Correspondence to Yukiko Gotoh: ygotoh{at}iam.u-tokyo.ac.jp
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Introduction |
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The 14-3-3 proteins are a family of phospho-Ser/phospho-Thrbinding molecules that play essential roles in many biological processes, including the regulation of cell death (van Hemert et al., 2001; Masters et al., 2002; Yaffe, 2002). 14-3-3 promotes cell survival by sequestering and inactivating several proapoptotic proteins, including Bad and FOXO3a, after their phosphorylation by survival-inducing kinases such as Akt (Zha et al., 1996; Datta et al., 1997; del Peso et al., 1997; Biggs et al., 1999; Brunet et al., 1999; Kops et al., 1999; Zhang et al., 1999; Masuyama et al., 2001; Masters et al., 2002; Basu et al., 2003). Indeed, the reduction of available 14-3-3 sensitizes cells to apoptotic signals, and overexpression of 14-3-3 renders cells resistant to apoptotic signals (Chan et al., 1999; Zhang et al., 1999; Xing et al., 2000; Masters and Fu, 2001; Samuel et al., 2001; Nomura et al., 2003; Tsuruta et al., 2004). The level of available 14-3-3 may, therefore, be crucial for determining the threshold above which apoptotic signals can initiate the program.
14-3-3 appears to be modulated by phosphorylation. Phosphorylated forms of 14-3-3ß and at Ser186 and 184, respectively, are abundantly present in brain tissue (Aitken et al., 1995). We found that 14-3-3
and
isoforms are phosphorylated at their respective phosphorylation sites by c-Jun NH2-terminal kinase (JNK; Tsuruta et al., 2004). This finding prompted us to examine whether phosphorylation of 14-3-3 may have any impact on the regulation of cell death because JNK has been suggested to play a key role in stress-induced apoptosis in a context-dependent manner (Yang et al., 1997; Tournier et al., 2000; Lei et al., 2002; Deng et al., 2003; Kuan et al., 2003). It is important to note that phosphorylation of 14-3-3
causes the dissociation of Bax from 14-3-3, leading to Bax translocation to the mitochondria and to apoptosis (Tsuruta et al., 2004). The expression of mutants lacking phosphorylation sites (Ser184A of 14-3-3
or Ser186A of 14-3-3
) effectively attenuates cell death that is induced by stress-activated JNK, suggesting that 14-3-3 is a major target of JNK in the induction of cell death. However, Bax is not a typical 14-3-3 ligand in the sense that this binding does not require the common ligand-binding groove and does not depend on ligand (Bax) phosphorylation (Nomura et al., 2003). Thus, it is unclear whether phosphorylation of 14-3-3 also affects the association of "typical" 14-3-3 ligands, which usually contain the 14-3-3binding motif RSXpSXP or RXXXpSXP (Muslin et al., 1996; Yaffe et al., 1997). Because Ser184 is located near the ligand-binding groove, the phosphorylation of this residue might regulate the association of many typical 14-3-3 ligands, including the aforementioned proapoptotic proteins. To investigate the hypothesis that the JNK-mediated phosphorylation of 14-3-3 may contribute to cell death by regulating typical 14-3-3 ligands in addition to Bax, we examined the effect of 14-3-3 phosphorylation on the association of Bad, which binds to the common ligand-binding groove of 14-3-3 upon Bad phosphorylation.
Bad is a "BH3-only" member of the Bcl-2 family (Danial and Korsmeyer, 2004). Survival factors suppress the proapoptotic function of Bad by phosphorylation at Ser112, 136, and 155 (Zha et al., 1996; Datta et al., 2000; Lizcano et al., 2000; Zhou et al., 2000). Phosphorylation of Ser112 and 136 creates the 14-3-3binding motifs RHSpS112YP and RSRpS136AP, respectively, and the consequent interaction with 14-3-3 leads to the cytoplasmic sequestration and inactivation of Bad. Akt, Pak-1, and p70S6 kinase appear to mediate the survival factorinduced phosphorylation of Bad at Ser136, whereas Rsk, mitochondria-associated protein kinase A, Pak-1 and -5, and Pim-1 are responsible for phosphorylation at Ser112 (Datta et al., 1997; del Peso et al., 1997; Blume-Jensen et al., 1998; Bonni et al., 1999; Harada et al., 1999, 2001; Tan et al., 1999; Schurmann et al., 2000; Shimamura et al., 2000; Cotteret et al., 2003; Aho et al., 2004). The dephosphorylated form of Bad is targeted to the mitochondria, where it causes apoptosis by binding and inactivating Bcl-2/Bcl-xL (Wang et al., 1999). Thus, survival factors regulate the interaction between Bad and 14-3-3 by regulating Bad phosphorylation. However, it remains to be determined whether the interaction between Bad and 14-3-3 is also regulated by the phosphorylation state of 14-3-3. Because Bad and JNK are both implicated in stress-induced cell death (Yang et al., 1997; Tournier et al., 2000; Datta et al., 2002; Lei et al., 2002; Deng et al., 2003; Kuan et al., 2003), we focused on Bad as a potential target for JNK-mediated apoptotic signals.
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Results |
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JNK promotes Bad translocation to mitochondria
Bad is localized mostly in the cytoplasm but redistributes to the mitochondria in response to survival factor deprivation (Li et al., 2004). Because 14-3-3 is regarded as a cytoplasmic anchor for Bad (Muslin and Xing, 2000) and we found that the JNK-mediated phosphorylation of 14-3-3 releases Bad from 14-3-3, we hypothesized that JNK might regulate the localization of Bad. To investigate this, we used Myc-tagged Bad (Myc-Bad). In all of the experiments with Myc-Bad, p35 was cotransfected to prevent caspase activity induced by the overexpression of Myc-Bad. In healthy cells, a large proportion of Myc-Bad localized in the cytoplasm, whereas a small proportion colocalized with MitoTracker CMXRos, as judged by immunocytochemical analysis (Fig. 3 a). We found that the expression of MKK7-JNK (WT), but not MKK7-JNK (KN), resulted in the translocation of Myc-Bad to the mitochondria. To confirm the effect of JNK on promoting Bad translocation, we examined the distribution of endogenous Bad by subcellular fractionation after transfection of COS-1 cells with MKK7-JNK constructs (Fig. 3 b). The amount of endogenous Bad that was detected in the mitochondrial fraction was increased by the expression of MKK7-JNK (WT). In contrast, the expression of MKK7-JNK (KN) did not have these effects. In this assay, the abundance of the mitochondrial marker F1F0-ATPase subunit and that of the cytosolic marker
-tubulin in these fractions were unaffected by the expression of either construct. Although MKK7-JNK (WT) resulted in the activation of caspase-3 (Lei et al., 2002; Tsuruta et al., 2004), the coexpression of p35 failed to inhibit Bad translocation to the mitochondrial fraction, suggesting that MKK7-JNK (WT) promoted the translocation of endogenous Bad to mitochondria independently of caspase activation.
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If JNK promotes Bad translocation to the mitochondria through phosphorylation of 14-3-3 proteins, the expression of a 14-3-3 mutant lacking the phosphorylation site should block JNK-induced Bad translocation because the unphosphorylated form of 14-3-3 should retain Bad in the cytoplasm. As expected, the coexpression of 14-3-3 Ser184A, and to a lesser extent of 14-3-3
(WT), inhibited MKK7-JNK (WT)induced Bad translocation to the mitochondrial fraction (Fig. 3 d). These results strongly suggest that 14-3-3 is an essential target for JNK in promoting Bad translocation to the mitochondria.
JNK promotes the association of Bad with Bcl-2/Bcl-xL
The BH3-only subfamily of Bcl-2 family proteins, including Bad, has been postulated to promote apoptosis by antagonizing the antiapoptotic functions of Bcl-2/Bcl-xL (Yang et al., 1995; Danial and Korsmeyer, 2004). Because we found that active JNK can induce Bad translocation to the mitochondria through phosphorylation of 14-3-3, we next asked whether JNK also induces Bad association with Bcl-2/Bcl-xL. When endogenous Bad in healthy cells was immunoprecipitated with anti-Bad antibody, small amounts of Bcl-2/Bcl-xL were coimmunoprecipitated (Fig. 4 a). In contrast, when MKK7-JNK (WT), but not MKK7-JNK (KN), was expressed in these cells, the amounts of endogenous Bcl-2/Bcl-xL that was associated with endogenous Bad greatly increased, corresponding to the reduction of 14-3-3 associated with Bad (Fig. 4 a). If JNK promotes the association of Bad with Bcl-2/Bcl-xL through phosphorylation of 14-3-3, the expression of 14-3-3 Ser184A should block the JNK-induced interaction between Bad and Bcl-2/Bcl-xL. Indeed, the expression of 14-3-3
Ser184A inhibited the increase of Bad protein that interacted with Bcl-2/Bcl-xL (Fig. 4 b). Altogether, these results suggest that the activation of JNK induces the activation of Bad at the mitochondria through phosphorylation of 14-3-3.
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Therefore, JNK appears to induce both the release of Bad from 14-3-3 and the dephosphorylation of Bad. Is there any causal relationship between these two events? To examine this, we treated COS-1 cells with the phosphatase inhibitor okadaic acid (OA) to prevent dephosphorylation of Bad at Ser112 and 136 (Fig. 4 d). We confirmed that OA maintained the level of Bad phosphorylation, yet we observed that the expression of MKK7-JNK (WT) still reduced the amount of 14-3-3 that is associated with Bad (Fig. 4 d). This suggests that dephosphorylation of Bad is not a prerequisite for the JNK-mediated dissociation of Bad from 14-3-3. On the other hand, the expression of 14-3-3 Ser184A suppressed Bad dephosphorylation at Ser112 and 136 (Fig. 4 e). This strongly supports the notion that Bad release from 14-3-3 is necessary for Bad dephosphorylation after JNK activation. Because Chiang et al. (2003) has shown that 14-3-3 and protein phosphatase 2A compete for Bad and that 14-3-3 maintains the phosphorylated state of Bad, it is conceivable that the JNK-mediated release from 14-3-3 makes Bad accessible to phosphatases and that it is dephosphorylated as a result.
JNK-mediated 14-3-3 phosphorylation regulates Bad-dependent cell death
We next examined the relative contribution of the JNK-induced phosphorylation of 14-3-3 to cell death regulation in a cellular context in which cell death is dependent on Bad and is counteracted by Akt. In HCT116 cells, UV-induced apoptosis, which is dependent on JNK (Fig. 5, a and b), and MKK7-JNK (WT)induced apoptosis were suppressed by small interference RNA (siRNA) against Bad (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200409117.DC1), suggesting that they are dependent on endogenous Bad, at least in part. It is important to note that the expression of 14-3-3 Ser186A, and to a lesser extent the expression of 14-3-3
(WT), suppressed both UV-induced apoptosis (Fig. 5, c and d) and MKK7-JNK (WT)induced apoptosis (Tsuruta et al., 2004). The ectopic expression of a low level of Bad caused enhanced UV-induced apoptosis; and, again, this enhancement was suppressed, in part, by the expression of 14-3-3
Ser186A and, to a lesser extent, by the expression of 14-3-3
(WT; Fig. 5 e). These results clearly indicate that 14-3-3 suppresses Bad-mediated apoptosis in a phosphorylation-dependent manner.
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Discussion |
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Recent studies have shown that JNK directly phosphorylates Bad at Ser128 and Thr201 (Donovan et al., 2002; Konishi et al., 2002; Yu et al., 2004). Phosphorylation of the former residue reduces the affinity of Bad for 14-3-3 and activates Bad, whereas phosphorylation of the latter reduces the affinity of Bad for Bcl-2/Bcl-xL and inactivates Bad. We found that the JNK-mediated phosphorylation of 14-3-3 reduced its interaction with WT and Ser128A Bad proteins to a similar extent. This suggests that JNK can cause the release of Bad from 14-3-3 independently of phosphorylation at Bad Ser128. However, it is possible that these two JNK-mediated phosphorylations (on Bad and 14-3-3) act in parallel or in a concerted fashion to effectively activate Bad. Thr201, on the other hand, exists in mouse Bad but not in human Bad and may not be generally involved in Bad regulation.
There appears to be some threshold below which apoptotic signaling can occur but cannot lead to cell death. In this context, 14-3-3 can function to raise this hypothetical threshold by acting as a "buffer" that sequesters several proapoptotic proteins in response to survival signals, including those mediated by Akt. This is also consistent with the observation that increasing or decreasing the amount of available 14-3-3 can shift the balance between survival and apoptosis, in effect raising or lowering the threshold for apoptotic signaling. There would be at least three ways to override the threshold set by 14-3-3. The first and probably the simplest way is to increase the total amount of proapoptotic proteins so that they exceed the threshold level. The second way is to reduce the amount of survival signals, which would result in dephosphorylation and release of proapoptotic proteins from 14-3-3 (Fig. 8 b). The third way is to reduce the level of available 14-3-3 by the JNK-mediated phosphorylation of 14-3-3, resulting in the release of proapoptotic proteins (Fig. 8 c). In this scenario, JNK functions to lower this hypothetical threshold, thus rendering cells more susceptible to apoptotic signals.
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Materials and methods |
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Cell culture and transfection
COS-1 cells were maintained in DME (Sigma-Aldrich) supplemented with 10% FBS and 1% penicillin/streptomycin. HCT116 cells were maintained in McCoy's 5A Medium (Sigma-Aldrich) with 10% FBS, 1% penicillin/streptomycin, and 1.5 mM L-glutamine. COS-1 cells were transfected with plasmids by the use of Fugene6 (Roche Diagnostics), and HCT116 cells were transfected with LipofectAMINE 2000 (Invitrogen).
Plasmid construction
The constructs encoding mice Bad (full length) and FOXO3a (aa 1525) were provided by M.E. Greenberg (Harvard Medical School, Boston, MA). We obtained the constructs encoding 14-3-3 from H. Fu (Emory University School of Medicine, Atlanta, GA) and obtained 14-3-3
from B. Vogelstein (The Johns Hopkins University School of Medicine, Baltimore, MD). For the Myc-tagged Bad expression vector, the Bad cDNA fragment was inserted into the BglII site of pCS4-Myc. Site-directed mutagenesis was performed with the QuikChange kit (Stratagene) to generate the Ser128 and Thr201 into Ala change in Bad and to generate the Lys49 into Glu change in 14-3-3
. WT, Ser128A, Thr201A, and Ser128A-Thr201A Bad cDNA were cloned into the BamHI sites of pET28a (Novagen), and K49E 14-3-3
cDNA was cloned into the BglII site of pCS4-Myc. pcDNA3-MKK7-FLAG-JNK (WT and KN), pET28a-MKK7-FLAG-JNK (WT and KN), pGEX6P-114-3-3
(WT and Ser184A), pCS4-Myc14-3-3
(WT and Ser184A), pCS4-Myc14-3-3
(WT and Ser186A), the constructs encoding p35 (a gift from M. Miura, University of Tokyo, Tokyo, Japan), CA-Akt, DN-Akt, WT-Akt, DN-JNK, and DN-JBD (a gift from R. Davis, University of Massachusetts Medical School, Worcester, MA) have been described previously (Masuyama et al., 2001; Tsuruta et al., 2002, 2004).
Antibodies
Antibodies to 14-3-3 (K-19; Santa Cruz Biotechnology, Inc.), 14-3-3 (C-18; Santa Cruz Biotechnology, Inc.), Bad (C-20; Santa Cruz Biotechnology, Inc.), phospho-Bad (Ser112; Cell Signaling), phospho-Bad (Ser136; Cell Signaling), Bcl-2 (N-19; Santa Cruz Biotechnology, Inc.), Bcl-xL (S-18; Santa Cruz Biotechnology, Inc.), cleaved caspase-3 (Cell Signaling), F1F0-ATPase subunit
(7H10; Molecular Probes),
-tubulin (DM1A; Sigma-Aldrich), GAPDH (MAB374; Chemicon), GST (B-14; Santa Cruz Biotechnology, Inc.), c-Jun (Cell Signaling), phosphoc-Jun (Cell Signaling), Akt (Cell Signaling), phospho-Akt (Ser473; 58F11; Cell Signaling), Rsk (Upstate Biotechnology), or phospho-p90Rsk (Thr359-Ser363; Cell Signaling) were used for immunoblot analysis. An antibody to phospho-Bad (Ser155) was provided by M.E. Greenberg (Datta et al., 2000). Antibodies to Bad (C-20; Santa Cruz Biotechnology, Inc.), 14-3-3
(C-18; Santa Cruz Biotechnology, Inc.), HA (Y-11; Santa Cruz Biotechnology, Inc.), Flag (M2; Sigma-Aldrich), normal rabbit IgG (Santa Cruz Biotechnology, Inc.), or normal goat IgG (Santa Cruz Biotechnology, Inc.) were used for immunoprecipitation.
Immunoblot analysis
Cells were washed with PBS and were lysed in an extraction buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM ß-glycerophosphate, 5 mM EGTA, 1 mM sodium pyrophoshate, 5 mM NaF, 1 mM Na3VO4, 0.5% Triton X-100, and 1 mM DTT) supplemented with protease inhibitors (1 mM PMSF, 5 µg/ml leupeptin, 5 µg/ml pepstatin A, and 5 µg/ml aprotinin). Proteins were separated by SDS-PAGE and were electrically transferred to a polyvinylidene difluoride membrane. The membrane was probed with the appropriate primary antibody and with an HRP-conjugated secondary antibody. Blots were visualized by Western Lightning (PerkinElmer).
Protein purification
His6-tagged Bad (WT, Ser128A, Thr201A, or Ser128A-Thr201A) was expressed in Escherichia coli BL21 by pET28a-based vectors. After IPTG was added, induction occurred; and His-tagged proteins were purified with Ni2+-bound column (ProBond resin; Invitrogen). The purification of GST14-3-3, GST-FOXO3a (aa 1525), and His6-tagged MKK7-JNK was performed as described previously (Brunet et al., 1999; Tsuruta et al., 2004).
Subcellular fractionation
Cells were washed with PBS, suspended in 300 µl of ice-cold isotonic buffer (200 mM mannitol, 70 mM sucrose, 1 mM EDTA, 10 mM Hepes-NaOH, pH 7.4, and 1 mM DTT) supplemented with the protease inhibitors described above, and homogenized with a Potter-Elvehjem homogenizer (model Mazela Z; Eyela). Nuclei and unbroken cells were removed by centrifugation at 500 g for 10 min, and the supernatant was further centrifuged at 100,000 g for 60 min. The resulting supernatant was saved as the cytosolic fraction, and the pellet was washed with isotonic buffer, resuspended in extraction buffer supplemented with protease inhibitors, and centrifuged at 20,000 g for 5 min to remove debris. The resulting supernatant was saved as the mitochondrial fraction.
Coimmunoprecipitation assay
COS-1 cells were lysed in lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM ß-glycerophosphate, 5 mM EGTA, 1 mM sodium pyrophoshate, 5 mM NaF, 1 mM Na3VO4, 0.05% Triton X-100, and 1 mM DTT). The lysates were then incubated with antibodies to Bad (C-20) for 1 h and were subsequently incubated with protein ASepharose beads (GE Healthcare) for 1 h. HCT116 cells were lysed in the lysis buffer described above, and the lysates were precleared with protein GSepharose beads (GE Healthcare). The lysates were incubated with antibodies to 14-3-3 (Santa Cruz Biotechnology, Inc.) and with protein GSepharose beads for 2 h. The proteinantibody complexes that were recovered on beads was subjected to immunoblot analysis after separation by SDS-PAGE.
GST pull-down assay
Recombinant GST14-3-3 (WT or Ser184A) was incubated with or without purified MKK7-JNK (WT or KN) in the presence of a kinase reaction buffer (100 µM ATP, 20 mM Tris-HCl, pH 7.5, and 15 mM MgCl2) for 30 min at 30°C. His6-Bad (WT, Ser128A, Thr201A, or Ser128A-Thr201A) protein was preincubated with or without active Akt immunoprecipitate in a kinase reaction buffer. Subsequently, Bad protein was mixed with GST14-3-3
and glutathioneSepharose 4B beads for 1 h at 4°C, and the bead-bound proteins were subjected to immunoblot analysis with antibodies to Bad and GST.
Immunofluorescence analysis
COS-1 cells were grown on poly-D-lysinecoated coverslips and were transfected with a plasmid expressing Myc-tagged Bad and p35 and/or expression plasmids encoding MKK7-JNK proteins. Cells were fixed in PBS containing 4% PFA for 10 min at RT. The fixed coverslips were permeabilized in PBS containing 0.5% Triton X-100 for 10 min, washed twice in PBS, and incubated in a blocking solution (PBS containing 2% BSA) for 30 min. The cells were then incubated in the blocking solution with anti-Myc antibody (9E10; Santa Cruz Biotechnology, Inc.) for 1 h and with AlexaFluor488 antimouse IgG antibody (Molecular Probes) for 1 h in the blocking solution. Where indicated, cells were stained with CMXRos before fixation to visualize mitochondria. Fluorescence images were recorded by a confocal laser-scanning microscope (model LSM-510; Carl Zeiss MicroImaging, Inc.). All images were captured at 40x by using objective lenses (1.4 NA; C-Apochromat). Pictures were analyzed by using LSM5 Image Browser (Carl Zeiss MicroImaging, Inc.).
RNAi experiment
The siRNA for human Bad mRNA was obtained from Japan Bio Services Co., Ltd. The siRNA sequences used in this study for Bad were sense (5'-UGAGUGACGAGUUUGUGGAdTdT-3') and antisense (5'-UCCACAAACUCGUCACUCAdTdT-3'). The 5' terminus of the sense sequence was labeled with Texas red, and the sequences were randomized for the control siRNA (sense, 5'-AGUUCGAUUUUAGGGGGGAdTdT-3'; antisense, 5'-UCCCCCCUAAAAUCGAACUdTdT-3'). Transfection to HCT116 cells was performed using Oligofectamine reagent (Invitrogen) according to the manufacturer's instructions except that the incubation time with transfection reagents was 120 min. The transfection efficiency was >95%, as assessed by Texas red.
Apoptosis assay
HCT116 cells were seeded into 60-mm culture dishes and were transfected with a plasmid expressing GFP together with expression plasmids encoding JBD, Bad, MKK7-JNK (WT), and various Akt or 14-3-3 proteins. After 24 h, cells were irradiated by UV for 6 h when necessary in the presence of 10% serum for 6 h. After staining with Hoechst 33342 for 10 min, the percentage of GFP-positive cells with a pyknotic nucleus was determined. For the RNAi experiments, 2.5 x 105 cells were seeded into 35-mm dishes and were transfected with 100 pmol siRNA. After 36 h, cells were transfected with 0.625 µg of expression vectors for GFP, MKK7-JNK (WT), or various Akt proteins or, after 48 h, cells were irradiated by UV. Then, they were stained with Hoechst 33342.
Statistical analysis
Results were expressed as the mean ± SD and were analyzed by using the unpaired t test.
Online supplemental material
Fig. S1 shows that the JNK-mediated phosphorylation of 14-3-3 can induce the dissociation of Bad regardless of the Ser128 and Thr201 phosphorylation of Bad in vitro. Fig. S2 shows that the expression of active JNK results in the reduction of Bad phosphorylation at Ser112, 136, and 155 without inhibiting phosphorylation of Akt and Rsk. Fig. S3 shows that UV- and active JNK-induced cell death is partially dependent on Bad. Fig. S4 shows that the introduction of siRNA against Bad suppressed cell death, which is induced by serum deprivation or DN-Akt. Fig. S5 shows that the JNK-mediated phosphorylation of 14-3-3 results in the release of FOXO3a from 14-3-3
in vitro. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200409117.DC1.
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Acknowledgments |
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This work was supported by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan and by Precursory Research for Embryonic Science and Technology 21 of the Japan Science and Technology Corporation.
Submitted: 20 September 2004
Accepted: 8 June 2005
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