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Originally published In Press as doi:10.1074/jbc.M710282200 on March 3, 2008

J. Biol. Chem., Vol. 283, Issue 19, 12981-12991, May 9, 2008
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GSK-3β Regulates Proper Mitotic Spindle Formation in Cooperation with a Component of the {gamma}-Tubulin Ring Complex, GCP5*Formula

Nanae Izumi{ddagger}, Katsumi Fumoto{ddagger}, Shunsuke Izumi§, and Akira Kikuchi{ddagger}1

From the {ddagger}Department of Biochemistry, Graduate School of Biomedical Sciences, and §Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan

Received for publication, December 18, 2007 , and in revised form, February 11, 2008.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Glycogen synthase kinase-3β (GSK-3β) is known to play a role in the regulation of the dynamics of microtubule networks in cells. Here we show the role of GSK-3β in the proper formation of the mitotic spindles through an interaction with GCP5, a component of the {gamma}-tubulin ring complex ({gamma}TuRC). GCP5 bound directly to GSK-3β in vitro, and their interaction was also observed in intact cells at endogenous levels. Depletion of GCP5 dramatically reduced the GCP2 and {gamma}-tubulin in the {gamma}TuRC fraction of sucrose density gradients and disrupted {gamma}-tubulin localization to the spindle poles in mitotic cells. GCP5 appears to be required for the formation or stability of {gamma}TuRC and the recruitment of {gamma}-tubulin to the spindle poles. A GSK-3 inhibitor not only led to the accumulation of {gamma}-tubulin and GCP5 at the spindle poles but also enhanced microtubule nucleation activity at the spindle poles. Depletion of GCP5 rescued this disrupted organization of spindle poles observed in cells treated with the GSK-3 inhibitor. Furthermore, the inhibition of GSK-3 enhanced the binding of {gamma}TuRC to the centrosome isolated from mitotic cells in vitro. Our findings suggest that GSK-3β regulates the localization of {gamma}TuRC, including GCP5, to the spindle poles, thereby controlling the formation of proper mitotic spindles.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Serine/threonine kinase glycogen synthase kinase-3 (GSK-3) was first described in a glycogen metabolic pathway (1). There are two GSK-3 isoforms, GSK-3{alpha} and GSK-3β, in mammalian cells, and both GSK-3 proteins regulate various physiological responses by phosphorylating many substrates, including protein synthesis, gene expression, subcellular localization of proteins, and protein degradation (2, 3). GSK-3 has been highly conserved during evolution and plays a fundamental role in cellular responses. For example, there are four genes, MCK1 (meiosis and centromere regulatory kinase-1), MDS1/RIM11, MRK1, and YOL128c, that encode homologs of mammalian GSK-3 in Saccharomyces cerevisiae. Mck1 stabilizes Rog1 (revertant of glycogen synthase kinase mutation protein 1) (4) and stimulates gene expression by Msn2 (multicopy suppressor of SNF1 protein 2) (5) in yeasts.

Evidence has been accumulating showing that GSK-3 plays a role in the dynamics of microtubules (2, 6). GSK-3, which is inactivated on the plus ends of microtubules, mediates Par6-atypical protein kinase C-dependent promotion of polarization and cell protrusion through microtubules (7). The binding of the adenomatous polyposis coli gene product (APC) to microtubules increases the stability of microtubules, and their interaction is decreased by the phosphorylation of APC by GSK-3β (8). We have found recently that GSK-3β binds and phosphorylates Bicaudal-D (BICD) (9). BICD is a human homologue of Drosophila Bicaudal-D (10), and there are two homologues in mammals, BICD1 and BICD2 (11). It has been reported that BICD proteins are involved in dynein-mediated minus end-directed transport from the Golgi apparatus to the endoplasmic reticulum (12, 13). In addition to these roles, we showed that GSK-3β functions in transporting centrosomal proteins to the centrosome by stabilizing the BICD and dynein complex, resulting in the regulation of a focused microtubule organization (9). Thus, GSK-3 plays a role at both the plus and minus ends of microtubules to regulate elongation, anchoring, and stability of microtubules in interphase.

Microtubule dynamics and organization of the microtubule network need to be controlled during mitosis in order to assemble a spindle apparatus capable of properly segregating the chromosomes. The rapidly growing and shrinking microtubules in the mitotic phase are captured and stabilized by attachment to the kinetochores, hence allowing the formation of a bipolar mitotic spindle. These changes in microtubule dynamics occur concomitantly with the phosphorylation of many proteins, through a number of mitotic kinases, including CDK1 (cyclin-dependent kinase 1), polo kinase, and aurora kinase (14). It has also been reported that GSK-3 is associated with spindle microtubules and spindle poles and that the inhibition of GSK-3 induces abnormality in astral microtubule length and chromosomal alignment (15, 16). However, the mechanism by which GSK-3 is involved in the regulation of mitotic spindle dynamics is not known.

{gamma}-Tubulin is essential for microtubule nucleation, and there are two {gamma}-tubulin complexes, the {gamma}-tubulin small complex ({gamma}TuSC)2 and the {gamma}-tubulin ring complex ({gamma}TuRC) (17). The simplest {gamma}TuSC, which is conserved among many organisms, including yeast, flies, and humans, contains {gamma}-tubulin, Spc97p/Dgrip84/GCP2 ({gamma}-tubulin complex protein-2), and Spc98p/Dgrip91/GCP3 and displays low microtubule nucleation activity in vitro (18). The functions of {gamma}TuSC have been studied extensively by genetic approaches, and deletion of either corresponding gene is lethal, resulting in an accumulation of cells in mitosis (19, 20). The larger complex, {gamma}TuRC, contains additional cap subunits Dgrip75/GCP4, Dgrip128/GCP5, Dgrip163/GCP6, and Dgp71WD/GCP-WD/NEDD1 (neural precursor cell-expressed, developmentally down-regulated protein 1), which hold multiple {gamma}TuSC subcomplexes together (21). Studies in Xenopus and Drosophila have shown that {gamma}TuRC associates with microtubule minus ends, possesses higher microtubule nucleation activity in vitro compared with {gamma}TuSC, and is required for the assembly of fully functional spindles (22–24). In interphase, most {gamma}TuRCs are in the cytoplasm; however, at the onset of mitosis, three to five times more {gamma}-tubulin is recruited to the centrosome (21). How {gamma}TuRC is targeted to the centrosome in a cell cycle-dependent manner is not well understood. In this study, we identified GCP5 as a novel binding protein of GSK-3. With the use of a small molecule GSK-3 inhibitor combined with an RNAi approach, we demonstrated that GSK-3 regulates the localization of GCP5 and {gamma}TuRC to the spindle poles in order to control proper mitotic spindle formation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials and Chemicals—HeLa S3 cells, U2OS cells, human GSK-3β cDNA, and KIAA1899 (human GCP5) cDNA were kindly provided by Dr. K. Matsumoto (Nagoya University, Nagoya, Japan), H. Saya (Keio University, Tokyo, Japan), Dr. J. R. Woodgett (Samuel Lunenfeld Research Institute, Toronto, Canada), and Kazusa DNA Research Institute (Chiba, Japan), respectively. Human GCP2 and GCP3 cDNAs and anti-GCP2 antibody were kind gifts from Dr. Y. Ono (Kobe University, Kobe, Japan). Anti-centrin 3 antibody was provided by Dr. M. Bornens (Institut Curie, Paris, France). Anti-GCP5 antibody was prepared in rabbits by immunization with recombinant full-length GCP5 protein. Anti-Myc antibody was prepared from 9E10 cells. Mouse monoclonal anti-GSK-3β and anti-EB1 antibodies were purchased from Transduction Laboratories. Mouse monoclonal anti-FLAG M2 antibody-conjugated agarose and anti-{gamma}-tubulin antibody and a rabbit polyclonal anti-{gamma}-tubulin antibody were from Sigma. Rabbit polyclonal anti-GFP and mouse monoclonal anti-HA (16B12) antibodies were from Invitrogen and COVANCE, respectively. GSK-3 inhibitors, SB216763 and SB415286, were from TOCRIS and Sigma, respectively. Other materials were from commercial sources.

Plasmid Construction—pCGN/GSK-3β, pCGN/GSK-3β (K85R), pEGFP/GSK-3β, pEF-BOS-Myc/GSK-3β, and pGEX-4T/GSK-3β were constructed as described previously (25–27). Standard recombinant DNA techniques were used to construct the following plasmids: pCGN/GCP5, pCGN/GCP5-(1–567), pCGN/GCP5-(568–1024), pEGFP/GCP5, and pMAL-C2/GCP5.

Cell Culture—HeLa S3, U2OS, and HEK-293T cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% calf serum or 10% fetal bovine serum. HeLa cells used for large scale spinner cultures to isolate GSK-3β complexes were grown in Joklik medium supplemented with 10% calf serum. Cells were transfected with plasmid DNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. When necessary, HeLa cells were incubated with 30 µM SB415286 for 16 h.

Immunoaffinity Purification of GSK-3β Protein Complexes— HeLa cells were transduced with a recombinant retrovirus expressing a bicistronic mRNA coding FLAG-HA-tagged human GSK-3β linked to an interleukin-2 receptor as a surface marker. The transduced subpopulation was purified by repeated cycles of affinity cell sorting (28). The cytoplasmic extracts were prepared as follows. Twelve liters of HeLa S3 cells (a total of ~9 x 109 cells) were centrifuged at 2,246 x g for 8 min at 4 °C, and the cell pellet was washed once with phosphate-buffered saline (PBS). The cell pellet was resuspended with 6x cell volume of hypotonic buffer (10 mM Tris-HCl, pH 7.3, 10 mM KCl, 1.5 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, and 10 mM β-mercaptoethanol) and shaken for several min. The suspended cells were centrifuged at 980 x g for 5 min at 4 °C, and the supernatant was discarded. The pellet was resuspended again with 1x cell volume of hypotonic buffer. After 10 min of incubation on ice, the swollen cells were homogenized with a Dounce homogenizer. The homogenate was centrifuged at 2,385 x g for 15 min at 4 °C, and the resulting supernatant (cytoplasmic extracts) was supplemented with one-tenth volume of 10x buffer (300 mM Tris-HCl, pH 7.3, 1.5 M KCl, 30 mM MgCl2) and incubated for 1 h at 4 °C. The extract was ultracentrifuged at 90,800 x g for 1 h at 4 °C. The obtained cytoplasmic extract was immediately frozen in liquid nitrogen before storage at –80 °C.

The GSK-3β-containing complexes in the cytoplasmic extracts (10 ml, 100 mg of total protein) were incubated with anti-FLAG M2 antibody-conjugated agarose (500 µl of beads). As a control, mock purification was performed from nontransduced HeLa cells through all purification steps. After an extensive wash with IP buffer (20 mM Tris-HCl, pH 8.0, 100 mM KCl, 5 mM MgCl2, 0.2 mM EDTA, 10% glycerol, 0.1% Tween 20, 1 mM β-mercaptoethanol, and a protease inhibitor mix (1 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, 20 µg/ml aprotinin, and 10 µg/ml pepstatin A)), the bound protein complexes were eluted from M2-agarose by incubation for 30 min with the 0.1 mg/ml FLAG peptide (Sigma) in 1 ml of IP buffer. The eluates were further purified by immunoprecipitation with anti-HA (12CA5) antibody conjugated to protein A-Sepharose (General Electric) (500 µl of beads). After washing with IP buffer, the bound complexes were eluted with 500 µl of 100 mM glycine-HCl, pH 2.5.

Mass Spectrometry Analysis—The GSK-3β complex purified from cytoplasmic extracts of HeLa cell was concentrated by StrataClean Resin (Stratagene). Resin-bound proteins were eluted with Laemmli buffer. The sample was separated by SDS-PAGE and silver-stained. Each band was excised from the SDS-polyacrylamide gel, digested with trypsin, and analyzed by mass spectrometry. The digest was lyophilized and dissolved in 50 µl of 0.1% trifluoroacetic acid solution, desalted using ZipTip µC18, and subjected to MALDI-TOF MS analysis. All MALDI-TOF mass spectra were obtained in the reflector positive mode using {alpha}-cyano-4-hydroxycinnamic acid (saturated solution in 50% acetonitrile with 0.1% trifluoroacetic acid) as the matrix. Analytes were prepared by mixing 0.5 µl of peptide sample with 0.5 µl of matrix solution on a MALDI plate and allowed to air-dry at room temperature in a hood before being inserted into the spectrometer. External calibration was conducted using ACTH-(1–7) and bradykinin. Peptides were identified using the Mascot search program (Matrix Science) to perform theoretical trypsin digests. All visible bands were analyzed, but some bands were not hit by the Mascot search program search. In addition to that, there were bands excised as a single band that consisted of multiple molecules. Only the consistent results through three independent experiments were described.

Complex Formation Assay—To show the interaction between endogenous GCP5 and GSK-3β, HeLa S3 cells were washed once with PBS and lysed in IP buffer. The lysates were immunoprecipitated with anti-GCP5 antibody, and the immunoprecipitate was probed with the indicated antibodies. To show the complex formation of overexpressed proteins, the indicated proteins were expressed in HEK-293T cells. The cells were washed once with Hepes-buffered saline (50 mM HEPES-NaOH, pH 7.4, 150 mM NaCl) and lysed in lysis buffer (50 mM HEPES-NaOH, pH 7.4, 150 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 0.5% Triton X-100, and a protease inhibitor mix). The lysates were centrifuged at 16,000 x g for 10 min at 4 °C, and then the obtained supernatants were immunoprecipitated with the indicated antibodies. The experiments were repeated at least three times, and representative data are shown.

Immunocytochemistry—HeLa S3 and U2OS cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with PBS containing 0.5% Triton X-100 for 10 min. Alternatively, the cultured cells were fixed with 100% methanol at –20 °C for 5 min and blocked in 2% bovine serum albumin in PBS for 10 min. To measure the accumulation area of {gamma}-tubulin and GCP5 at the spindle poles, each staining area at the spindle pole was measured manually using LSM510 software. For microtubule nucleation activity measurement, quantification of fluorescence intensity of EB1 staining at the spindle poles (1 µm2 circular area) was performed. To show colocalization of two proteins, more than 50 cells were observed carefully, and representative images are shown in the figures.

RNAi—The following double-stranded RNA oligonucleotides were synthesized using a CUGA in vitro small interference RNA (siRNA) synthesis kit (Nippon Gene), and the targeted sequences were as follows: human GCP5, 5'-GGAACATCATGTGGTCCATCA-3'; control (scrambled siRNA), 5'-CAGTCGCGTTTGCGACTGG-3'. HeLa S3 cells were transfected with each siRNA at 100 nM using Oligofectamine (Invitrogen), and the cells were used for experiments at 72 h post-transfection.

Sucrose Density Gradient Centrifugation Analysis—HeLa S3 cell extracts were prepared accordingly (29) with slight modifications. In brief, confluent cells (10-cm diameter dish) were lysed in 0.4 ml of HEPES-S buffer (50 mM HEPES-NaOH, pH 7.4, 100 mM NaCl, 1 mM EGTA, 1 mM MgCl2, 1 mM β-mercaptoethanol, and a protease inhibitor mix). The cell extract was homogenized by passing 30 times through a 291/2-gauge needle. After centrifugation for 10 min at 12,000 x g at 4 °C, the supernatant was loaded onto a 3.3-ml 5–40% discontinuous sucrose gradient consisting of 0.66 ml each of 5, 10, 20, 30, and 40% sucrose in HEPES-S buffer. The gradient was then centrifuged in a RPS56T-swing (Hitachi, Tokyo, Japan) rotor for 4 h at 314,000 x g at 4 °C. Fractions were collected from top to bottom (14 fractions) and analyzed by immunoblotting.

Centrosome Isolation—Centrosome was prepared from mitotic HeLa S3 cells according to the methods described previously (9, 30). To obtain mitotic cells, HeLa cells were treated with double thymidine blocks with the addition of 2 mM thymidine. After the second thymidine block, cells were released for 6 h before adding 50 ng/ml nocodazole. At 6–8 h after the addition of nocodazole, cells arrested in mitosis were harvested by shake-off. After centrifugation of HeLa S3 cell lysates by a discontinuous gradient consisting of 1 ml of 40%, 1 ml of 50%, and 1.6 ml of 70% sucrose solutions, fractions (200 µl/each fraction) were collected from top to bottom. The centrosome fractions were determined by immunoblotting with anti-{gamma}-tubulin and anti-GCP5 antibodies. The fractions that contained centrosome were then prepared accordingly as described (31). Centrosome fractions (400 µl) were diluted with 5x volume of PIPES buffer (10 mM PIPES-NaOH, pH 7.2) and centrifuged for 15 min at 24,000 x g at 4 °C. Pelleted centrosome was then resuspended in 200 µl of PIPES buffer.

Purification of HA-GCP5/{gamma}TuRC—Forty-eight hours after transfection, lysates of HEK-293T cells expressing HA-GCP5 (three 10-cm diameter dishes) were prepared to purify HA-GCP5/{gamma}TuRC as described (32). The lysates were immunoprecipitated with anti-HA (12CA5) antibody-conjugated Sepharose (300 µl of beads), and the beads were washed three times with HEPES-A buffer (50 mM HEPES-NaOH, pH 7.4, 150 mM NaCl, 1 mM dithiothreitol, 1 mM EGTA, 1 mM MgCl2, 0.25 mM GTP, and a protease inhibitor mix) containing 0.5% Triton X-100, once with HEPES-A buffer containing 250 mM NaCl instead of 150 mM NaCl, and once with HEPES-A buffer. The complex was eluted by incubation with 300 µl of 1 mg/ml HA peptide (Roche Applied Science) in HEPES-A buffer for 3 h at 4 °C. The presence of HA-GCP5 and {gamma}-tubulin complex in the eluates was confirmed by immunoblotting with anti-GCP5, anti-GCP2, and anti-{gamma}-tubulin antibodies.

Co-sedimentation Assay of GCP5/{gamma}TuRC and Centrosome— An In vitro sedimentation assay was performed by mixing equal volumes (10 µl each) of centrosome fractions (0.8 µg) and purified HA-GCP5/{gamma}TuRC (35 ng). To inhibit GSK-3 activity, SB415286 was added in the mixture at the beginning of incubation. After incubation for 30 min at 30 °C, the mixture was separated into the pellet and supernatant by centrifugation at 20,000 x g for 10 min at 4 °C. The pellet was washed once with PIPES buffer and resuspended in Laemmli buffer. The samples were subjected to SDS-PAGE, followed by immunoblotting with the indicated antibodies.

Statistical Analysis—All of the chromosome alignment counts, GCP5 and {gamma}-tubulin area measurements, and microtubule nucleation activity quantifications were performed for at least three independent experiments. At least 20 cells in each of prophase, prometaphase, and metaphase were analyzed in a single experiment. Five hundred cells were analyzed in a single chromosome alignment count. The results shown indicate means ± S.D. Statistical analysis was performed using StatView software (SAS Institute Inc.). An unpaired t test with a p value of <0.05 was used to determine statistical significance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of GCP5 as a GSK-3-binding Protein—To understand how GSK-3 is involved in mitotic spindle formation, GSK-3-binding proteins were purified from cytoplasmic extracts of HeLa cells stably expressing FLAG and HA epitope-tagged GSK-3β. As a first step, the proteins were purified by affinity chromatography with anti-FLAG antibody-conjugated agarose, and the bound polypeptides were eluted with the FLAG peptide. As a control, we performed a mock purification from cytoplasmic extracts prepared from nontransduced HeLa cells. The FLAG affinity-purified materials were further purified by immunoaffinity chromatography with an anti-HA antibody. After the second affinity purification, these proteins were subjected to protein identification by mass spectrometry. Twelve proteins were identified (Fig. 1, A and B). Among them, we were interested in GCP5, because it is one of the subunits comprising {gamma}TuRC (32). GCP5 belongs to the GCP family (GCP2 to -6) and has two conserved regions, called Spc (spindle pole body component), in common with other members of this family (Fig. 1C).

GSK-3β and GCP5 formed a complex at their endogenous levels in HeLa S3 cells when GCP5 was immunoprecipitated with a specific antibody (Fig. 2A). {gamma}-Tubulin was also observed in the immunocomplexes (Fig. 2A). When HA-GCP5 and Myc-GSK-3β were co-expressed in HEK-293T cells, HA-GCP5 was observed in the Myc-GSK-3β immunocomplexes (Fig. 2B). Reciprocally, enhanced green fluorescence protein (EGFP)-fused GSK-3β (EGFP-GSK-3β) was immunoprecipitated with HA-GCP5 (Fig. 2B). In addition, EGFP-GSK-3β also formed a complex with HA-GCP2 and HA-GCP3, which are the other components of {gamma}TuRC (Fig. 2B). In vitro binding studies using recombinant proteins demonstrated that GST-GSK-3β binds directly to MBP-GCP5 at a Kd value of ~125 nM (Fig. 2C). Both wild-type and a kinase-inactive mutant (K85R) of GSK-3β formed a complex with EGFP-GCP5 (supplemental Fig. S1). These results indicate that GCP5 binds directly to GSK-3β in intact cells and that the kinase activity of GSK-3 is not necessary for the formation of the complex.

To identify which region of GCP5 is important for binding to GSK-3β, two deletion mutants of HA-GCP5 were expressed in HEK-293T cells (Fig. 2D). Myc-GSK-3β formed a complex with GCP5-N (GCP5-(1–567)), the N-terminal region of GCP5, but interacted little with HA-GCP5-C (GCP5-(568–1024)), the C-terminal region of GCP5 (Fig. 2D). HA-GCP5-C formed a complex with {gamma}-tubulin slightly, and HA-GCP5-N did not. These results suggest that GCP5 has different sites for interaction with GSK-3β and {gamma}-tubulin. Consistent with these observations, HA-GCP5 formed a complex with {gamma}-tubulin and Myc-GSK-3β (Fig. 2D). Therefore, it is likely that GSK-3β is associated with the {gamma}-tubulin complex.


Figure 1
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  		FIGURE 1.
Identification of GCP-5 as a GSK-3β-binding protein. A, FLAG-HA epitope-tagged GSK-3β (GSK-3β) was purified from cytoplasmic extracts of HeLa cells expressing GSK-3β by immunoprecipitation with an anti-FLAG antibody (lane 2), followed by an anti-HA antibody (lane 3). As a control, mock purification was performed from nontransduced HeLa cells (lanes 1 and 4). The asterisks indicate the proteins that were identified by mass spectrometry. B, the GSK-3β complexes purified by the anti-FLAG and anti-HA antibodies, corresponding to lane 3 in A, were subjected to protein identification by mass spectrometry. C, schematic representation of the deletion mutants of GCP5 used in this study. SPC, spindle pole body component. WT, wild type.

 


Figure 2
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  		FIGURE 2.
Interaction of GCP5 with GSK-3β. A, lysates of HeLa S3 cells were probed with the indicated antibodies (lane 1). The same lysates were immunoprecipitated (IP) with anti-GFP (lane 2; control) or anti-GCP5 antibody (lane 3), and the immunoprecipitates were probed (IB) with the indicated antibodies (Ab). The arrow and arrowhead indicate the positions of GSK-3β and Ig, respectively. B, top panels, lysates of HEK-293T cells co-expressing Myc-GSK-3β and HA-GCP5 were probed with the indicated antibodies (lanes 1 and 2). The same lysates were immunoprecipitated with anti-Myc antibody, and the immunoprecipitates were probed with the indicated antibodies (lanes 3 and 4). The arrow and arrowhead indicate the positions of HA-GCP5 and immunoglobulin, respectively. Bottom panels, the lysates of HEK-293T cells co-expressing EGFP-GSK-3β and HA-GCP2, HA-GCP3, or HA-GCP5 were probed with the indicated antibodies (lanes 1–5). The same lysates were immunoprecipitated with anti-HA antibody, and the immunoprecipitates were probed with the indicated antibodies (lanes 6–9). C, the indicated concentrations of GST-GSK-3β were incubated with MBP or MBP-GCP5 (25 pmol) conjugated to the resin in 200 µl of 20 mM Tris-HCl, pH 7.5, containing 1 mM EDTA, 1 mM dithiothreitol, and 100 mM NaCl for 2 h at 4° C. The samples were recovered with centrifugation, and the precipitates were proved with anti-GST antibody (top). The intensity of each band was measured in the NIH Image software and expressed as an arbitrary unit. The results shown are representative of three independent experiments. D, left panels, lysates of HEK-293T cells co-expressing HA-GCP5 deletion mutants and Myc-GSK-3β were probed with anti-HA, anti-GSK-3β, or anti-{gamma}-tubulin antibodies. Right panels, the same lysates were immunoprecipitated with anti-HA antibody, and the immunoprecipitates were probed with the indicated antibodies. WT, wild type.

 
Roles of GCP5 in the Recruitment of {gamma}-Tubulin to the Spindle Poles—Since it has been shown that GCP5 is localized with {gamma}-tubulin as {gamma}TuRC to the centrosome (32), we examined the role of GCP5 in the formation of {gamma}TuRC, to determine the physiological relevance of the function of a complex between GSK-3β and GCP5. When the lysates of HeLa S3 cells were fractionated by sucrose density gradient centrifugation, {gamma}-tubulin, GCP2, and GCP5 co-sedimented in fraction 11 (Fig. 3A). {gamma}TuRC has been shown to have a size of ~32 S (32, 33); thus, the heavy fraction (fraction 11 in Fig. 3A) in the HeLa S3 cell lysates could correspond to {gamma}TuRC. GCP2 and {gamma}-tubulin were also present in light fractions (fraction 5), which presumably correspond to {gamma}TuSC (18, 34). The light fractional entity of GCP5 (fraction 4) was presumed to correspond to monomeric GCP5 (35). GCP5, GCP2, and {gamma}-tubulin in fraction 3 may be breakdown products of {gamma}TuRC during sucrose density gradient centrifugation (33).

When GCP5 was depleted by RNAi, endogenous GCP5 was reduced to less than 20% (Fig. 3A). This depletion was specific to GCP5 as determined by examining the protein levels of GCP2, β-tubulin, {gamma}-tubulin, GSK-3β, and β-actin (Fig. 3A). When extracts from GCP5-depleted cells were subjected to the sucrose density gradient centrifugation, the amounts of {gamma}-tubulin and GCP2 present in the {gamma}TuRC fraction were greatly reduced (Fig. 3A). Since the total amounts of {gamma}-tubulin and GCP-2 were not changed by depletion of GCP5, GCP5 appears to be involved in the formation or stability of {gamma}TuRC. These results are consistent with the observation that depletion of Dgrip128 (Drosophila {gamma}-tubulin ring protein 128; fly homolog of GCP5) shows a decrease in {gamma}TuRC content (24).

To show a role of GSK-3 in the regulation of mitotic spindle dynamics, we focused on mitotic cells. It has been shown that GSK-3β associates with the mitotic spindle and that a kinase-inactive form is present at the spindle poles (15). Immunostaining with each specific antibody showed that GCP5 is co-localized with {gamma}-tubulin to the spindle poles (Fig. 3B). Furthermore, transiently expressed HA-GCP5 and the N-terminal region of GCP5 were localized to the mitotic spindles and spindle poles, where GCP2 was also present. On the other hand, overexpressed HA-GCP5-C was observed neither along the spindles nor spindle poles (Fig. 3C). These findings suggest that the N-terminal region of GCP5 is required for the localization of GCP5 to the spindle poles. The amount of {gamma}-tubulin that accumulated at the spindle poles in mitotic cells was reduced by depletion of GCP5 (Figs. 3D and S2). The results from Fig. 3, A and D, suggest that depletion of GCP5 not only reduced the cellular {gamma}TuRC content but also impaired the recruitment of {gamma}-tubulin to the spindle poles in HeLa S3 cells. Therefore, GCP5 may be necessary for the recruitment of {gamma}-tubulin to the spindle poles.


Figure 3
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  		FIGURE 3.
Roles of GCP5 in the recruitment of {gamma}-tubulin to the spindle poles. A, left panels, HeLa S3 cells were transfected with control (scrambled) or GCP5 siRNA for 72 h, and the cell lysates were probed with the indicated antibodies. Right panels, the same lysates were fractionated by sucrose density gradient centrifugation and probed with the indicated antibodies. The relative molecular mass marker shown is bovine serum albumin (4.3 S). The results shown are representative of three independent experiments. B, HeLa S3 cells were stained with anti-GCP5 (red) and anti-{gamma}-tubulin (green) antibodies. In the merged images, co-localization of GCP5 and {gamma}-tubulin is shown in yellow. Insets, magnifications of the spindle pole areas (arrowheads) in the merged images. The results shown are representative of 50 mitotic cells. C, U2OS cells expressing HA-GCP5, HA-GCP5-N, or HA-GCP5-C were stained with anti-HA (green) and anti-GCP2 (red) antibodies. In the merged images, co-localization of HA-GCP5 and GCP2 is shown in yellow. D, HeLa S3 cells transfected with control or GCP5 siRNA for 72 h were stained with anti-GCP5 (green) and anti-{gamma}-tubulin (blue) antibodies. DNA was stained using propidium iodide (PI) (red). Insets, magnifications of the spindle pole areas (arrowheads) where centrosomal {gamma}-tubulin is present. Right panel, the area of the {gamma}-tubulin staining around the spindle poles in 20 metaphase cells was measured. The results shown are means ± S.D. of three independent experiments. Scale bars, 10 µm.

 
Involvement of GSK-3 in the Recruitment of {gamma}TuRC to the Spindle Poles during Mitosis—To clarify the role of GSK-3 in the dynamics of mitotic spindles, HeLa S3 cells were treated with SB415286, a GSK-3 inhibitor. As compared with control cells, the GCP5 and {gamma}-tubulin staining areas around the spindle poles seemed to be enlarged in the cells treated with SB415286 (Fig. 4A). These observations imply that there was an increase in GCP5 and {gamma}-tubulin recruitment to the spindle poles. Although the increases in GCP5 and {gamma}-tubulin accumulation at the spindle poles seemed to be small, the results were reproducible and statistically significant (Fig. 4, B and C). In contrast, SB415286 did not affect localization of centrin 3, a centriole marker (Fig. 4A). We also observed the chromosomal misalignment and extended astral microtubules in SB415286-treated mitotic cells irrespective of the establishment of normal bipolarity with bipolar spindles (Fig. 4, A and D). It has been reported that the inhibition of GSK-3 impairs the chromosomal alignment and that SB415286-treated cells often progress through the mitotic phase with the presence of unaligned lagging chromosomes (15, 16). In order to clarify the timing of the mitotic stage, in which the recruitment of GCP5/{gamma}TuRC to the spindle poles is regulated by GSK-3, we analyzed the time course of the accumulation of GCP5 from the onset of the mitotic stage to metaphase. We looked for prophase, prometaphase, and metaphase in control and SB415286-treated cells and measured the GCP5 staining area at the spindle poles. The accumulation of GCP5 at the spindle poles was enhanced at the onset of mitosis, and its elevated levels continued until metaphase (Fig. S3, A and B). Therefore, the recruitment of GCP5/{gamma}TuRC to the spindle poles may be regulated by GSK-3 at the onset of mitotic phase when the recruitment of {gamma}TuRC is presumed to be enhanced during the maturation of the centrosome (21, 24). When extracts from cells treated with SB415286 were subjected to sucrose density gradient centrifugation, co-sedimentation of GCP5, GCP2, and {gamma}-tubulin as part of {gamma}TuRC in the heavy fractions was unchanged as compared with control extracts (Fig. 4E), suggesting that GSK-3 activity is not involved in the complex formation of {gamma}TuRC. These findings suggest that GSK-3 may regulate the recruitment of {gamma}TuRC to the spindle poles.


Figure 4
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  		FIGURE 4.
Involvement of GSK-3 in the recruitment of {gamma}TuRC to the spindle poles during mitosis. A, HeLa S3 cells were treated with DMSO (control) or 30 µM SB415286 (SB) for 16 h and stained with anti-β-tubulin, anti-GCP5, anti-centrin 3, and {gamma}-tubulin antibodies. DNA was stained using PI. The magnified images of the spindle pole areas (arrowheads) where GCP5, centrin 3, or{gamma}-tubulin is present are shown in the insets. The arrow indicates the abnormally elongated astral microtubules. Scale bars, 10 µm. B and C, the areas of GCP5 (B) and {gamma}-tubulin (C) around the spindle poles in 20 mitotic cells were measured. The results shown are means ± S.D. of three independent experiments. D, mitotic cells with properly aligned metaphase chromosome with bipolar spindles and the cells with at least one chromosome not present in the center of the spindle, as seen in A, were counted as aligned and misaligned cells, respectively, for 500 cells in total. The results shown are means ± S.D. of three independent experiments. E, lysates of HeLa S3 cells treated with DMSO (control) or 30 µM SB415286 for 16 h were fractionated by sucrose density gradient centrifugation and probed with the indicated antibodies (Ab). The results shown are representative of three independent experiments.

 
Rescue of Abnormal Phenotypes Induced by GSK-3 Inhibition by GCP5 Depletion—To determine whether the accumulation of GCP5 to the spindle poles is involved in the abnormal phenotypes induced by the inhibition of GSK-3, GCP5 was depleted by RNAi in these cells. Depletion of GCP5 reduced the level of {gamma}-tubulin accumulation at the spindle poles in the cells treated with SB415286 (Fig. 5, A and B). Moreover, the SB415286-induced chromosomal misalignment was not completely but was partially rescued by depletion of GCP5 (Fig. 5, A and C). Although the result showed the statistical significance, the rescue effect of GCP5 depletion was not fully effective in the chromosome alignment. We speculate that {gamma}TuRC could play a part in the formation of proper chromosomal alignment, but there are other molecules regulated downstream of GSK-3, which are involved in the alignment of chromosome during mitotic phase.

Since elongated astral microtubules were observed in SB415286-treated cells (see Fig. 4A, arrow) (15), we suspected the overactivation of the microtubule nucleation at the spindle poles. The microtubule nucleation activity was measured by quantifying the fluorescence intensity of EB1 staining around the spindle poles, because EB1 has been reported to represent the marker for the rate of microtubule nucleation activity (36). Treatment of the mitotic cells with SB415286 increased the fluorescence intensity of the EB1 staining area around the spindle poles (Fig. 5D). Statistical analyses indicated that this increase of EB1 intensity in SB415286-treated cells was significant. Depletion of GCP5 reduced the intensity of EB1 staining in the control cells (Fig. 5E). Furthermore, SB415286-induced increase of nucleation activity was also dramatically reduced in GCP5-depleted cells (Fig. 5E). Therefore, microtubule nucleation activity at the spindle poles is, in part, regulated by GSK-3, and this regulation for the recruitment of {gamma}TuRC including GCP5 to the spindle poles might be important for proper spindle formation. These results suggest that GSK-3 is involved in the regulation of mitotic spindle outgrowth by controlling the amount of {gamma}TuRC targeted to the spindle poles in mitotic cells.

Enhanced Association of {gamma}TuRC with Centrosome by GSK-3 Inhibition in Vitro—To examine whether the inhibition of GSK-3 is the major cause of the increase in {gamma}TuRC recruitment to the spindle poles, we purified HA-GCP5/{gamma}TuRC from HEK-293T cells and mixed it with isolated centrosome from mitotic HeLa S3 cells with or without GSK-3 inhibitor in vitro. It has been reported that purified CDK2-cyclin E mixed with isolated centrosome in vitro can phosphorylate and dissociate nucleophosmin-B23 from the centrosome (31). We applied a similar method to examine the effect of SB415286 on the association of {gamma}TuRC with the centrosome. First, we tested whether purified HA-GCP5/{gamma}TuRC associates with the isolated centrosome. After incubation for 30 min, HA-GCP5 was indeed precipitated in the presence of isolated centrosome but not its absence (Fig. 6A). From our prior study, we found that 1 µM SB415286 is sufficient to inhibit the 30 ng of purified GST-GSK-3 kinase activity (data not shown), and at the cell culture level, 30 µM SB415286 has been used in previous studies (15, 16), including our study described here. Hence, we performed the in vitro sedimentation assay of HA-GCP5/{gamma}TuRC by mixing it with isolated centrosome in the presence of various concentration of SB415286. By inhibiting GSK-3 activity, the amounts of HA-GCP5 co-sedimented with the centrosome were increased (Fig. 6B). In order to show the specificity of the effects of SB415286 on the association of GCP5 with the centrosome, we used inhibitors for other kinase; Rho-kinase, phosphatidylinositol 3-kinase, and casein kinase I (Fig. 6C). The binding of HA-GCP5 to the centrosome was enhanced specifically by SB415286 but not by the other kinase inhibitors, although high doses (10-fold higher than the Ki value in vitro) were used. These results suggest that GSK-3 activity may regulate the association of HA-GCP5 with the centrosome.


Figure 5
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  		FIGURE 5.
Rescue of abnormal phenotypes induced by GSK-3 inhibition by GCP5 depletion. A, after HeLa S3 cells had been transfected with control or GCP5 siRNA for 56 h, the cells were further incubated with DMSO (control) or 30 µM SB415286 (SB) for 16 h. The cells were then stained with anti-{gamma}-tubulin antibody. DNA was stained using PI. B, the area of {gamma}-tubulin staining at the spindle poles in 20 mitotic cells was measured as in Fig. 4C. The results shown are means ± S.D. of three independent experiments. C, cells with misaligned chromosomes were counted as described in the legend to Fig. 4D. D, HeLa S3 cells were treated as in Fig. 4A and were stained with anti-EB1 antibody. DNA was stained using PI. The magnified images of EB1 at the spindle poles (arrowheads) are shown as enlarged images. The fluorescence intensity of EB1 staining in a circular region around spindle poles in 20 cells was quantified. The results shown are means ± S.D. of three independent experiments. E, HeLa S3 cells were treated as in A and were stained with anti-EB1 antibody. DNA was stained with PI. The fluorescence intensity of EB1 staining was quantified as in D. The results shown are means ± S.D. of three independent experiments. Scale bars, 10 µm.

 


Figure 6
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  		FIGURE 6.
In vitro association of HA-GCP5/{gamma}TuRC with centrosome. A, HA-GCP5/{gamma}TuRC purified from HEK-293T cells was mixed with or without the isolated centrosome from mitotic HeLa S3 cells. The mixtures were incubated for 30 min at 30 °C. The pellet containing centrosome and the supernatant with unbound HA-GCP5/{gamma}TuRC were separated by centrifugation, and samples were probed with indicated antibodies (Ab). The results shown are representative of three independent experiments. B, purified HA-GCP5/{gamma}TuRC and the isolated centrosome were incubated in the presence of various concentrations of SB415286 (SB). None, no treatment. DMSO was used as the control buffer in which SB415286 was dissolved. The samples were probed with anti-HA antibody (top). The results shown are representative of three independent experiments. The band intensity in the pellet (ppt) or supernatant (sup) fraction and the sum of the intensity in the pellet and supernatant fraction were measured in NIH Image software, and the former value was divided by the latter. The ratio of the amount of HA-GCP5 in pellet and supernatant fractions was expressed in arbitrary units (bottom). The results shown are means ± S.D. of three independent experiments. C, the binding of HA-GCP5 to the centrosome was assayed as in A and B, in the presence of various kinase inhibitors. DMSO was used as a control. Y, 2 µM Y27632 (Rho-kinase inhibitor); LY, 15 µM LY294002 (phosphatidylinositol 3-kinase inhibitor); CKI-7, 100 µM CKI-7 (casein kinase I inhibitor).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
It has been reported that small molecule inhibitors of GSK-3 cause increases in the length of the astral microtubules and the rate of chromosomal misalignment in mitotic cells (15, 16). These results raised the possibility that GSK-3 is involved in the regulation of microtubule dynamics and spindle pole organization in the mitotic phase. However, the mechanism by which the inhibition of GSK-3 activity causes chromosomal instability was not addressed. In this study, we showed that GSK-3β forms a complex with GCP5, GCP2, and {gamma}-tubulin, which are components of {gamma}TuRC. Furthermore, the inhibition of GSK-3 activity enhances the recruitment of GCP5 and {gamma}-tubulin to the spindle poles in mitotic cells, as a consequence leading to the increase in the microtubule nucleation activity at the spindle pole.

Roles of GCP5 in {gamma}TuRC—Each {gamma}TuRC consists of multiple copies of {gamma}-tubulin, GCP2, GCP3, and additional proteins GCP4, GCP5, GCP6, and GCP-WD/NEDD1 (21, 24, 32). The stoichiometry of the {gamma}-tubulin complex components indicated that the {gamma}-tubulin-GCP2-GCP3 subcomplex and GCP4 are present in multiple copies (29, 32). GCP5 has been indicated to be present as a single copy per complex (32). Taken together, the {gamma}-tubulin-GCP2-GCP3 subunits are thought to interact with tubulin heterodimers, and GCP4, GCP5, and GCP6 might be involved in the association with the centrosomes (21). Our results suggest that GSK-3 regulates the recruitment of {gamma}TuRC to the spindle poles at least by binding to GCP5. It has been shown that phospho-GSK-3, which is an inactive form, localizes to the spindle poles (15). Although the degree of inhibition of GSK-3 activity in mitotic spindle poles is not known, we speculate that the site-specific suppression of GSK-3 could be required for the proper recruitment of {gamma}TuRC to the spindle poles but that severe inhibition by SB415286 may impair the fine balancing of {gamma}TuRC content at the spindle poles to organize proper spindle formation.

We also found that GCP5 is necessary for the formation or stability of {gamma}TuRC, but GSK-3 activity is not required. Our conclusion is based on biochemical analyses of the distribution of {gamma}TuSC and {gamma}TuRC in the sucrose density sedimentation and immunocytochemical analyses of subcellular localization of {gamma}-tubulin. Depletion of GCP5 showed a marked decrease in {gamma}TuRC content and led to the impaired localization of {gamma}-tubulin to the spindle poles. The former result is consistent with the roles of Dgrip128 (GCP5) of Drosophila in the assembly of {gamma}TuRC (24). It has also been reported that {gamma}TuRC is dispensable for the targeting of {gamma}-tubulin to the centrosome in Drosophila and that depletion of Dgrip128 reduces the {gamma}-tubulin localization to the spindles but not to the spindle poles (24, 37). In contrast, our results suggest that GCP5 may play essential roles in the recruitment of {gamma}-tubulin to the spindle poles in mammalian mitotic cells (HeLaS3 cells). Therefore, it is possible that the recruitment of {gamma}TuRC to the spindle poles may be regulated in the cell context manner.

Involvement of GSK-3 in Microtubule Nucleation Activity in {gamma}TuRC—Phenotypes induced by the inhibition of GSK-3 might, in part, result from overactivation of microtubule nucleation due to the enhanced accumulation of {gamma}TuRC to the spindle poles. Our data also showed that reduction of the {gamma}-tubulin accumulation at the spindle poles was accompanied by rescue of the abnormal chromosome alignment in GCP5-depleted cells. It is possible that enhanced recruitment of {gamma}-tubulin to the spindle poles causes the increase in the microtubule nucleation activity, consequently leading to extended astral microtubules. However, it is not known how GSK-3 regulates the recruitment of {gamma}TuRC to the spindle poles at present. One possibility is that GSK-3 phosphorylates GCP5 or components of {gamma}TuRC and that the phosphorylation suppresses the recruitment or association of {gamma}TuRC to the spindle poles. Our preliminary results suggest that GCP5 is phosphorylated by GSK-3β (Fig. S4A). When immunoprecipitated HA-GCP5 from mitotic HeLa S3 cell lysates was incubated with GST-GSK-3β, the phosphorylation of HA-GCP5 was detected. The phosphorylation was inhibited by the addition of SB216763. The phosphorylation of GCP5, prepared from asynchronous cells, by GSK-3β was not detected. The amounts of endogenous GCP5 or ectopically expressed GCP5 were unchanged in the interphase and mitotic phase (Fig. S4B). Therefore, GSK-3 may phosphorylate GCP5 in mitotic cells in order to inhibit the association of {gamma}TuRC with or the targeting of {gamma}TuRC to the centrosome. Of course, GSK-3 may phosphorylate other components of {gamma}TuRC. Collectively, these results suggest the GSK-3 activity is somehow involved in the regulation of {gamma}TuRC recruitment and association with the centrosome. We cannot exclude the possibility that there are alternative factors influenced by SB-treatment that could modify the {gamma}TuRC affinity with the centrosome. Further studies need to be done to clarify the correlation between GSK-3 activity and the mechanism controlling the recruitment of {gamma}TuRC to and association of {gamma}TuRC with the centrosome at the onset of mitosis.

To coordinate the appropriate localization of {gamma}TuRC to the spindle poles, GSK-3 activity might be inhibited by protein kinase B (15). It has been reported that GSK-3 is present as an inactive form on the plus ends of microtubules and promotes polarization and cell protrusion through microtubules in interphase (7). We have shown that inhibition of GSK-3 does not affect nucleation of microtubules at the centrosomes but inhibits the anchoring of microtubules to the centrosome during interphase (9). Therefore, GSK-3 could be active in the centrosomes during interphase. It is intriguing to speculate that GSK-3 has different functions in the regulation of microtubule dynamics, and its activity is exquisitely regulated in a cell cycle-dependent manner.


   FOOTNOTES
 
* This work was supported by grants-in-aid for scientific research and for scientific research on priority areas from the Ministry of Education, Science, and Culture, Japan (2005, 2006, and 2007). 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

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S4. Back

1 To whom correspondence should be addressed. Tel.: 81-82-257-5130; Fax: 81-82-257-5134; E-mail: akikuchi{at}hiroshima-u.ac.jp.

2 The abbreviations used are: {gamma}TuSC, {gamma}-tubulin small complex; {gamma}TuRC, {gamma}-tubulin ring complex; RNAi, RNA interference; HA, hemagglutinin; MALDI, matrix-assisted laser desorption/ionization; TOF, time of flight; PBS, phosphate-buffered saline; siRNA, small interference RNA; HEK, human embryonic kidney; EGFP, enhanced green fluorescence protein; GST, glutathione S-transferase; HA, hemagglutinin; IP, immunoprecipitation; PIPES, 1,4-piperazinediethanesulfonic acid; MS, mass spectrometry; PI, propidium iodide; DMSO, dimethyl sulfoxide. Back


   ACKNOWLEDGMENTS
 
We thank Drs. K. Matsumoto, H. Saya, J. R. Woodgett, Y. Ono, and M. Bornens for donating cells and plasmids; Drs. K. Ueda-Hamao and T. Ikura for technical help and advice on immunoaffinity purification; and Dr. K. Kikuchi for technical help and helpful discussion.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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