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Originally published In Press as doi:10.1074/jbc.M403029200 on August 16, 2004

J. Biol. Chem., Vol. 279, Issue 45, 47201-47211, November 5, 2004
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Direct Association with Inner Centromere Protein (INCENP) Activates the Novel Chromosomal Passenger Protein, Aurora-C*{boxs}

Xiangyu Li{ddagger}§, Gyosuke Sakashita{ddagger}§, Hideki Matsuzaki¶, Kenji Sugimoto||, Keiji Kimura**, Fumio Hanaoka**{ddagger}{ddagger}, Hisaaki Taniguchi¶, Koichi Furukawa{ddagger}, and Takeshi Urano{ddagger}§§

From the {ddagger}Department of Biochemistry II, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-8550, Membrane Dynamics Project, Synchrotron Radiation Research Network, Harima Institute at Spring-8, RIKEN, Kouto, Mikazuki, Sayo, Hyogo 679-5148, ||Division of Applied Biochemistry, Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531, **Cellular Physiology Laboratory, Discovery Research Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, and {ddagger}{ddagger}Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan

Received for publication, March 18, 2004 , and in revised form, August 12, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A family of serine/threonine kinase Aurora constitutes a key regulator in the orchestration of mitotic events. The human Aurora paralogues Aurora-A, Aurora-B, and Aurora-C have a highly conserved catalytic domain. Extensive studies on the role of Aurora-A and Aurora-B have revealed distinct localizations and functions in regulating mitotic processes, whereas little is known about Aurora-C. The present study shows that human Aurora-C is a chromosomal passenger protein that forms complexes with Aurora-B and inner centromere protein (INCENP), which are known passenger proteins. We show that INCENP binds and activates Aurora-C in vivo and in vitro. Furthermore, Aurora-C co-expressed with INCENP elicits the phosphorylation of endogenous histone H3 in mammalian cells, even though this phosphorylation is not sufficient to establish chromosome condensation in interphase cells. We therefore suggest that Aurora-C is a novel chromosomal passenger protein that cooperates with Aurora-B to regulate mitotic chromosome dynamics in mammalian cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The regulation of mitotic processes is complex and precisely controlled. Post-translational ubiquitin-mediated proteolysis and a substantial wave of protein phosphorylation play important roles in signaling events that coordinate mitotic processes and ensure accurate chromosome segregation (reviewed in Refs. 1 and 2). Maturation promoting factor, a complex of the serine/threonine kinase Cdc2 and its partner cyclin B, is the most studied of the kinase complexes that are essential for mitotic progression. In addition to Cdc2, the polo-like kinases, NimA-related (Nek), Bub1, LATS, and the Aurora families are implicated in many mitotic processes (2). The characterization of mutations in the prototype of the Aurora family, Drosophila melanogaster aurora, revealed that Aurora is important for centrosome separation and bipolar spindle formation (3). A screen for temperature-sensitive conditional mutants for chromosomal missegregation identified increase-in-ploidy 1 (Ipl1), the sole essential Aurora of Saccharomyces cerevisiae (4).

The mammalian Aurora family constitutes a closely related subgroup of three serine/threonine protein kinases referred to as Aurora-A, Aurora-B, and Aurora-C (2, 5–6). Although not identical, the structure of the Aurora-A catalytic domain has many conformational similarities with that of cAMP-dependent protein kinase and Cdk2 (7–9). Aurora-A and Aurora-B have very distinct localizations and functions even though they share a high degree of sequence similarity within the catalytic domain (74% identity and 85% similarity). Aurora-A is up-regulated during mitosis (10) and degraded by ubiquitin-mediated proteasome activity triggered by the hCdh1-activated anaphase-promoting complex/cyclosome after metaphase (11–14). Immunostaining of endogenous Aurora-A by using a monoclonal antibody and time-lapse analysis of the ectopic expression of green fluorescent protein (GFP)1-tagged Aurora-A have demonstrated that Aurora-A localizes to centrosomes and regions of microtubules that are proximal to centrosomes during mitosis (11, 15). These findings suggest that Aurora-A is involved in the regulation of microtubule nucleation at centrosomes, a process that includes phosphorylation, dephosphorylation, and association with TPX2 or Ajuba (16). Aurora-B is essential for cytokinesis, chromosome condensation, kinetochore function, chromosome segregation, spindle-assembly checkpoint, and microtubule dynamics (17). Aurora-B is also regulated by phosphorylation (18) and dephosphorylation (19) and by association with the chromosomal passenger proteins INCENP and survivin, which are important for both targeting and activation of the kinase (6).

The dynamic distribution of a class of chromosomal passenger proteins that includes Aurora-B, INCENP, survivin, TD-60, and Orc6 is similar during mitosis (20). Among them, at least Aurora B, INCENP, and survivin interact as a complex. These passenger proteins localize on centromeres early during mitosis, transfer to the central spindle midzone in anaphase, and finally remain associated with the midbody during cytokinesis. The dramatic movements of these proteins during mitosis have generated the notion that they play important roles in mitotic spindle dynamics during the coordination of chromosomal and cytoskeletal events associated with mitosis (21).

Histone modifications, including acetylation, methylation, phosphorylation, ubiquitination, and combinations of these processes, have been characterized (22–23). Mitotic chromosome condensation and segregation in a variety of organisms are closely linked with mitotic phosphorylation of histone H3 at serine residues 10 and 28 (24–26). Aurora-B is responsible for mitotic phosphorylation of histone H3 on serine residues 10 and 28 (19, 27–28). In response to extracellular signals, more localized histone H3 phosphorylation is mediated by RSK2, MSK1, mitogen-activated protein kinases, and IKK{alpha} (29–31), thereby inducing immediate-early gene expression.

Aurora-C is little understood. It has been found only in mammals, is specifically expressed at high levels in the testis, and localizes to centrosomes from anaphase to telophase, and its expression levels are elevated in several cancer cell lines (32–34). Here we show that human Aurora-C forms complexes with Aurora-B and INCENP, known passenger proteins. We expressed a human GFP-tagged Aurora-C plasmid to determine its localization, and we found instead that it acts as a passenger protein. We also demonstrated that human Aurora-C phosphorylates histone H3 in vitro, whereas the catalytically inactive form of this kinase had no effect. Furthermore, we showed that INCENP binds and activates Aurora-C in vivo and in vitro and that Aurora-C co-expressed with INCENP elicits histone H3 phosphorylation of interphase cells. These observations suggest that Aurora-C is a novel chromosomal passenger protein and that human Aurora-C has important functions in regulating mitotic chromosome dynamics in coordination with Aurora-B in human cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Polyclonal antibodies specific to phosphorylated serine residues 10 or 28 in histone H3 and MPM-2 monoclonal antibody were obtained from Upstate Biotechnology, Inc. Polyclonal antibody to MAPK was obtained from Cell Signaling Technology. Anti-FLAG mouse monoclonal antibody (M2) was purchased from Sigma. Rabbit polyclonal antibodies were raised against synthetic peptides corresponding to the C-terminal sequences of each human condensin subunit, as described (35). The anti-pan Aurora monoclonal antibody (K3-7) recognizes all of the human Aurora family (12). Anti-Glu and anti-Myc mouse monoclonal antibodies were gifts from Dr. Larry A. Feig (Tufts University, Boston).

Plasmids—Human Aurora-C (GenBankTM accession number AY714054 [GenBank] , see supplement Fig. 1) and INCENP (GenBankTM accession number AY714053 [GenBank] ) were cloned from a human testis cDNA library (Clontech) by using partial cDNAs obtained by the reverse transcription-PCR of total RNA from DLD-1 colon cancer cells. IN-box is a C-terminal domain (amino acids 783–918) of INCENP. We created a mammalian expression vector by inserting cDNA into an altered version of pcDNA3 (Invitrogen) that contained a FLAG epitope (MDYKDDDDK) 5' to the cloning site, into myc/pcDNA3, into glu/pMT3 (36), or into pEGFP-C1 (Clontech) to encode a protein tagged with GFP at the N terminus. Point mutations within Aurora-C were engineered by standard double PCR mutagenesis. All PCR-amplified cDNA products were fully sequenced to confirm mutations and to verify the absence of secondary point mutations. The plasmids glu-Aurora-A/pMT3 and glu-Aurora-B/pMT3 have been described (11, 19).

Transient Transfection—COS-7 and human MDA-435 cells were maintained in Dulbecco's modified Eagle's medium (Sigma) containing 10% fetal calf serum (Sigma) at 37 °C in a 5% CO2 atmosphere. COS-7 (3 x 105 cells in 60-mm culture dishes) and MDA-435 cells were transfected with mammalian expression vectors using DEAE-dextran (36) and LipofectAMINE 2000 (Invitrogen), respectively.

Monoclonal Antibody Generation—We immunized mice with recombinant GST-tagged Aurora-B or IN-box, and serum titers were monitored by immunoblotting using lysates of COS cells transfected with glu-Aurora-B/pMT3 or myc-INCENP/pcDNA3, respectively. After several injections, splenic lymphocytes were isolated and fused to the myeloma cell line NS-1. Clonal populations of fusion cells were screened for antibody production by enzyme-linked immunosorbent assay, and productive cells were cloned to monoclonal lines by serial dilution screening (37). Two monoclonal antibodies for Aurora-B and three for INCENP were prepared that also detected protein in immunoblots. Highly concentrated H7-4 monoclonal antibodies for Aurora-B and 7.2.2 for INCENP were obtained from murine ascites after an intraperitoneal injection of hybridoma cells.

Indirect Immunofluorescence Microscopy—MDA-AF8-A2 cells (38) and HeLa cells were seeded on poly(D-lysine)-coated coverslips at ~25% confluence. The following day the cells were washed once with ice-cold phosphate-buffered saline (PBS) and fixed with methanol at -20 °C for 10 s. The cells were then washed three times with ice-cold PBS and incubated for 5 min at room temperature in PBS containing 0.05% Triton X-100. The permeabilized cells were washed three times with ice-cold PBS and then covered with a solution of 10% bovine serum albumin in PBS and incubated for 30 min at room temperature. Diluted monoclonal antibody was placed as a drop on the coverslips and incubated for 45 min at 37 °C in a humidified chamber. The coverslips were then washed six times with PBS and covered with a solution containing goat anti-mouse Alexa 488 (Invitrogen) for 30 min in the dark at 37 °C in a humidified chamber. The coverslips were washed six times with PBS, mounted (mounting medium; Vector Laboratories) with DAPI, and attached to slides with clear nail polish.

Establishment of Stable Cell Lines and Confocal Time-lapse Microscopy—Stable cell lines, MDA-GFP-Aurora-B, MDA-GFP-Aurora-C, and MDA-GFP-INCENP, were generated by transfecting MDA-435 cells using LipofectAMINE 2000 reagent (Invitrogen) with expression vectors Aurora-B/pEGFP-C1, Aurora-C/pEGFP-C1, and INCENP/pEGFP-C1, respectively. The transfected cells were selected in 750 µg/ml of G418 (Invitrogen) and collected 7–10 days later. Single colonies of stably transfected cells were selected after 2 weeks. Three independent cell lines for each construct were analyzed. Confocal time-lapse fluorescence images were acquired using an FV500 laser-scanning confocal unit coupled to an inverted microscope (model IX81; Olympus) equipped with an oil-immersion objective (PLAPO60x; Olympus) and Fluoview software (Olympus). Cells were maintained at 37 °C and analyzed on 35-mm glass-based dishes (IWAKI) in CO2-independent medium (Invitrogen) to avoid medium acidification in the CO2-free atmosphere. Time-lapse recording was established by monitoring green fluorescence, and the images were captured every 1.5 or 3 min.

Cell Cycle Synchronization—Tissue culture dishes (10 cm) were seeded at a density of 3x106 with exponentially growing HeLa cells. For S phase synchronization, HeLa cells were synchronized as follows. On the following day, thymidine (Sigma) was added to the media to a final concentration of 2.5 mM, and the plates were incubated for 16 h at 37 °C. The plates were then washed three times with PBS, and normal growth medium was added. After 8 h at 37 °C, the cells were exposed to thymidine for an additional 16 h. For M phase synchronization, cells were incubated with medium containing 400 ng/ml nocodazole for 8 h. The cells were released from nocodazole by three PBS washes, and normal growth medium was added. To monitor the cell cycle distribution, the cellular DNA content was assayed by standard techniques using propidium iodide staining. Events were analyzed by a FACScan (BD Biosciences).

In Vitro Translation—The cell-free expression of cDNAs was analyzed in the T7 TNT-coupled transcription/translation system (Promega). Briefly, cDNAs in myc/pcDNA3 or in FLAG/pcDNA3 (1.5 µg of circular plasmid each) were transcribed/translated alone or co-translated in rabbit reticulocyte lysate. The coupled transcription/translation reactions (total volume, 50 µl) were incubated at 30 °C for 1.5 h, and translated proteins were immunoprecipitated as described below.

Immunoprecipitation—HeLa or transfected COS cells in 60-mm dishes were lysed by rocking in 500 µl of RIPA buffer (PBS with 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 0.1 µM okadaic acid) for 15 min at 4 °C. The lysates were clarified by centrifugation at 16,500 x g for 5 min and then immunoprecipitated with primary monoclonal antibody bound to protein G-Sepharose (Amersham Biosciences) at 4 °C for 75 min. The beads were washed five times with 1 ml of RIPA buffer and finally resuspended in 15 µl of 2x SDS sample buffer. The precipitated proteins were separated by SDS-PAGE and then immunoblotted. Prior to immunocomplex kinase assays, glu-Aurora-C immunoprecipitates were washed twice with kinase buffer without dithiothreitol.

In Vitro Kinase Assay—Reaction mixtures (30 µl) containing substrates, enzymes, 50 µM ATP, and 3µCi of [{gamma}-32P]ATP in kinase activity buffer (20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 0.5 mM dithiothreitol, 0.1 mM EDTA) were incubated for 20 min at 30 °C. The reaction products separated by 12% SDS-PAGE were visualized by Coomassie Brilliant Blue R-250 staining. The gels were dried, and 32P was detected using a MacBAS-1500 imaging analyzer (Fuji Film).

Subcellular Fractionation—HeLa cells (1 x 106) were lysed with 200 µl of CSK buffer (10 mM PIPES (pH 6.8), 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA) containing 0.3% Triton X-100 at 4 °C for 30 min. The soluble and insoluble fractions were separated by centrifugation for 10 min at 2000 x g. The supernatants were recovered as the chromatin-unbound fraction (Fig. 3A, fr. 1). The insoluble fraction was suspended in 200 µl of CSK buffer containing 200 units of DNase I (Roche Applied Science), incubated at room temperature for 30 min, and clarified by centrifugation for 10 min at 16,500 x g. The supernatants were recovered as the DNase-extractable fraction (Fig. 3A, fr. 2). The pellets were suspended in 200 µl of CSK buffer containing 2 M NaCl and 0.3% Triton X-100 and centrifuged for 10 min at 17,500 x g. The supernatants were saved as the high salt extractable fraction (Fig. 3A, fr. 3), and the pellets were saved as the high salt-resistant fraction (Fig. 3A, fr. 4). We considered that the DNase-extractable (Fig. 3A, fr.2) and high salt-extractable (Fig. 3A, fr.3) fractions contained chromatin-bound fractions (39).



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  		FIG. 3.
Intracellular distribution of Aurora-C. A, left panel, schematic representation of cell fractionation. See "Experimental Procedures" for details. Fr, fraction. Right panel, chromatin-unbound (lane 1), DNase-extractable (lane 2), high salt-extractable (lane 3), and high salt-resistant (lane 4) fractions prepared from HeLa cells incubated with nocodazole were immunoblotted (IB) with anti-Pan Aurora antibody. B, localization of GFP-Aurora-C (green) in transiently transfected MDA cells. Meta, metaphase; ana, anaphase; telo, telophase. Cells are counterstained with DAPI (blue). Scale bar, 5 µm. C, live-cell imaging of GFP-Aurora-C during mitosis. MDA-GFP-Aurora-C cells were cultured in 35-mm glass-based dishes. Live cells were observed using a confocal microscope. Time-lapse imaging was established by monitoring green fluorescence, and images were captured every 3 min. See QuickTime Movie 1 in Supplemental Material.

 
In Vivo Histone H3 Phosphorylation Assay—COS cells were seeded on poly(D-lysine)-coated coverslips at ~25% confluence. The following day COS-7 cells were transfected with myc-Aurora-C/pcDNA3 (3 µg) either alone or together with INCENP/pEGFP-C1 (0.6 µg) using DEAE-Dextran. Thirty six hours later, the cells were fixed for indirect immunofluorescence described above with anti-phosphohistone H3 (Ser(P)-10) antibody. We also stained cells with anti-Myc monoclonal antibody to detect exogenous Aurora-C. We simultaneously detected INCENP expression by monitoring a green fluorescence tag. We counted more than 200 cells by using a fluorescence microscope.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Human Aurora-B Is a Chromosomal Passenger Protein— Immunostaining with polyclonal antibodies sometimes produces variable results. We therefore generated monoclonal antibodies against human Aurora-B to study its distribution and function. We tested the specificity of the H7-4 antibody using immunoblots of transfected COS cell lysates. The antibody recognized a single protein of the predicted molecular weight in whole cell lysates from COS cells transfected with Glu-tagged Aurora-B (Fig. 1A). The H7-4 antibody did not recognize either human Aurora-A or Aurora-C. Exponentially growing MDA-AF8-A2 cells (38) stably expressed GFP-CENP-A, with which we examined Aurora-B localization during the cell cycle by using indirect immunofluorescence microscopy. GFP-CENP-A localized to the centromere throughout the cell cycle. In contrast, Aurora-B localization was entirely punctate between the two GFP-CENP-A dots until metaphase (Fig. 1, B–D). Entry into anaphase led to Aurora-B redistribution to the central spindle midzone (Fig. 1, E and F) and then final accumulation at the midbody during telophase and cytokinesis (data not shown). Aurora-B immunostaining was consistent with that detected using polyclonal antibodies (40–42) and with the live-cell imaging of MDA-GFP-Aurora-B cells stably expressing GFP-Aurora-B (data not shown). Therefore, human Aurora-B behaves as a passenger protein whose localization changes dynamically with the progression of mitosis.



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  		FIG. 1.
Subcellular localization of Aurora-B. A, specificity of monoclonal anti-Aurora-B antibody H7-4. COS cells, transfected with pMT3 mammalian expression plasmids encoding glu-Aurora-A, glu-Aurora-B, or glu-Aurora-C, were lysed and equalized for protein content. Cell lysates were separated by 10% SDS-PAGE and processed for immunoblotting (IB) with anti-Aurora-B (H7-4) (left panel) or anti-Glu (right panel) monoclonal antibodies. B–F, dynamic redistribution of endogenous Aurora-B investigated by immunofluorescence microscopy. Asynchronous MDA-AF8-A2 cells growing on coverslips were fixed with methanol and stained for Aurora-B. GFP-CENP-A (green) localizes to centromeres throughout the cell cycle. Aurora-B (red) concentrates at the inner centromere in the middle of two GFP-CENP-A dots until metaphase (B–D). Entry into anaphase led to Aurora-B redistribution to central spindle midzone (E and F). Cells are counterstained with DAPI (blue). Scale bar, 5 µm.

 
Aurora-C Binds Aurora-B in Vivo—While characterizing Aurora-B function, the monoclonal antibody against Aurora-B showed that this protein co-immunoprecipitates with Aurora-C but not with Aurora-A from HeLa cell extracts (Fig. 2A). To confirm this finding, we transfected glu-Aurora-B either alone or together with Aurora-C (Myc-tagged) into COS cells and immunoprecipitated it with the anti-Glu monoclonal antibody. We then immunoblotted the immunoprecipitates by using the anti-Myc antibody to determine whether stable complexes had formed. glu-Aurora-B immunoprecipitates contained not only the wild type but also a catalytically inactive mutant (KN) of Aurora-C (Fig. 2B, lanes 7 and 8). On the other hand, anti-Glu immunoprecipitates from cells transfected with Myc-tagged Aurora-C alone did not contain Aurora-C (Fig. 2B, lane 6), demonstrating the specificity of the interaction. However, these experiments with in vitro translated proteins did not detect interactions between Aurora-B and Aurora-C (Fig. 2C, lane 6), suggesting that the association between Aurora-B and Aurora-C observed in vivo may require other protein(s) or post-translational modification of these proteins.



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  		FIG. 2.
Interaction of Aurora-B with Aurora-C in vivo. A, endogenous Aurora-B interacts with Aurora-C in vivo. Aurora-C, but not Aurora-A, was detected in Aurora-B immunoprecipitates (IP) from nocodazole (noco)-treated HeLa. B, Myc-Aurora-C or glu-Aurora-B was transiently expressed either alone or together in COS cells. Transfection conditions for each lane are indicated at the top of the panels. Immunoblots (IB) of total cellular lysates (left) and glu-Aurora-B immunoprecipitates (right) probed with anti-Glu and anti-Myc antibodies. C, Aurora-C and Aurora-B translated in vitro do not associate with each other. Myc-Aurora-B/pcDNA3 or FLAG-Aurora-C/pcDNA3 (1.5 µg of circular plasmid each) was transcribed/translated alone or together using T7 TNT-coupled system. Translated proteins were immunoprecipitated using mouse anti-Myc monoclonal antibody. Asterisks represent heavy chains of mouse monoclonal antibody used for immunoprecipitation.

 
Aurora-C Is a Chromosomal Passenger Protein—To determine the subcellular distribution of Aurora family proteins, mitotic HeLa cells were successively treated with CSK buffer containing each of the following: 0.3% Triton X-100, DNase I, and 2 M NaCl (Fig. 3A, left panel). Aurora-A was found in both chromatin-unbound (Fig. 3A, Fr. 1) and DNase I-treated (Fr. 2) fractions, whereas both Aurora-B and Aurora-C were located in high salt-soluble (Fr. 3) and -insoluble (Fr. 4) fractions, demonstrating that the subcellular distribution of Aurora-C coincides with that of Aurora-B and not of Aurora-A and that Aurora-C was tightly bound to mitotic chromosomes at prometaphase.

To locate intracellular Aurora-C, we transiently expressed Aurora-C attached with GFP to the N terminus in human MDA cells. GFP-Aurora-C was found on chromosomes and not at centrosomes during metaphase (Fig. 3B, meta). When cell division entered anaphase, GFP-Aurora-C translocated into the central spindle midzone (Fig. 3B, ana and telo). To validate these observations, we generated stable cell clones expressing GFP-Aurora-C in MDA cells and further analyzed the dynamics of its distribution during the cell cycle. During mitosis, time-lapse imaging of GFP-tagged Aurora-C showed that it translocated from metaphase chromosomes (Fig. 3C, 15 min and QuickTime movie 1 in Supplemental Data) to the central spindle midzone (45 min) and then remained in the midbody (57 min). These data demonstrated that Aurora-C is a chromosomal passenger protein like Aurora-B.

INCENP as a Passenger Protein—Several lines of evidence indicate that the association of INCENP with Aurora-B correlates with enhanced Aurora-B kinase activity (43–45). We generated monoclonal antibodies against human INCENP that recognized a single protein of the predicted molecular weight in whole lysates from exponentially growing HeLa cells (Fig. 4A). The electrophoretic mobility of accumulating INCENP protein became retarded during the G2/M phase and declined during G1. INCENP proteins are expressed throughout the cell cycle. The level of control MAPK protein did not significantly change during cell cycle progression.




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  		FIG. 4.
Protein expression and subcellular translocation of INCENP. A, HeLa cells were synchronized at G1/S boundary by double thymidine block, and then cells were collected at the indicated times after release from arrest. Protein levels of INCENP, Aurora family, and MAPK were determined by immunoblotting (IB) with anti-INCENP (upper panel), anti-Pan Aurora (middle panel), or with anti-MAPK antibodies (lower panel), respectively. Flow cytometry confirmed cell cycle synchronization (data not shown). B, subcellular distribution of endogenous INCENP investigated by immunofluorescence microscopy. Exponentially growing HeLa cells on coverslips were fixed with methanol and visualized by staining with anti-INCENP monoclonal antibody followed by Alexa 488-conjugated goat anti-mouse secondary antibody. Prometa, prometaphase; meta, metaphase; ana, anaphase. Scale bar, 10 µm. C, localization of GFP-INCENP in transiently transfected MDA cells. Meta, metaphase; ana, anaphase; telo, telophase. Scale bar, 10 µm. D, live-cell imaging of GFP-INCENP during mitosis. MDA-GFP-INCENP cells were cultured in 35-mm glass-based dishes. Live cells were observed by confocal microscopy. Time-lapse imaging was established by monitoring green fluorescence, and images were captured every 1.5 min. See QuickTime Movie 2 in Supplemental Material.

 
Indirect immunofluorescence analysis of fixed HeLa cells with monoclonal antibodies against INCENP showed that it is confined to centromeres during prometaphase, to the central spindle midzone in anaphase, and to midbodies during late telophase. These findings confirmed that the distribution of INCENP precisely corresponds with that of Aurora-B and Aurora-C throughout all phases of mitosis (Fig. 4B). By contrast, the nuclear distribution of INCENP at interphase was punctate, which did not coincide with DAPI staining (Fig. 4B).

To confirm these findings by using monoclonal antibodies, we cloned cells that stably expressed GFP-INCENP in MDA cells and further analyzed the dynamics of its distribution during the cell cycle. The profile of GFP-INCENP expression was similar to that of the endogenous protein as shown above (Fig. 4C). Time-lapse imaging of GFP-tagged INCENP during mitosis showed that it translocated from metaphase chromosomes (Fig. 4D, 0 min, and QuickTime Movie 2 in Supplemental Material) to the central spindle midzone (13 min) and then remained in the midbody (28 min). These data substantiated the notion that INCENP is a chromosomal passenger protein in addition to Aurora-B and Aurora-C.

Characterization of the Biochemical Properties of Aurora-C— We demonstrated that protein phosphatases associated with human Aurora-B function as negative regulators of kinase activation (19). To investigate whether okadaic acid affects human Aurora-C activity, we incubated transfected COS cells with okadaic acid and performed in vitro kinase assays with glu-Aurora-C immunoprecipitates and GST-H3-(5–15) as the substrate. Okadaic acid potently inhibits PP1, PP2A, and PP5, and GST-H3-(5–15) is an appropriate in vitro substrate for Aurora-B (19). We found that human Aurora-C phosphorylated histone H3 and that okadaic acid increased Aurora-C kinase activity (Fig. 5A, compare lanes 1 with 3 and 7 with 9), whereas the expression level of Aurora-C did not significantly change. We confirmed that Aurora-C phosphorylates GST-H3-(23–33) in vitro, and by using phospho-specific antibodies, we also showed that Aurora-C phosphorylates histone H3 at serine residues 10 and 28 (data not shown). Aurora-C expression is regulated by the cell cycle (12, 32). To determine whether the microtubule-depolymerizing drug nocodazole influences Aurora-C activity, we transfected glu-Aurora-C into COS cells, and we then incubated them with nocodazole for harvesting at prometaphase. We performed kinase assays in vitro by using glu-Aurora-C immunoprecipitates and GST-H3-(5–15) as the substrate. In contrast to the effect of okadaic acid, Aurora-C was barely activated by nocodazole (Fig. 5A, compare lanes 1 with 2 and 7 with 8). These findings demonstrated that inhibition of okadaic acid-sensitive phosphatase(s) is involved in Aurora-C activation and that prometaphase arrested by nocodazole is not sufficient to activate Aurora-C.



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  		FIG. 5.
Aurora-C activation through direct binding with INCENP. A, biochemical properties of Aurora-C. Left panel, wild type (wt) or catalytically inactive (KN) forms of Aurora-C (Glu-tagged) transiently expressed in COS cells. Right panel, Glu-tagged Aurora-B or mutant form in activation loop (T195A) transiently expressed in COS cells. Transfected cells were incubated for 30 min with either 0.4 µM okadaic acid (OA) or 400 ng/ml nocodazole (N) for 14 h. glu-Aurora-C was immunoprecipitated (IP) by using a mouse anti-Glu monoclonal antibody, and immunocomplexes were analyzed by using in vitro kinase (IVK) assays. After termination, reaction mixtures were resolved by SDS-PAGE and visualized by autoradiography (upper panels). Coomassie Brilliant Blue R-250 staining (lower panels) shows equal amounts of immunoprecipitated Aurora-C and recombinant GST-H3-(5–15) substrate. All data from in vitro kinase assays are representative of multiple independent studies. Left, molecular mass standards. Asterisk indicates immunoglobulin. Aurora-C phosphorylation was further increased, and its mobility was shifted by okadaic acid (lanes 3 and 9). B, INCENP binds and activates Aurora-C in vivo. Myc-tagged INCENP was transiently expressed either alone or together with wild-type (wt) or catalytically inactive (KN) forms of Aurora-C (Glu-tagged) in COS cells. C, endogenous INCENP associates with Aurora-B and Aurora-C in vivo. Reciprocal immunoprecipitates obtained using anti-Aurora-B (lanes 3 and 4) or anti-INCENP (lanes 5 and 6) from exponentially growing (-) or nocodazole-treated (noco) HeLa cells were immunoblotted (IB) against anti-INCENP (upper panel) or anti-Pan Aurora antibodies (lower panel). D, Aurora-C activation by direct binding with INCENP. Proteins translated in vitro were immunoprecipitated using anti-Myc monoclonal antibody, and immunocomplexes were analyzed using in vitro kinase assays.

 
We assessed Aurora-C kinase activity by introducing single substitutions into Aurora-C. In contrast to the wild type, the mutant of an invariant lysine residue (Lys-69) replaced with arginine in the ATP-binding motif of the catalytic domain of Aurora-C (KN) displayed no GST-H3-(5–15) kinase activity regardless of nocodazole or okadaic acid (Fig. 5A, lanes 4–6). Threonine 195 is in the activation loop of the catalytic domain between subdomains VII and VIII (46), and phosphorylation of the equivalent residue within this loop activates several kinases including cAMP-dependent kinase and Cdc2 (47). We monitored the effect of mutating Thr-195 to alanine (T195A) on the kinase activity of Aurora-C with or without nocodazole or okadaic acid. Kinase activity in the T195A mutant of Aurora-C was substantially lost along with Aurora-C (KN) (Fig. 5A, lanes 10–12). In the presence of okadaic acid, Glu-immunoprecipitated Aurora-C was phosphorylated to an increased extent and its mobility shifted (Fig. 5A, lower panels, compare lanes 1 with 3 and 7 with 9).

INCENP Activates Aurora-C in Vivo and in Vitro through Direct Binding—The catalytic domain of Aurora-C is more similar at the amino acid level to the corresponding region of Aurora-B (92%) than to that of Aurora-A (84%). We therefore examined whether INCENP binds and activates Aurora-C. Aurora-C co-expressed with INCENP produced a substantial increase in Aurora-C activity over the level induced by okadaic acid (Fig. 5B, lanes 2–4). Aurora-C (KN) and the wild type bound to INCENP (Fig. 5B, lanes 3 and 6), but this mutant had no detectable kinase activity toward substrate GST-H3-(5–15) even when co-expressed with INCENP (Fig. 5B, lane 6). These data demonstrated that INCENP binds and activates Aurora-C in vivo. Co-immunoprecipitated INCENP as well as Aurora-C was phosphorylated (Fig. 5B, lane 3). We tested whether endogenous human INCENP immunoprecipitated from extracts of HeLa cells forms complexes with Aurora-B and Aurora-C. Fig. 5C, lanes 5 and 6, show that Aurora-B and Aurora-C both co-immunoprecipitated with INCENP, whereas Aurora-A did not. Reciprocal immunoprecipitation using anti-Aurora-B demonstrated that Aurora-B associated with INCENP and Aurora-C (Fig. 5C, lanes 3 and 4). These data confirmed that INCENP forms complexes with Aurora-B and Aurora-C in vivo. Nocodazole induced two migrating forms of INCENP (Fig. 5C, lanes 2, 4, and 6). Treating the immunoprecipitates of cells incubated with nocodazole with phosphatase before immunoblotting increased INCENP mobility, indicating that the slower migrating band was the phosphorylated form (data not shown).

We investigated whether the interaction with INCENP was sufficient to activate Aurora-C in vitro. Myc-tagged wild type or mutant (KN) Aurora-C cDNA with either a FLAG-tagged IN-box (a conserved motif in the C terminus of INCENP) cDNA or a control vector was co-expressed in an in vitro translation system, immunoprecipitated using anti-Myc antibody coupled to beads, and assayed for activation. Wild type and mutant (KN) of Aurora-C both co-immunoprecipitated with IN-Box (Fig. 5D, lanes 3 and 4, lower panel), whereas only the wild type showed substantial kinase activity when co-expressed with IN-Box (Fig. 5D, compare lanes 1 with 3 and 3 with 4, top panel). These results indicated that direct binding to the IN-box was indispensable and sufficient for Aurora-C activation. Co-immunoprecipitated IN-box as well as Aurora-C was also phosphorylated under these conditions (Fig. 5D, lane 3, top panel).

Co-expression of Aurora-C with INCENP Elicits Histone H3 Phosphorylation of Interphase Cells—We examined whether Aurora-C co-expressed with INCENP elicits the phosphorylation of endogenous histone H3 in mammalian cells as follows. We transiently transfected COS cells with Myc-tagged Aurora-C alone or together with EGFP-INCENP and then detected phosphorylation by immunofluorescence by using an anti-phospho-histone H3 (Ser(P)-10) antibody. We also stained cells with anti-Myc monoclonal antibody to detect exogenous Aurora-C. Over 90% of COS cells expressing both Aurora-C and INCENP had anti-phospho-histone H3 antibody immunoreactivity (Fig. 6, A and B), and about 5% of cells expressing cDNA for one of them expression was immunoreactive. The staining intensity and distribution of Ser(P)-10 and of both cDNAs expressed in cells during interphase were identical to those at the G2/M phase of nontransfected cells. Expression of Aurora-C (KN), even with INCENP, reduced the immunoreactivity to about 1% of cells, suggesting that Aurora-C (KN) works as a dominant negative regulator in mammalian cells. Trends in these data using COS cells were similar to those found in HeLa cells (data not shown). Thus, Aurora-C can phosphorylate histone H3 in coordination with INCENP in vivo.



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  		FIG. 6.
Phosphorylation of histone H3 is not sufficient to establish chromosome condensation in interphase cells. A, Aurora-C co-expressed with INCENP elicits endogenous histone H3 phosphorylation in interphase COS cells. COS cells were transiently transfected with Myc-tagged Aurora-C or catalytically inactive (KN) form alone or together with EGFP-INCENP and then detected by immunofluorescence using anti-phospho-histone H3 (Ser(P)-10) antibody. We also stained cells with anti-Myc monoclonal antibody to detect exogenous Aurora-C. B, anti-phospho-histone H3 (Ser(P)-10)-positive cells were counted. Results are shown as mean ± S.E. of over 200 cells from at least three separate experiments. C and D, histone H3 phosphorylation does not induce targeting of condensin complex to chromatin (C) and MPM-2 phospho-epitope (D) in interphase cells. Mock or myc-Aurora-C with EGFP-INCENP were transiently expressed in COS cells. Immunoblots of cell-fractionated samples were probed with anti-hCAP-D2 (C, upper panels), anti-hCAP-E (C, lower panels), and phospho-specific MPM-2 (D) antibodies. See "Experimental Procedures" for details. Fr, fraction.

 
Although controversial (48), histone H3 phosphorylation at serine 10 has been implicated in both chromosome condensation and segregation during mitosis in many animal species (24–26). Therefore, we investigated whether introducing histone H3 phosphorylation into interphase cells could mimic the status of mitotic cells ("pseudo-mitotic state"). We initially checked the condensation state of chromosomes by using DAPI staining. Both the nuclear morphology and levels of DAPI staining were similar to nontransfected interphase cells (Fig. 6A and data not shown). We then examined whether histone H3 phosphorylation induces targeting of the condensin complex to chromatin, because the condensin complex is a key component of the molecular condensation machinery (48). Cell fractionation assays revealed that the subcellular localization of all five components of human condensin was not affected by histone H3 phosphorylation (Fig. 6C and data not shown). In contrast, chromosomal targeting of the condensin complex was stimulated by nocodazole (positive control). We finally analyzed the appearance of the MPM-2 phospho-epitope because its presence on mitotic chromosomes is closely associated with the condensed state (49). The results showed that the MPM-2 epitope was not created even when both Aurora-C and INCENP were expressed (Fig. 6D and data not shown). Thus, histone H3 phosphorylation is not sufficient to establish chromosome condensation in interphase cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Over the past few years, the Aurora protein kinase family has emerged as a major regulator of mitotic events (2, 5–6). Aurora-A and Aurora-B are the most extensively studied among this family. Aurora-C has been detected in mammals (human, rat, and mouse), but whether nonmammalian species possess aurora-c genes remains obscure. S. cerevisiae and Schizosaccharomyces pombe contain a single gene encoding Aurora ipl1p and Ark1p, respectively, suggesting that all human Auroras were derived from a single ancestral gene through a series of duplication events. With regard to the higher degree of sequence similarity (85% identity and 92% similarity) within the catalytic domains between Aurora-B and Aurora-C, Aurora-C arose from more recent gene duplication of aurora-b. However, the three Auroras differ in the length and sequence of the N-terminal domain, which might be important for targeting the kinases and for binding other molecules. A LIM domain containing the protein Ajuba binds and activates human Aurora-A through the N-terminal noncatalytic domain of Aurora-A (50). One challenge will be to define the function of the N-terminal domain of each of the Auroras.

We developed monoclonal antibodies against the known human chromosomal passenger proteins Aurora-B and INCENP, and we applied them to immunofluorescence, immunoblotting, and immunoprecipitation. Immunofluorescence analysis using these antibodies confirmed that Aurora-B and INCENP are chromosomal passenger proteins, (Fig. 1, B–F, and Fig. 4B). Reciprocal immunoprecipitation using these antibodies revealed that the passenger proteins, Aurora-B, INCENP, and Aurora-C, form a complex (Fig. 5C). Moreover, this study was facilitated by the initial observation that this antibody against human Aurora-B co-immunoprecipitated Aurora-C from HeLa cell lysate. These results indicate that the antibodies will be useful tools with which to study the functions of passenger proteins and to find physiological binding partners using a combination of biochemical approaches (e.g. mass spectrometric analysis).

To address the dynamics of Aurora-B and INCENP in terms of their intracellular location during mitosis more directly, we used time-lapse imaging, and we monitored the motion of these proteins tagged with GFP when stably expressed in human cells. The results revealed the dynamic translocation of these GFP fusion proteins from metaphase chromosomes to the central spindle midzone at anaphase in the same manner as chromosomal passenger proteins (Fig. 4D and data not shown). Therefore, the selective targeting of chromosomal passenger proteins was not affected by the N-terminal GFP tag in this system. In contrast, GFP-tagged Aurora-A associates with centrosomes and regions of microtubules that are proximal to centrosomes under the same conditions (15). Our observations of endogenous Aurora-A behavior using a monoclonal antibody concurred with these findings (11). Consequently, the time-lapse findings of at least two human Auroras using GFP-tagged proteins closely agreed with immunolocalization using monoclonal antibodies, suggesting that the N-terminal GFP tag does not affect the proper localization of human Auroras. Because we could not generate monoclonal antibodies that recognize endogenous Aurora-C, we used time-lapse imaging to localize GFP-tagged Aurora-C in human cells. The data demonstrated that the behavior of Aurora-C, like Aurora-B, is typical of that of chromosomal passenger proteins. Biochemical data also supported this observation. Live-cell imaging of cells expressing GFP-tagged chromosomal passenger proteins is a powerful tool with which to analyze subcellular translocation and the role of these proteins during mitosis.

The movements of chromosomal passenger proteins during mitosis suggest that they play important roles in coordinating spindle mechanics during the cell cycle (21). Our observations indicated that Aurora-C falls into this category of proteins. Among the chromosomal passenger proteins, only Aurora-B and Aurora-C are serine/threonine kinases, and they form a complex with the passenger proteins, INCENP, and survivin. These proteins mutually influence the assembly and localization of the complexes, and so perturbation of their functions causes chromosome alignment, segregation, and cytokinesis to fail in mammalian cells (40, 51–55). In addition, the reversible protein phosphorylation of passenger proteins mediated by these kinases might ensure the correct assembly, spatial localization, and functions of the complex.

We showed that the biochemical properties of human Aurora-C resemble those of Aurora-B (18, 19). Aurora-C is tightly bound to mitotic chromosomes, and it phosphorylates serine residues 10 and 28 on histone H3. The activation of Aurora-C is observed following inhibition of okadaic acid-sensitive phosphatase(s) and, most importantly, can be mediated by INCENP through direct association. INCENP has been implicated in Aurora-B activation (44, 45). Whether the activation of Aurora-B and Aurora-C by INCENP resembles the recently described mode of activation of Aurora-A by TPX2 remains to be clarified (9). Details of the relationship between Aurora-C and other targets of Aurora-B such as MgcRacGAP (56), PRC1 (57), myosin II regulatory light chain (58), and intermediate filament proteins (59, 60) remain to be explored. Finally, we confirmed that Aurora-C phosphorylates histone H3 in coordination with INCENP in vivo. The development of this in vivo system showed that histone H3 phosphorylation is not sufficient to establish chromosome condensation in interphase cells. This result is in concert with the observation that simply stimulating H3 phosphorylation by inhibiting PP1 is insufficient for chromosome condensation in the Xenopus system (61). However, our results do not exclude the important contribution of histone H3 phosphorylation to mitosis (62).

While this manuscript was in revision, Dasra A and Dasra B/Borealin were identified as part of the vertebrate chromosomal passenger complex containing INCENP, Survivin, and Aurora-B (63, 64). This complex is required for microtubule stabilization and spindle formation. Dasra B/Borealin shows limited similarity to the Caenorhabditis elegans chromosomal passenger protein CSC-1 (65). The precise roles of Aurora-C in the complex will be elucidated.

In summary, Aurora-C is a novel chromosomal passenger protein that coordinates with Aurora-B to regulate mitotic chromosome dynamics in mammalian cells. The next objective will be to explore the molecular mechanisms of the process and to determine how it regulates subcellular translocation involving Aurora-B and Aurora-C in passenger protein complexes during mitosis.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY714054 [GenBank] and AY714053 [GenBank] .

* This work was supported by a grant-in-aid for scientific research and the 21st Century COE Program from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. 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

{boxs} The on-line version of this article (available at http://www.jbc.org) contains Figs. S1 and S2 and Movies 1 and 2. Back

§ These two authors contributed equally to this work. Back

§§ To whom correspondence should be addressed. 81-52-744-2071; Fax: 81-52744-2069; E-mail: turano{at}med.nagoya-u.ac.jp.

1 The abbreviations used are: GFP, green fluorescent protein; INCENP, inner centromere protein; KN, catalytically inactive mutant; PBS, phosphate-buffered saline; DAPI, 4',6-diamidino-2-phenylindole; CSK buffer, cytoskeleton buffer; PIPES, 1,4-piperazinediethanesulfonic acid; GST, glutathione S-transferase; EGFP, enhanced GFP. Back


   ACKNOWLEDGMENTS
 
We thank Larry A. Feig (Tufts University, Boston) for anti-Glu and anti-Myc monoclonal antibodies and Kazunori Ida (Olympus) for technical assistance with the confocal time-lapse microscopy.



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