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1 Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Myodaiji, Okazaki, Aichi 444-8787, Japan
2 Kondoh Differentiation Signaling Project, ERATO/Kondoh Research Team, SORST, JST, Yoshidakawaracho, Sakyo-ku, Kyoto 606-8305, Japan
3 Graduate School of Frontier Biosciences, Osaka University, Yamadaoka, Suita 565-0871, Japan
4 Department of Molecular Biomechanics, The Graduate University for Advanced Studies (SOKENDAI), Myodaiji, Okazaki, Aichi 444-8787, Japan
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Abstract |
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by double-stranded RNA-mediated translational interference. These observations indicate that both Wnt proteins can interact with the majority of Fz receptors and elicit signaling reactions exemplified by Dsh phosphorylation but that the stabilization of ß-catenin/armadillo proteins in the Wnt/ß-catenin signaling occurs only when specific combinations of Wnt and Fz meet. |
Introduction |
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In the Wnt/ß-catenin pathway, a signal is transferred from the Fz receptor to the nucleus through a series of components, including Dishevelled (Dsh/Dvl), glycogen synthase kinase 3ß (GSK-3ß), Axin, ß-catenin/armadillo and Lef-1/Tcf. In the absence of the Wnt signal, GSK-3ß phosphorylates ß-catenin on a scaffold provided by Axin, targeting it for degradation by proteasomes. Upon binding of Wnt to its receptors, ß-catenin is stabilized by the antagonizing action of Dsh on GSK-3ß as well as by the destabilization of Axin through LRP 5/6 (Mao et al. 2001; Li et al. 2002). Although the mechanism of this antagonism stimulated by Wnt signals is not yet fully understood, it has been shown that Wnt signals elevate the phosphorylation level of Dsh (Yanagawa et al. 1995; Gonzalez-Sancho et al. 2004). Stabilized ß-catenin forms a complex with the Lef-1/Tcf transcription factors, which then activates the transcription of target genes.
This signaling pathway has been shown to be preferentially activated by particular members of the Wnt family. In Xenopus early embryos, misexpression of a subclass of the Wnt family, referred to as the Wnt-1 subclass, which includes Wnt-1, Wnt-3, Wnt-3a and Wnt-8, causes axis duplication by enhancing the Wnt/ß-catenin signaling (McMahon & Moon 1989; Sokol et al. 1991; Torres et al. 1996). In contrast, misexpression of another subclass, referred to as the Wnt-5a subclass, which includes Wnt-5a and Wnt-11, does not cause the axis duplication, but rather results in defective convergent extension movement by activating another signaling pathway, non-canonical Wnt pathway (Torres et al. 1996; Heisenberg et al. 2000). However, the molecular basis to cause difference in activation of the Wnt/ß-catenin pathway has still been uncertain.
For understanding of the molecular mechanism responsible for the activation of the Wnt/ß-catenin pathway, the receptor specificity of these Wnt ligands should be defined. So far, several studies have shown physical and functional associations between particular Wnt and Fz molecules (Hsieh et al. 1999; Boutros et al. 2000; Rulifson et al. 2000; Strapps & Tomlinson 2001; Holmen et al. 2002; Wu & Nusse 2002). For instance, the binding analysis between membrane-tethered forms of Drosophila Wnts and the ligand-binding domains of its Fz receptors showed a wide-spectrum of binding affinities (Rulifson et al. 2000; Wu & Nusse 2002). However, the receptor specificity of different mammalian Wnts has not yet been systematically examined by analyzing interactions between intact Wnt proteins and Frizzled receptors. Here, we focused on Wnt-3a and Wnt-5a, typical members of the Wnt-1 and the Wnt-5a subclasses, respectively, and examined their specificity toward various members of the Fz receptor family under the same conditions. To examine their effects on the components of the Wnt signaling, we monitored the steady-state level of armadillo/ß-catenin proteins, as well as the phosphorylation level of Dsh. Our results indicate that both of these Wnt signals can be transmitted to the cytoplasm and elevate the Dsh phosphorylation level in combination with the majority of Fz receptors, but that specific combinations of Wnt and Fz members are required for efficient stabilization of armadillo/ß-catenin proteins.
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Results |
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Prior to examining the functional interaction of Wnt-3a and Wnt-5a proteins with various members of the Fz family of receptors, we first established a series of stable transfectants each exogenously expressing a single member of this family. For establishing these transfectants, we considered it desirable to use a cell line that expresses no or only a small amount of Fz protein, because it was strongly anticipated that a high level expression of the endogenous Fz proteins would cover or mask the function of exogenously expressed Fz proteins. However, all mammalian culture cell lines that we had examined, including mouse L and NIH3T3 cells, expressed large amounts of various Fz proteins; for example, L cells highly expressed at least Fz1, 2, 4, 5 and 7 (data not shown). In contrast, Drosophila S2 cells have been shown to express only a trace level of endogenous Fz. Thus, we generated various Drosophila S2 transfectants, in which a single type of Fz receptor was exogenously expressed. In this study, a series of S2 cell lines expressing mouse Fz3, Fz4, Fz6 or Fz8, or human Fz5, as well as a control transfectant expressing Drosophila Fz2 (Dfz2), which is known to induce the Wnt/ß-catenin signaling with Wingless, were used. The expression level of the exogenous Fz genes was changeable under the control of the methallothionein promoter, whose function is enhanced by the addition of metals such as Cu2+ (Supplemental Fig. S1B). To compare the effect of the exogenous proteins properly between the Fz members, we selected cell lines expressing each Fz mRNA at almost the same level under the same culture conditions and used them for the following experiments (Supplemental Fig. S1A).
First of all, the stimulation level of the Wnt/ß-catenin pathway through various Wnt–Fz interactions was examined by monitoring the amount of Arm, a Drosophila homolog of ß-catenin. To avoid a possibility that fractionation procedure of cytosolic components may cause artificial variation in recovery of the cytosolic Arm, we monitored its amount in the whole cell, which includes both cytosolic and membrane-bound Arm. As a control, cocultivation of Wingless-expressing S2 cells with Dfz2-expressing cells caused an increase in the level of Arm proteins at both non-induced (–Cu2+) (Fig. 1A, lanes 13, 14) and induced (+Cu2+) levels (Fig. 1B, lanes 13, 14) of Dfz2 proteins; whereas no increase in Arm was observed in S2 cells transfected with the control vector (Fig. 1A,B; lanes 11, 12). Wg also increased the Arm protein level through Fz3, Fz4, Fz5, and Fz8, but not through Fz6, at both the basal and induced levels of Fzs (Fig. 1A,B). Thus, these Fz proteins can transmit the Wg signaling even in Drosophila S2 cells. In addition, even in the absence of Wingless-expressing cells, the levels of Arm proteins were increased depending on the amount of these Fzs (Fig. 1A,B). Thus, expression of Fzs themselves may also result in an increase in the level of Arm proteins in this system.
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Both Wnt-3a and Wnt-5a proteins elevate the Dsh phosphorylation level in combination with the majority of Fz receptors
Upon the stimulation of Wnt signaling, the phosphorylation level of Dsh is increased prior to the accumulation of Arm/ß-catenin proteins (Yanagawa et al. 1995). Thus, an increase in the Dsh phosphorylation level seems to be an early indicator responsive to the Wnt stimuli, although the role of this phosphorylation in the Wnt signaling has not yet been revealed. Interestingly, in addition to Wg and Wnt3a, Wnt-5a has also been shown to trigger phosphorylation of Dsh in mammalian cells (Gonzalez-Sancho et al. 2004). Thus, both the Wnt-1 and the Wnt-5a subclass ligands can induce Dsh phosphorylation. However, it remained totally unknown whether these different Wnt ligands can induce Dsh phosphorylation through common or different sets of receptors. So, we next examined which members of the Fz receptor family are able to mediate the increase in the Dsh phosphorylation level by interacting with Wnt-3a or Wnt-5a.
As previously reported, Dsh phosphorylation, indicated by the mobility shift of Dsh, was detected in a transfectant expressing Dfz2 even in the absence of Wg, and its level was increased by the addition of a Wg signal (data not shown) (Yanagawa et al. 1995). The level of Dsh phosphorylation in the Dfz2-expressing cells was also increased by the addition of Wnt-3a C.M. (Fig. 2A,B; lanes 11–14). Upon stimulation with Wnt-3a proteins, the Dsh phosphorylation level was also increased in most of the Fz-expressing cells (Fig. 2A,B). All Fz members that induced the stabilization of Arm/ß-catenin proteins when interacting with Wnt-3a, Fz4, Fz5, and Fz8 showed an increased level of Dsh phosphorylation in response to the Wnt-3a stimulus, although the basal level of phosphorylation was not consistent among them. In addition, Fz3, which did not induce Arm/ß-catenin stabilization upon Wnt-3a treatment, also showed the mobility shift of Dsh by Wnt-3a (Fig. 2A,B; lanes 1, 2). Thus, Fz3 can mediate Wnt-3a signaling to increase the Dsh phosphorylation level, although this action is not sufficient to stabilize Arm/ß-catenin. In contrast, Fz6-expressing cells showed no increase in the Dsh phosphorylation level in response to the Wnt-3a stimulus, while the mobility shift of Dsh occurred depending on the amount of Fz6. Interestingly, as in the case of Wnt-3a, Wnt-5a also increased the Dsh phosphorylation level in cells expressing most Fz receptors except Fz6 under conditions where the level of Fz expression was basal or induced (Fig. 2C,D). Therefore, both Wnt-3a and Wnt-5a proteins can interact with the majority of Fz receptors and elicit early signaling reactions exemplified by Dsh phosphorylation.
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is required for the Wnt-induced Dsh phosphorylation
It has been indicated that Dsh is phosphorylated by a number of protein kinases, i.e. casein kinase I (CKI) (Peters et al. 1999; Hino et al. 2001; McKay et al. 2001; Matsubayashi et al. 2004), casein kinase II (Willert et al. 1997; Kuhl et al. 2000), Par-1 (Sun et al. 2001), and PKC (Kuhl et al. 2001; Kinoshita et al. 2003). Among these kinases, CKI has been shown to be required for the Dsh phosphorylation induced by Wg (Matsubayashi et al. 2004). However, it has not yet been shown whether the same or a different kinase is involved in phosphorylation induced by another Wnt ligand, especially in the case where it is induced by the Wnt-5a-subclass ligand. Thus, we next examined whether CKI was also required for the Wnt-3a- or Wnt-5a-dependent phosphorylation of Dsh observed in the various Fz-expressing cells (Fig. 3). In this experiment, we focused on those producing Fz3, Fz4, and Fz8, because the Wnt-dependent mobility shift of Dsh was more clearly observed in these cells than in others. To decrease the activities of casein kinase I , we performed double-stranded RNA-mediated translational interference. Each S2 cell line was cultured with synthetic double-stranded RNA with a sequence identical to a part of CKI mRNA and treated with Wnt-3a or Wnt-5a C.M. As a control, each S2 cell line was also cultured with synthetic double-stranded RNA specific for bacterial LacZ mRNA. In Fz3-, Fz4- and Fz8-expressing cells, Dsh phosphorylation induced by the addition of Wnt-3a C.M. was almost totally abrogated by treatment with double-stranded RNA for CKI , as previously observed with Wg. Furthermore, that induced by the addition of Wnt-5a C.M., as well as that occuring in the absence of Wnt C.M., was also almost completely abolished by CKI -RNAi, but not by control RNAi. Thus, not only the Wnt-3a- but also the Wnt-5a-induced Dsh phosphorylation through Fz3, Fz4 and Fz8, as well as the basal phosphorylation dependent on Fz4 and Fz8, required the activity of CKI .
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Recently, it was reported that nuclear localization of Dsh is crucial for activation of the Wnt canonical pathway (Itoh et al. 2005). We therefore examined whether the nuclear localization of Dsh would be specifically enhanced by the addition of Wnt-3a to cultures of Fz3, Fz4 and Fz8-expressing cells. However, no obvious enhancement of nuclear accumulation was found by the addition of Wnt-3a or Wnt-5a proteins (Fig. 4). Thus, neither Wnt-3a nor Wnt-5a protein enhanced the nuclear accumulation of Dsh at least in S2 cells. On the other hand, we found that the phosphorylated forms of Dsh preferentially remained in the cytoplasm, suggesting that the phosphorylated Dsh may function there.
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Discussion |
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To understand the molecular basis for generating the functional specificity between Wnt and Fz members, it is important to identify factors that contribute to the specificity. Some characteristics specific to Wnt-3a must contribute to the efficient signal transduction of the Wnt/ß-catenin pathway, because Wnt-3a, but not Wnt-5a, could induce the stabilization of ß-catenin under the conditions used in this study (Fig. 1). One factor that may cause this ligand specificity is the possible preferential in interaction of Wnt-3a with some other Wnt signaling component, aside from Fz, that plays a crucial role in the ß-catenin stabilization. It has already been described that the interaction of LRP5/6, a co-receptor of Wnt, with Axin is also involved in the stabilization of ß-catenin in parallel with the Dsh-dependent pathway (Mao et al. 2001; Li et al. 2002). Thus, the LRP5/6 co-receptor is a possible candidate to cause the difference in function between Wnt-3a and Wnt-5a; and their ability to interact with Arrow (Wehrli et al. 2000), a Drosophila homolog of LRP5/6, might be different between them. In support of this idea, a dominant-negative form of LRP6 and Dickkopf-1, which inhibits the Wnt/ß-catenin signaling by binding to LRP5/6, cannot inhibit the Dsh phosphorylation induced by Wnt-3a or Wnt-5a (Gonzalez-Sancho et al. 2004). Thus, LRP5/6 does not appear to be required for the Dsh phosphorylation, which is commonly induced by Wnt-3a and Wnt-5a, whereas it is required for efficient signal transduction of the Wnt/ß-catenin pathway, which is specifically induced by Wnt-3a, but not by Wnt-5a. To reveal further the molecular mechanism underlying the specificity toward LRP5/6, quantitative analysis of the physical interaction between these Wnts and LRP5/6 should be informative. As another possible factor that may affect the ligand specificity, preferential binding to Fz receptors themselves can also be considered. In Drosophila, it has been shown that binding constants with respect to soluble forms of Fzs are different among Wnt proteins, such as Wg, DWnt2, DWnt3, and DWnt8 (Wu & Nusse 2002). Thus, we cannot exclude the possibility that some possible difference in binding affinity of Wnt-3a and -5a for various Fzs may influence the efficiency of the transduction of the Wnt signaling pathway.
Some intrinsic characteristic specific to particular Fz molecules may also be another possible factor affecting the signal transduction in the Wnt/ß-catenin pathway. Under the conditions used in this study, only specific Fzs could induce the ß-catenin stabilization depending on the Wnt-3a signals (Fig. 1). Phylogenic analysis indicates that, in terms of the amino acid sequences in both their extracellular and intracellular domains, Fz4, Fz5, Fz8 and Dfz2, which can mediate a Wnt-3a-induced increase in ß-catenin, are closely related to each other, but different from Fz3 and Fz6, which did not cause ß-catenin stabilization by Wnt-3a (Huang & Klein 2004). Thus, some characteristic structures in the extracellular and/or intracellular domains specific to the former Fzs may be needed for efficient transmission of Wnt-3a signaling to the downstream components of the Wnt/ß-catenin pathway. Previous evidence suggests that some characteristics of the former Fzs’ domains may be involved in efficient signal transduction. For instance, the extracellular CRD domain of Dfz2 shows higher affinity toward Wg than that of Dfz1, which activates the Wnt/ß-catenin less efficiently than Dfz2 (Rulifson et al. 2000). Furthermore, mouse Fz4, mouse Fz8 and human Fz5, all of which activated the stabilization of armadillo with both Wg and Wnt-3a ligands in our systems, were shown to bind evidently to Wg through their CRD domains; whereas the CRD domains of mouse Fz3 and Fz6 showed less evident binding to Wg in comparison with the former Fz members. Thus, ligand affinity is likely to be one factor for the efficient transmission, and the characteristic structures in particular Fzs appear to contribute to affinity for particular ligands. On the other hand, the cytoplasmic sequence of Dfz2 has also been shown to be responsible for efficient activation of the Wnt/ß-catenin pathway (Boutros et al. 2000). Thus, the cytoplasmic structure characteristic for the particular Fzs may also contribute to efficient transmission of the Wnt/ß-catenin signaling. Further extensive studies, including quantitative analysis to examine the binding affinity between Wnt and Fz proteins, will be required for identification of the factors involved in the specificity of interaction between Wnts and Fzs.
In contrast to our results, co-injection of Wnt-5a and human Fz5 mRNA into Xenopus embryos induces the canonical signaling. In comparison with cells expressing other Fz receptors, Fz5-expressing cells showed higher levels of armadillo proteins and Dsh phosphorylation even under the condition without the addition of Wnt proteins (Figs 1 and 2). This indicates that Fz5 appears to be potentially more active for transmitting the signal to the cytoplasmic components than other Fz receptors. We therefore speculate that some specific factor in Xenopus embryos may enhance the weak activity of Wnt-5a in the canonical signaling and that this enhanced activity may be specifically evident in the combination with a potentially active receptor, Fz5. To reveal the whole mechanism underlying Wnt-Fz specificity, we must identify and characterize other factors involved in the canonical signaling.
Several studies have indicated that Dsh is phosphorylated upon the stimulation of the Wnt signaling pathway induced by different Wnt ligands. In addition, a number of kinases that catalyze Dsh phosphorylation have been found. However, it has been unknown whether different Wnt ligands induce Dsh phosphorylation through the same or different molecular machinery. Our results clearly show that both Wnt-3a and Wnt-5a can increase the Dsh phosphorylation level through common receptors and kinase. Both Wnt-3a and Wnt-5a proteins shared common Fz receptors for their induction of Dsh phosphorylation (Fig. 2), and required CKI activity for this phosphorylation (Fig. 3). Thus, common mediators appear to be involved in Dsh phosphorylation induced by a typical member representing each of the two different subclasses of the Wnt family. One of the important issues about this phosphorylation is whether or not, and if so, how this phosphorylation event affects signal transmission in the individual Wnt pathways. However, it is likely to be difficult to show that CKI -mediated Dsh phosphorylation is involved in the signal transmission of the canonical Wnt pathway simply by inhibiting the activity of this kinase, because this kinase also plays another role as a priming kinase, which is required for GSK-dependent ß-catenin phosphorylation in the canonical Wnt pathway in Drosophila S2 cells. To elucidate the role of Dsh phosphorylation in the Wnt pathways, we must identify the site phosphorylated in response to these Wnt proteins and examine its requirement in these.
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Experimental procedures |
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L cells were cultured in a 1 : 1 mixture of Dulbecco's Modified Eagle Medium and Ham's F12 medium supplemented with 8.3% fetal bovine serum and antibiotics at 37 °C. For establishment of L cells transfected with Wnt-3a cDNA, or Wnt-5a cDNA, the mouse Wnt-3a cDNA or Wnt-5a cDNA was inserted into pPGKneo, containing the neomycin phosphotransferase gene (neo) driven by the PGK promoter (pPGKWnt-3a or pPGKWnt-5a). The expression of Wnt-3a and Wnt-5a was driven by a promoter of the rat phosphoglycerokinase gene (PGK promoter) and terminated at a transcriptional terminator sequence of the bovine growth hormone gene in these constructs.
pPGKWnt-3a and pPGKWnt-5a were separately introduced into L cells, which had been plated in 60-mm culture dishes at a density of 1.5 x 1054 cells/plate 1 day before the DNA addition, by the calcium phosphate method. To these cultures, 400 µg/mL of G418 was added 2 days after transfection; and stably transfected clones were then selected.
Preparation of active and soluble Wnt proteins
We have already reported cell lines that secrete active and soluble Wnt-3a protein efficiently into their culture medium (Shibamoto et al. 1998). According to the same method used for Wnt-3a, we established new cell lines that secrete soluble Wnt-5a protein into their culture medium and prepared conditioned medium containing active Wnt-5a protein (Yamanaka et al. 2002) (Supplemental Fig. S2).
Cell cultures and transfections of S2 cells
Cells of the Drosophila Schneider S2 cell line were cultured with Schineider's Drosophila medium (Invitrogen) supplemented with 10% fetal bovine serum and antibiotics at 25 °C as described (van Leeuwen et al. 1994; Yanagawa et al. 1995).
Drosophila S2 cells, in which only a trace level of endogenous Fz expression is detected, were used for establishing a series of stable tranfectants expressing various Fz proteins. pMK33-based expression vectors, in which mouse Fz3, Fz4, Fz8, and human Fz5 cDNAs were driven by the metallothionein promoter, were constructed, and introduced into S2 cells by using Effectine reagent (Qiagen). The stable transfectants were selected with hygromycin B (200 µM), and expression of the introduced Fz genes was confirmed by RT-PCR using gene-specific primers for each Fz gene. Specific primers used for RT-PCR were as follows: mouse Fz3, sense primer 5'-CCAGTTGCAGTGCAGAGGGACTATG-3', anti-sense primer 5'-CATTGCAGGCTACTCGGTCCTCCAG-3'; mouse Fz4, sense primer 5'-GCAGAGCCCGTTCTCATCCAAGAAG-3', anti-sense primer 5'-AGGAGCCACCACAAAGCCAGTGAGG-3'; human Fz5, sense primer 5'-GTTCGTGTGCAAGTGTCGCGAGCCC-3', anti-sense primer 5'-GGCTGCAGGCCACGCTGGCATGGCC-3'; mouse Fz6, sense primer 5'-TCTGGGGAGGTGTGGACTTCTGTTG-3', anti-sense primer 5'-TCTCTGCGGTCTTCTGGGTTGGAAG-3'; mouse Fz8, sense primer 5'-GTCGCTGTTCCGAATCCGTTCAGTC-3', anti-sense primer 5'-ACACGCCCGATGTGATGCCCACTAC-3'; Drosophila Fz2, sense primer 5'-TTCCAAACTGCGGCATACCGTGCAA-3', anti-sense primer 5'-TAGGTGAGCAGGAAGACCAGGGTGC-3'. Since each Fz gene was driven by the metallothionein promoter, highly extended expression of Fz genes could be induced by adding 0.5 mM CuSO4.
Other Fz-expressing S2 cells, i.e. mouse Fz6/S2, Drosophila Fz2/S2, and control pMK33 vector-transfected S2 cell lines, were kindly donated by Dr S. Yanagawa (Yanagawa et al. 1998). S2 cells transfected with a construct containing the heat-shock promoter driving Wg expression (S2-HS-wg cells) were described earlier (Cumberledge & Krasnow 1993) and were kindly provided by Dr Yanagawa.
Treatment of Fz-S2 cells with Wnt proteins
Lysates of cells treated for 180 min with medium conditioned by Wnt-3a- or Wnt-5a-expressing cells, or with that conditioned by control L cells, were subjected to immunoblotting with anti-Arm antibody for comparison of Arm protein levels in several S2 cell lines, or with anti-Dsh antibody to examine the extent of phosphorylation of Dsh proteins in S2 cell lines. Wg treatment of S2 cell lines was performed by cocultivation for 180 min with heat-pretreated S2-HS-wg cells. Whereas the S2 cell line used for establishing Fz expressing transfectants adhered to cell culture plates, S2-HS-wg cells did not adhere to cell culture plates. Thus, after the cocultivation, S2-HS-wg cells were washed out with PBS. For each assay, more than 2 independent stable clones of pMK-Fz3, 4, 5 and 8 transfectants were used; and we confirmed that the results obtained were basically the same.
dsRNA production and RNAi procedures
The RNAi experiments in Drosophila S2 were performed as previously described (3 x 106 S2 cells were incubated for 3 days with 15 µg of dsRNA in each well of a 6-well plate; (Clemens et al. 2000)). Individual dsRNAs were generated by using a Megascript T7 transcription kit (Ambion) and the DNA templates, which were generated by PCR using sets of primers with T7 RNA polymerase binding sites. Primer sequences used to generate specific dsRNA were obtained from the GENBANK database: Drosophila CKI (GENBANK accession No. U55848), sense primer 457–480, anti-sense primer 1138–1161; LacZ (accession No. E00696), sense primer 399–420, anti-sense primer 1138–1162. Specificity of CKI -RNAi was examined by Northern blot analysis (Yanagawa et al. 2002).
Nuclear and cytoplasmic extracts preparation
A nuclear extract kit (Active Motif) was used to prepare nuclear and cytoplasmic extracts from S2 cells according to the instruction manual that the manufacture recommended.
Antibodies and immunoblotting
Preparation of anti-Wnt-3a (3a#3–1) or anti-Wnt-5a (5a#5–5D) antibody will be described elsewhere (R. T and S. T., unpublished observation). Briefly, we injected mice with bacterially expressed recombinant Wnt-3a or Wnt-5a proteins, and screened for anti-Wnt-3a- or anti-Wnt-5a-producing hybridomas.
Anti-Armadillo antibody (N2 7A1 ARMADILLO) was obtained from Developmental Studies Hybridoma Bank. Rat anti-Dsh polyclonal antibody was kindly donated by Dr T. Uemura of Kyoto University (Shimada et al. 2001). Anti-actin antibody was purchased from Chemicon.
For immunoblotting, proteins separated by SDS-polyacrylamide gel electrophoresis were electrophoretically transferred onto polyvinylidene fluoride membranes by means of a semidry blotting apparatus (Bio Craft, Japan). The membranes were blocked in TBS (50 mM Tris-HCl [pH 7.6], 150 mM NaCl, 1 mM CaCl2) with 5% skim milk for 1 h at room temperature, and then incubated with primary antibodies. The membranes were next incubated with horseradish peroxidase-conjugated antibodies for 1 h, and developed by use of the reagents from an Enhanced Chemiluminescent Detection System (Amersham).
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: stakada{at}nibb.ac.jp
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Received: 16 February 2005
Accepted: 21 June 2005
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1.1 fold, 1.3
