was loaded on the column and incubated for 1 h, and the flow-through fraction was collected. The column was washed with 10× volume of incubation buffer, and wash fraction was collected. Finally, the column was eluted with elution buffer (50 mm Tris-HCl, pH 7.5, 1 m NaCl, 0.1% Triton X-100, 0.01% phenylmethylsulfonyl fluoride), and elution fraction was collected. Non-bound glutathione-agarose beads and GST protein-bound glutathione-agarose beads were used as negative control. All collected fractions were resolved on SDS-PAGE and were analyzed by immunoblot analysis using GST mouse antibody (NeoMarkers). Transient Transfection and Immunoprecipitation—293T cells were grown to ∼50% confluency in Dulbecco's modified Eagle's medium with 10% fetal bovine serum at 37 °C in 10% CO2 and were co-transfected with GFP-NEK2A and FLAG-MAD1 by GeneJammer (Stratagene, La Jolla, CA), according to the manufacturer's protocol. Cells were collected 24–36 h after transfection, and proteins were solubilized in lysis buffer (50 mm HEPES, pH 7.4, 150 mm NaCl, 2 mm EGTA, 0.1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, and 10 g/ml pepstatin A). Lysates were clarified by centrifugation at 16,000 × g for 10 min at 4 °C. GFP-tagged fusion proteins were incubated with anti-GFP monoclonal antibody bound to protein-A/G beads (Pierce). Beads were washed five times with lysis buffer and then boiled in protein sample buffer for 2 min. After SDS-PAGE, proteins were transferred to nitrocellulose membrane. The membrane was divided into three strips and probed with antibodies against the GFP epitope, MAD1, and tubulin, respectively. Immunoreactive signals were detected with ECL kit (Pierce) and visualized by autoradiography on Kodak BioMax film. siRNA Treatment and Assay for Knock-down Efficiency—The siRNA sequence used for silencing of NEK2A corresponds to the coding region 260–280 (relative to the start codon). As a control, either a duplex targeting cyclophilin or scramble sequence was used (5Yao X. Abrieu A. Zheng Y. Sullivan K.F. Cleveland D.W. Nat. Cell Biol. 2000; 2: 484-491Crossref PubMed Scopus (307) Google Scholar). The 21-mer oligonucleotide RNA duplexes were synthesized by Dharmacon Research, Inc. (Boulder, CO). In the trial experiments, different concentrations of siRNA oligonucleotides were used for different time intervals of treatment as detailed previously (5Yao X. Abrieu A. Zheng Y. Sullivan K.F. Cleveland D.W. Nat. Cell Biol. 2000; 2: 484-491Crossref PubMed Scopus (307) Google Scholar). In brief, HeLa cells were synchronized and transfected with 21-mer siRNA oligonucleotides or control scramble oligonucleotide, whereas the efficiency of the siRNA oligonucleotide on NEK2A protein knock-down was judged by Western blotting analysis (5Yao X. Abrieu A. Zheng Y. Sullivan K.F. Cleveland D.W. Nat. Cell Biol. 2000; 2: 484-491Crossref PubMed Scopus (307) Google Scholar). Immunofluorescence Microscopy—For immunofluorescence, cells were seeded onto sterile, acid-treated 18-mm coverslips in 6-well plates (Corning Glass). Double thymidine-blocked and -released HeLa cells were transfected with 2 μg/ml LipofectAMINE 2000 pre-mixed with various siRNA oligonucleotides as described above. In general, 36 h after transfection with siRNA or scrambled (control) oligonucleotides, HeLa cells were rinsed for 1 min with PHEM buffer (100 mm PIPES, 20 mm HEPES, pH 6.9, 5 mm EGTA, 2 mm MgCl2, and 4 m glycerol) and were permeabilized for 2 min with PHEM plus 0.2% Triton X-100 as described previously (5Yao X. Abrieu A. Zheng Y. Sullivan K.F. Cleveland D.W. Nat. Cell Biol. 2000; 2: 484-491Crossref PubMed Scopus (307) Google Scholar, 27Yao X. Anderson K.L. Cleveland D.W. J. Cell Biol. 1997; 139: 435-447Crossref PubMed Scopus (189) Google Scholar). Extracted cells were then fixed in freshly prepared 4% paraformaldehyde plus 0.05% glutaraldehyde in PHEM and rinsed three times in PBS. Cells on the coverslips were blocked with 0.05% Tween 20 in PBS (TPBS) with 1% BSA (Sigma). These cells were incubated with various primary antibodies in a humidified chamber for 1 h and then washed three times in TPBS. Monoclonal antibodies bound to MAD1, HEC1, and NEK2A were visualized using fluorescein-conjugated goat anti-mouse IgG), respectively, whereas binding of anti-centromere antibody was visualized using Texas Red-conjugated goat anti-human IgG + IgM. DNA was stained with DAPI (Sigma), and MAD2 was labeled with an affinity-purified rabbit antibody as described previously (5Yao X. Abrieu A. Zheng Y. Sullivan K.F. Cleveland D.W. Nat. Cell Biol. 2000; 2: 484-491Crossref PubMed Scopus (307) Google Scholar). Slides were examined with a Zeiss Axiovert-200 fluorescence microscope, and images were collected and analyzed with Image-5 (Carl Zeiss, Germany). In some cases, aliquots of oligonucleotide-treated HeLa cells were exposed to 100 ng/ml nocodazole for a period of 18 h to determine whether elimination of NEK2A abrogates the mitotic spindle checkpoint. These cells were then fixed and stained with DAPI for scoring the cell fate profiling under microscope. Far Western Assay—The purified soluble GST-fused MAD1-(380–680) was resolved on 10% SDS-PAGE and transferred onto nitrocellulose membrane (Amersham Biosciences). The membrane was blocked with 2% BSA in PBS containing Tween 20 and incubated with ∼10 μg of His-tagged NEK2A-(305–446) as a probe and BSA as a control. After a 2-h incubation, the membrane was washed four times with PBS and incubated with histidine antibody (Cell Signaling Co.) for 1 h. Horse-radish peroxidase-conjugated secondary antibody was used to detect primary antibody, and the blot was developed using an ECL kit (Amersham Biosciences). Cell Culture and Transfection—HeLa and 293T cells, from American Type Culture Collection (Manassas, VA), were maintained as subcon-fluent monolayers in Dulbecco's modified Eagle's medium (Invitrogen) with 10% fetal calf serum (HyClone, UT) and 100 units/ml penicillin plus 100 μg/ml streptomycin (Invitrogen). In some cases, thymidine-blocked and -released HeLa cells were transfected with 2 μg/ml LipofectAMINE 2000 pre-mixed with 2 μg/ml cDNA plasmid encoding GFP and GFP-NEK2A-KD (kinase-death mutant K37R), respectively, as described (28Zhang J. Fu C. Miao Y. Duo Z. Yao X. Sci. Bull. 2002; 119: 345-353Google Scholar). In general, 36 h after transfection, HeLa cells were pre-extracted with PHEM plus 0.2% Triton X-100 followed by fixation and immunocytochemistry as described above. Western Blot—Samples were subjected to SDS-PAGE on 6–16% gradient gel and transferred onto nitrocellulose membrane. Proteins were probed by appropriate primary and secondary antibodies and detected using ECL (Pierce). The band intensity was then scanned using a PhosphorImager (Amersham Biosciences). NEK2A Is a Novel Interactor for MAD1—To identify proteins that associate with MAD1, the full length of MAD1 cDNA was used as bait to screen a HeLa cDNA library by using GAL4 yeast two-hybrid system. Screening was performed on a total 1 × 105 clones with 31 positive clones. Following analysis by restriction digests of HaeIII and AluI, we got several novel interacting partners of MAD1. Nucleotide sequencing revealed that one of these interactors encodes the C terminus of NEK2A (amino acids from 305 to 446). Its sequence matches human NEK2A (GenBank™ accession number XM001878). To verify the specificity of interaction between NEK2A and MAD1 and to define further the domain(s) that mediates the interaction between MAD1 and NEK2A, we took advantage of yeast genetics and sought to perform additional yeast two-hybrid screens to map the binding interface(s) between NEK2A and MAD1. To this end, we used reverse transcriptase-PCR to obtain the full-length cDNA of NEK2A from a HeLa cell library, and we cloned it into pGAD T7 vector. To define better the domain responsible for the specific interaction between MAD1 and NEK2A, we employed a two-dimensional structure prediction analysis to search for protein-protein interaction modules in the two proteins. We found that both proteins contained a leucine zipper motif. The leucine zipper of MAD1 is located between amino acids 500 and 522, and the leucine zipper of NEK2A is located between amino acids 306 and 328. Since leucine zipper mediates protein-protein interaction, we sought to test if leucine zippers mediate the association between MAD1 and NEK2A-(305–446) (NEK2AD3). To this end, a series of deletion mutants were generated as illustrated in Fig. 1A. The NEK2A deletion mutant plasmids were co-transformed with full-length MAD1 cDNA into yeast cells, respectively. We used β-galactosidase activity (LacZ reporter) as a measure for protein-protein interaction. As shown in Fig. 1B, yeast genetic assay indicates full-length MAD1 binds to NEK2A C terminus (amino acids 329–446), a region adjacent to the leucine zipper. However, full-length NEK2A does not interact with MAD1, suggesting that a conformational change of NEK2A, which exposed its C terminus, may be required for exposing its MAD1 binding domain. To map the NEK2A binding interface on MAD1, deletion mutant cDNAs of MAD1 were co-transformed with NEK2AD3 into yeast cells. As shown in Fig. 1, C and D, NEK2A binds to the MAD1 central region (amino acids 380–532), which contains a leucine zipper motif. Thus, our yeast genetic assay indicates that the leucine zipper of MAD1 directly binds to the C-terminal 117 amino acids of NEK2A. NEK2A Forms a Complex with the Mitotic Checkpoint Component MAD1—To validate the interaction between MAD1 and NEK2A observed in our yeast two-hybrid assay and test if NEK2A forms a complex with MAD1, we carried out immunoprecipitation in FLAG-MAD1 and GFP-NEK2A co-transfected 293T cells using epitope tag antibodies. As shown in Fig. 2A, Western blot using GFP antibody confirmed that NEK2A is pulled down by FLAG immunoprecipitation of MAD1 (lane 8; FLAG intraperitoneal). Conversely, Western blot using FLAG antibody revealed that MAD1 is pulled down by GFP-NEK2A (lane 4; GFP intraperitoneal). No FLAG-tagged MAD1 was precipitated with control IgG (lane 3), and no actin was detected in any of the immunoprecipitates. Thus, we conclude that the interaction between MAD1 and NEK2A is specific. To test if NEK2A directly binds to MAD1 in vitro and to confirm the binding interface from the yeast screen, we employed GST-MAD1 D3 as affinity matrix and loaded equal amounts of bacterial lysates that contain His-NEK2AD3 fusion protein. Western blot, shown in Fig. 2B, using poly-histidine antibody validates that the NEK2A D3S binds to MAD1 via its C terminus. In fact, the interaction of NEK2AD3S and MAD1D3 is very strong and can sustain high salt wash (e.g. 1 m NaCl). An example of a far Western blot shown in Fig. 2C demonstrates that MAD1 C-terminal domain directly binds to the leucine zipper-containing region of NEK2A. Thus, we conclude that NEK2A interacts with MAD1. NEK2A Is Localized to the Kinetochore of Mitotic Cells— Previous studies (20Fry A.M. Meraldi P. Nigg E.A. EMBO J. 1998; 17: 470-481Crossref PubMed Scopus (343) Google Scholar) of NEK2A suggest that NEK2A is primarily found at centrosomes and is involved in centrosomal maturation. A recent report (17O'Connell M.J. Krien M.J. Hunter T. Trends Cell Biol. 2003; 13: 221-228Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar) claimed that NEK2A is also associated with the kinetochore of mitotic cells. Since MAD1 is a kinetochore protein conserved from yeast to human, biochemical interaction between MAD1 and NEK2A propelled us to test whether NEK2A is a centromere protein. To this end, we use a monoclonal NEK2A antibody raised against human protein to test if NEK2A protein is enriched in isolated HeLa cell chromosome fraction. As shown in Fig. 3A, Western blot analysis revealed that the NEK2A antibody specifically recognized a single protein band of ∼46 kDa in whole mitotic HeLa cell extracts, which closely matched the calculated molecular weight of NEK2A (Fig. 3A, left panel). To determine if NEK2A is a centromere-associated protein, we compared NEK2A protein levels in the interphase and mitotic cell lysates with that of isolated chromosome scaffold fraction by using Western blot analysis. As shown in Fig. 3A (right panel), the NEK2A signal is intensified in isolated chromosome scaffolds compared with that of mitotic cell lysates, a similar profile to CENP-E, indicating that NEK2A is enriched in the isolated chromosome fraction. To ascertain if NEK2A is indeed a kinetochore protein, we adopted a pre-extraction procedure that allows better labeling of kinetochore protein while preserving fine cyto-structure of mitotic cells (5Yao X. Abrieu A. Zheng Y. Sullivan K.F. Cleveland D.W. Nat. Cell Biol. 2000; 2: 484-491Crossref PubMed Scopus (307) Google Scholar, 27Yao X. Anderson K.L. Cleveland D.W. J. Cell Biol. 1997; 139: 435-447Crossref PubMed Scopus (189) Google Scholar). As shown in Fig. 3B, pre-extracted HeLa cells were stained using the NEK2A monoclonal antibody and a fluorescein-conjugated goat anti-mouse secondary antibody, whereas a human CREST anti-centromere antibody that reacts primarily with CENP-B followed by a rhodamine-conjugated goat anti-human secondary antibody was used to identify the actual centromere (Fig. 3B, red). In the prometaphase shown in Fig. 3B, NEK2A staining appears as pairs of clearly resolved double dots (green, arrows)