Casein Kinase Iepsilon Enhances the Binding of Dvl-1 to Frat-1 and Is Essential for Wnt-3a-induced Accumulation of beta -Catenin*

Shin-ichiro HinoDagger , Tatsuo Michiue§, Makoto Asashima§, and Akira KikuchiDagger

From the Dagger  Department of Biochemistry, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8551, Japan and the § Sorst Project and Department of Life Science (Biology), University of Tokyo, 3-8-1, Komaba, Meguro-ku, Tokyo 153-8902, Japan

Received for publication, December 30, 2002, and in revised form, January 19, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We demonstrate that Dvl-1, casein kinase Iepsilon (CKIepsilon ), and Frat-1 activate the Wnt signaling pathway cooperatively. The amino acid region 228-250 of Dvl-1 was necessary for its binding to Frat-1, and the interaction of Dvl-1 with Frat-1 was enhanced by CKIepsilon . Coexpression of Dvl-1 and Frat-1 caused accumulation of beta -catenin synergistically in L cells. Both proteins also activated the transcriptional activity of T-cell factor-4 (Tcf-4) synergistically in human embryonic kidney 293 cells, but coexpression of Dvl-1-(Delta 228-250), which lacks the amino acid region 228-250 from Dvl-1, and Frat-1 did not. Dvl-1, but not Dvl-1-(Delta 228-250), acted synergistically with CKIepsilon to activate Tcf-4. Depletion of CKIepsilon by double-stranded RNA interference in HeLa S3 cells led to the inhibition of Wnt-3a-induced phosphorylation of Dvl and the binding of Dvl-1 to Frat-1. Furthermore, depletion of CKIepsilon reduced the Wnt-3a-induced accumulation of beta -catenin, although it did not affect the basal level of beta -catenin. These results indicate that CKIepsilon -dependent phosphorylation of Dvl enhances the formation of a complex of Dvl-1 with Frat-1 and that this complex leads to the activation of the Wnt signaling pathway.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Wnt proteins constitute a large family of cysteine-rich secreted ligands that control development in organisms ranging from nematode worms to mammals (1). In vertebrates, the Wnt signaling pathway regulates axis formation, organ development, and cellular proliferation, morphology, motility, and fate (2, 3). The protein level of free cytoplasmic beta -catenin is controlled by the Wnt signal. In unstimulated cells, cytoplasmic beta -catenin is destabilized by a multiprotein complex containing Axin (or its homolog Axil/conductin), GSK-3beta ,1 and APC (4-8). Interaction of GSK-3beta with Axin in the complex facilitates efficient phosphorylation of beta -catenin by GSK-3beta . Phosphorylated beta -catenin forms a complex with Fbw1 (beta TrCP/FWD1), which resides in an E3 ubiquitin ligase complex (9, 10). Ubiquitination of beta -catenin induces its rapid proteosomal degradation (11).

When Wnt binds to the Frizzled/LRP co-receptor at the cell surface, a cytoplasmic protein, Dvl, antagonizes GSK-3beta -dependent phosphorylation of beta -catenin. Once the phosphorylation of beta -catenin is reduced, it dissociates from the Axin complex, and beta -catenin is no longer degraded, resulting in its accumulation in the cytoplasm. Stabilized beta -catenin is translocated into the nucleus, where it binds to transcriptional factors such as the Tcf/lymphoid enhancer binding factor and thereby stimulates the transcription of Wnt target genes (1, 12). Thus, the Wnt signal stabilizes beta -catenin, thereby regulating the expression of various genes. However, the precise molecular mechanism by which the signal is transmitted to stabilize beta -catenin after Wnt binds to the Frizzled/LRP co-receptor is unknown.

Dvl is a cytoplasmic phosphoprotein that acts downstream of Frizzled and is a key protein mediating the Wnt signal (1-3). Three Dvl genes, Dvl-1, -2, and -3, have been isolated in mammals. Dvl homologs are conserved in Drosophila (Dishevelled, abbreviated Dsh) and Xenopus (Xenopus dishevelled, abbreviated Xdsh). All Dvl and Dsh family members contain the following three highly conserved domains: an N-terminal DIX domain; a central PDZ domain; and a DEP domain. Expression of Dvl in cells induces the accumulation of beta -catenin and the activation of Tcf (13, 14). Although it is not known at present whether Dvl binds directly to the Frizzled/LRP co-receptor or whether intermediary proteins are involved in the signal transmission between Frizzled and Dvl, Dvl appears to bind to Axin and inhibit GSK-3beta -dependent phosphorylation of beta -catenin, APC, and Axin (13-17). Furthermore, Dvl has been shown to bind to CKIepsilon and Frat-1 (18-21).

CKI comprises a large family of related gene products, namely alpha , beta , gamma , delta , and epsilon  (22). They all share at least 50% amino acid identity within the protein kinase catalytic domain. Different CKI family members generally show different tissue distributions and subcellular localization and have distinct roles (22, 23). As for regulation of the Wnt signaling pathway by CKI, seemingly conflicting findings have been reported. CKIepsilon forms a complex with Dvl and Axin, and CKIepsilon and Dvl-1 activate Tcf-4 cooperatively in mammalian cells (18-20,24). Overexpression of CKIepsilon in Xenopus embryos induces expression of siamois, a Wnt-response gene, and axis duplication (18, 19, 25). These results suggest that CKIepsilon regulates the Wnt signaling pathway positively. It has also been shown that CKIalpha primes phosphorylation of beta -catenin by GSK-3beta and induces the degradation of beta -catenin (26-28). Furthermore, disruption of CKIalpha stabilizes beta -catenin in mammalian HEK-293T cells and Drosophila Schneider cells (26, 27). These results suggest that CKIalpha functions as a negative regulator of the Wnt signaling pathway. One possible explanation for these different findings may be that distinct CKI isoforms have opposite roles in the Wnt signaling pathway.

Frat is yet another Dvl-binding protein. Frat was originally isolated on the basis of its tumor-promoting activity in human lymphocytes (29) and shares three conserved regions with Xenopus GBP, which binds to GSK-3 and activates the Wnt signaling pathway (30). The Frat family consists of three members: Frat-1, -2, and -3. It has been shown that different sites of Frat-1 interact with GSK-3 and Dvl-1 and that Wnt-1 disintegrates the complex formation of Frat-1, Dvl-1, and Axin, resulting in the activation of the Wnt signaling pathway (21). However, how these three proteins, Dvl-1, CKIepsilon , and Frat-1, functionally interact with one another to regulate the Wnt signaling pathway is not known.

Here we demonstrate that CKIepsilon enhances the binding of Dvl-1 and Frat-1 and that the interaction of Dvl-1 with Frat-1 is important for the activation of the Wnt signaling pathway. Furthermore, we demonstrate that depletion of CKIepsilon , but not CKIalpha , is essential for Wnt-3a-induced beta -catenin accumulation by the use of ds RNAi.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Materials and Chemicals-- pRSETB/human CKIepsilon (hCKIepsilon ) and pRSETB/hCKIepsilon kinase negative (KN), pTOPFLASH and pFOPFLASH, pcDNA3/hFrat-1, pcDNA3-FLAG/rAxin, pPGK/Wnt-3a, and HeLa S3 cells were provided by Drs. D. M. Virshup (University of Utah, Salt Lake City, UT), H. Clevers (University Hospital, Utrecht, The Netherlands), S. Tanaka (Kyusyu University, Fukuoka, Japan), K. Miyazono (Tokyo University, Tokyo, Japan), S. Takada (Kyoto University, Kyoto, Japan), and K. Matsumoto (Nagoya University, Nagoya, Japan), respectively. Recombinant baculoviruses expressing GST-fused Frat-1 (GST-Frat-1) (wild type) were generated by Dr. Y. Matsuura (Research Institute of Microbial Diseases, Osaka University, Suita, Japan). GST-Frat-1 was purified from Spodoptera frugiperda 9 cells. MBP and His6 fusion proteins were purified from Escherichia coli according to the supplier's instructions. L cells stably expressing hDvl-1 or hDvl-1-(Delta 228-250) were generated by selection with G418 as described (31). Wnt-3a-conditioned medium was produced as described previously (32). COS and L, HeLa S3, and HEK-293 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% calf serum and 10% fetal bovine serum, respectively. The anti-Myc antibody was prepared from 9E10 cells. The anti-GST, anti-MBP, and anti-Dvl antibodies were prepared in rabbits by immunization with recombinant GST, MBP, and Dvl-1-(1-140) proteins, respectively. The anti-FLAG and polyclonal anti-beta -catenin antibodies, the monoclonal anti-beta -catenin, anti-GSK-3beta , the anti-CKIepsilon antibodies, and the anti-CKIalpha antibody were purchased from Sigma, Transduction Laboratories (Lexington, KY), and Santa Cruz Biotechnology, respectively. Cy5-labeled anti-mouse IgG was obtained from Amersham Biosciences. The Alexa 546-labeled anti-rabbit or mouse IgG and the anti-GFP antibody were from Molecular Probes, Inc. (Eugene, OR). Other materials were from commercial sources.

Plasmid Construction-- pCGN/hDvl-1 (wild type), pCGN/hDvl-1-(1-519), pCGN/hDvl-1-(140-670), pMAL-c2/hDvl-1 (wild type), pSP64T-Myc/mDvl-1 (wild type), pEF-BOS-HA/hTcf-4E, pCGN/hCKIepsilon , pCGN/hCKIepsilon (KN), pEGFP-C1/hCKIepsilon , pEGFP-C1/hCKIepsilon (KN), and pSP64T-Myc/hCKIepsilon were constructed as described (5, 7, 14, 20). Standard recombinant DNA techniques were used to construct the following plasmids: pCGN/hFrat-1 (wild type); pCGN/hFrat-1-(1-185); pEF-BOS-Myc/hFrat-1; pVIKS/hFrat-1; pEGFP-C1/hFrat-1; pEF-BOS-HA/hDvl-1-(1-250); pCGN/hDvl-1-(Delta 141-227); pCGN/hDvl-1-(Delta 228-250); pCGN/hDvl-1-(Delta 251-336); pCGN/hDvl-1-(337-670); pEF-BOS-Myc/hDvl-1 (wild type); pEF-BOS-Myc/hDvl-1-(Delta 228-250); pMAL-c2/hDvl-1-(Delta 228-250); and pSP64T-Myc/hDvl-1-(Delta 228-250). In these plasmids, some plasmid constructions were done by digesting the original plasmids with restriction enzymes and inserting the fragments into the vectors. Other constructions were done by inserting the fragments generated by PCR into the vectors. The entire PCR products were sequenced, and the structures of all plasmids were confirmed by restriction enzyme analysis.

Complex Formation of Frat-1 with Dvl-1-- To determine whether Frat-1 forms a complex with Dvl-1 in intact cells, COS cells (60-mm-diameter dishes) transfected with pCGN-, pEGFP-, pcDNA3-FLAG-, or pEF-BOS-Myc-derived plasmids were lysed in 200 µl of the lysis buffer (20 mM Tris/HCl, pH 7.5, 137 mM NaCl, 1% Nonidet P-40, 10% glycerol, 25 mM beta -glycerophosphate, 5 mM sodium orthovanadate, 5 mM NaF, 5 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, and 20 µg/ml aprotinin), and the lysates were centrifuged at 15, 000 × g for 10 min at 4 °C. The supernatant (20 µg of protein) was probed with the anti-Myc, anti-HA, anti-GFP, anti-FLAG, or anti-GSK-3beta antibody to detect the protein expression levels. The same lysates (200 µg of protein) were immunoprecipitated with the anti-Myc antibody, and then the immunoprecipitates were probed with the same antibodies.

To examine the direct interaction of Frat-1 with phosphorylated Dvl-1 using purified proteins in vitro, MBP-Dvl-1 and MBP-Dvl-1-(Delta 228-250) (2.6 µg of protein) immobilized on amylose resin were incubated with or without His6-CKIepsilon (0.3 µg of protein) in 15 µl of kinase reaction mixture (50 mM Tris/HCl, pH 7.5, 10 mM MgCl2, 1 mM dithiothreitol, and 50 µM ATP) for 30 min at 30 °C. Phosphorylated Dvl-1 (20 pmol) was incubated with GST-Frat-1 (wild type) (30 pmol) in 100 µl of reaction mixture (20 mM Tris/HCl, pH 7.5, and 1 mM dithiothreitol) for 1 h at 4 °C. After MBP fusion proteins were precipitated by centrifugation, the precipitates were probed with the anti-GST antibody.

Immunocytochemistry-- L cells grown on coverslips were fixed for 20 min in PBS containing 4% paraformaldehyde. The cells were washed with PBS three times and then permeabilized with PBS containing 0.1% Triton X-100 and 2 mg/ml bovine serum albumin for 2 h. The cells were washed and incubated with the anti-HA and polyclonal anti-beta -catenin antibodies for 1 h. After being washed with PBS, they were further incubated for 1 h with Alexa 546-labeled mouse IgG, Cy5-labeled anti-mouse IgG, or Alexa 546-labeled anti-rabbit IgG. The coverslips were washed with PBS, mounted on glass slides, and viewed with a confocal laser-scanning microscope (LSM510, Carl-Zeiss, Jena, Germany). All procedures were carried out at room temperature.

Luciferase Assay-- To observe Tcf-4 activity, the indicated amounts of pCGN/Dvl-1, pCGN/hDvl-1-(Delta 228-250), pCGN/hFrat-1, pCGN/hFrat-1-(1-185), pCGN/hCKIepsilon , pCGN/hCKIepsilon (KN), or pPGK/Wnt-3a were transfected into HEK-293 cells (35-mm-diameter dishes) with pTOPFLASH (0.5 µg), pEF-BOS-HA/hTcf-4E (0.1 µg), and pME18S/lacZ (0.5 µg) (31, 33). Forty-six hours after the transfection, the cells were lysed, and the luciferase activity was measured using a PicaGene (Toyo B-NET Co., Ltd., Tokyo, Japan) and a lumiphotometer TD4000 (Futaba Medical, Tokyo, Japan). To standardize the transfection efficiency, pME18S/lacZ carrying the SRalpha promotor linked to the coding sequence of the beta -galactosidase gene was used as an internal control.

Xenopus Injections and Analyses of Phenotypes-- Myc-tagged Dvl-1, Myc-Dvl-1-(Delta 228-250), and HA-CKIepsilon cDNA were subcloned into pSP64T (34). Sense mRNA was obtained by in vitro transcription of linearized templates using the SP6-mMESSAGE mMACHINE kit (Ambion, Austin, TX). Fertilized eggs were dejellied using 4.5% L-cysteine hydrochloride monohydrate, and mRNAs were injected into ventral blastomeres at the four-cell stage. After injection, embryos were cultured for 3 days (stage 40-41).

RNA Interference-- Two RNA interferences specific to human CKIalpha (sense) and human CKIepsilon (sense), 5'-CCAGGCAUCCCCAGUUGCUTT-3' and 5'-UGGCCAAGAAGUACCGGGATT-3', respectively, were synthesized, and double-stranded RNA oligonucleotides were annealed in vitro before transfection. Transfection was done with Oligofectamine (Invitrogen) on HeLa S3 cells (35-mm-diameter dishes). Ninety-six hours after the transfection, the cells were treated with Wnt-3a-conditioned medium or control medium for 1 h. Then the cells were washed in cold PBS and homogenized at 4 °C in 200 µl of PBS containing 25 mM beta -glycerophosphate, 5 mM sodium orthovanadate, 5 mM NaF, 20 µg/ml leupeptin, 20 µg/ml aprotinin, and 5 mM phenylmethylsulfonyl fluoride. The homogenates were centrifuged at 100,000 × g for 30 min at 4 °C, and the supernatant was used as the cytosolic extract. Aliquots (10 µl) of the cytosolic extract were probed with the anti-Dvl, anti-CKIepsilon , anti-CKIalpha , anti-GSK-3beta , and monoclonal anti-beta -catenin antibodies.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Enhancement of the Interaction of Dvl-1 with Frat-1 by CKIepsilon -- The constructions of Frat-1 and Dvl-1 used in this study are shown in Fig. 1. Although we showed in a previous report that CKIepsilon forms a complex with and phosphorylates Dvl-1 (20), the physiological significance of the phosphorylation of Dvl-1 by CKIepsilon remained unclear. Because the phosphorylation of Dvl-2 by CKII enhances the interaction of Dvl-2 with beta -arrestin1 (35), we tried to identify protein(s) that associate with Dvl-1 phosphorylated by CKIepsilon . It has been shown that CKIepsilon stimulates the binding of GBP to Dvl in Xenopus extracts in vitro (24). Human Frat-1 contains three regions that are well conserved with the corresponding regions in Xenopus GBP (30), and it binds to Dvl-1 (21). Therefore, we examined whether CKIepsilon enhances the binding of Dvl-1 to Frat-1. HA-Dvl-1 and Myc-Frat-1 were expressed with GFP-CKIepsilon or its kinase negative form, GFP-CKIepsilon KN, in COS cells. GFP-CKIepsilon , but not GFP-CKIepsilon KN, induced a mobility shift of HA-Dvl-1 on an SDS-PAGE gel, reflecting the phosphorylation of Dvl-1 (20) (Fig. 2A, lanes 2-4). When the lysates expressing HA-Dvl-1 and Myc-Frat-1 were immunoprecipitated with the anti-Myc antibody, a small amount of HA-Dvl-1 was detected in the Myc-Frat-1 immune complexes (Fig. 2A, lane 14). Expression of GFP-CKIepsilon , but not GFP-CKIepsilon KN, greatly enhanced the formation of the complex between HA-Dvl-1 and Myc-Frat-1 (Fig. 2A, lanes 15 and 16).


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Fig. 1.   Schematic representation of deletion mutants of human Frat-1 and Dvl-1 used in this study.


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Fig. 2.   Enhancement of the interaction of Dvl-1 with Frat-1 by CKIepsilon . A, interaction of the deletion mutants of Dvl-1 with Frat-1 in intact cells. Lysates (20 µg of protein) of COS cells expressing the indicated proteins were probed with the anti-HA, anti-GFP, or anti-Myc antibody (lanes 1-13 and 26-38). The same lysates (200 µg of protein) were immunoprecipitated with the anti-Myc antibody, and then the immunoprecipitates were probed with the same antibodies (lanes 14-25 and 39-50). WT, wild type; KN, kinase negative form; IP, immunoprecipitation; Ab, antibody. B, sequence alignment of Dvl. The region of residues 228-250 of human Dvl-1 is aligned to mouse Dvl-1, Xenopus dsh, and Drosophila Dsh. C, complex formation of GSK-3beta , Dvl-1, Frat-1, and CKIepsilon in intact cells. Lysates (20 µg of protein) of COS cells expressing the indicated proteins were probed with the anti-Myc, anti-GFP, anti-GSK-3beta , or anti-HA antibody (lanes 1-5). The same lysates (200 µg of protein) were immunoprecipitated with the anti-Myc antibody, and then the immunoprecipitates were probed with the same antibodies (lanes 6-9). D, direct interaction of Frat-1 with phosphorylated Dvl-1. After MBP-Dvl-1 (lanes 1-3) and MBP-Dvl-1-(Delta 228-250) (lanes 4-6) immobilized on amylose resin were incubated with (lanes 2, 3, 5, and 6) or without (lanes 1 and 4) His6-CKIepsilon in the presence (lanes 1, 2, 4, and 5) or absence (lanes 3 and 6) of ATP, the samples were further incubated with GST-Frat-1. MBP fusion proteins were precipitated by centrifugation, and the precipitates were probed with the anti-MBP and anti-GST antibodies. MBP-Dvl-1 (lane 7), MBP-Dvl-1-(Delta 228-250) (lane 8), and GST-Frat-1 (lane 9) (1 µg of protein) were stained with Coomassie Brilliant Blue. The arrowheads indicate MBP-Dvl-1 (WT), MBP-Dvl-1-(Delta 228-250), and GST-Frat-1, and the other bands are their degradation products. The results shown are representative of four independent experiments.

To clarify which region of Dvl-1 is necessary for the interaction with Frat-1, various deletion mutants of HA-Dvl-1 were expressed with Myc-Frat-1 and GFP-CKIepsilon (Fig. 2A, lanes 5-13 and 27-38). Among these deletion mutants, HA-Dvl-1-(1-250) and HA-Dvl-1-(337-670) did not form a complex with Myc-Frat-1 irrespective of the expression of GFP-CKIepsilon (Fig. 2A, lanes 17-19 and 48-50), but HA-Dvl-1-(1-519) and HA-Dvl-1-(140-670) associated with Myc-Frat-1 in a manner dependent on their phosphorylation by GFP-CKIepsilon (Fig. 2A, lanes 20-25). Therefore, the amino acid region 141-336 of Dvl-1 may be important for the interaction with Frat-1. These results are consistent with the previous observations that mouse Dvl-1-(201-375) interacts with Frat-1 (21). Furthermore, we deleted three amino acid regions, 141-227, 228-250, and 251-336 (PDZ domain), from HA-Dvl-1. HA-Dvl-1-(Delta 141-227) and HA-Dvl-1-(Delta 251-336) formed a complex with Myc-Frat-1 in a manner dependent on CKIepsilon , but HA-Dvl-1-(Delta 228-250) did not (Fig. 2A, lanes 27-35 and 39-47). These results clearly indicate that CKIepsilon -dependent phosphorylation of Dvl-1 enhances the interaction of Dvl-1 with Frat-1 and that amino acid region 228-250 of Dvl-1 is necessary for the interaction with Frat-1. As Dvl-1-(1-250) does not contain the CKIepsilon -binding region (20), the reason for the failure of this Dvl-1 mutant to bind to Frat-1 might be that CKIepsilon does not phosphorylate this mutant. The amino acid region 228-250 of Dvl-1 is evolutionarily conserved (Fig. 2B), suggesting that this region is functionally important.

The C-terminal region of GBP binds to GSK-3 (30), and Frat-1-(186-279) indeed interacted with GSK-3beta in intact cells (data not shown). When coexpressed with GFP-CKIepsilon , significant amounts of HA-Frat-1 were observed in the Myc-Dvl-1 immune complex in COS cells (Fig. 2C, lanes 2, 3, 6, and 7). Furthermore, endogenous GSK-3beta was also observed in the Myc-Dvl-1 immune complex when GFP-CKIepsilon was coexpressed (Fig. 2C, lane 6). GFP-CKIepsilon enhanced the formation of the complex of Myc-Dvl-1 with the N-terminal region of Frat-1 (HA-Frat-1-(1-185)), but endogenous GSK-3beta was not observed in this complex (Fig. 2C, lanes 4, 5, 8, and 9). These results suggest that CKIepsilon enhances the formation of the complex of GSK-3beta with Dvl-1 through Frat-1.

GST-Frat-1 bound to MBP-fused Dvl-1 (MBP-Dvl-1) when they were incubated with His6-CKIepsilon and ATP, although they did not interact with each other in the absence of His6-CKIepsilon (Fig. 2D, lanes 1 and 2). Incubation with His6-CKIepsilon in the absence of ATP resulted in a detectable interaction of MBP-Dvl-1 with GST-Frat-1 (Fig. 2D, lane 3). Therefore, the binding of CKIepsilon to Dvl-1 may induce a conformational change of Dvl-1, resulting in the interaction of Dvl-1 with Frat-1. GST-Frat-1 did not bind to MBP-Dvl-1-(Delta 228-250) irrespective of incubation with His6-CKIepsilon and ATP (Fig. 2D, lanes 4-6). Previously we showed that phosphorylation of GST-Dvl-1 by CKIepsilon in vitro exhibits a mobility shift (20). Although we do not know the reasons for the undetectable mobility shift of MBP-Dvl-1 and MBP-Dvl-1-(Delta 228-250), we confirmed that CKIepsilon indeed phosphorylates them by autoradiography (data not shown). CKIepsilon did not phosphorylate MBP-Dvl-1-(228-250) (data not shown). Taken together, Frat-1 prefers Dvl-1 phosphorylated by CKIepsilon , and the amino acid region 228-250 of Dvl-1 is necessary for the direct binding of Dvl-1 to Frat-1.

Subcellular Localization of Dvl-1 and Frat-1-- The importance of the amino acid region 228-250 of Dvl-1 for the binding to Frat-1 was confirmed by the immunocytochemical assay. When GFP-Frat-1 was expressed alone in L cells, Frat-1 was distributed throughout the cytoplasm (Fig. 3A). HA-Dvl-1 was observed as small particles, consistent with previous reports (13, 14). Coexpression with HA-Dvl-1 changed the localization of GFP-Frat-1 dramatically, and these two proteins were found to colocalize (Fig. 3, B-D). HA-Dvl-1-(Delta 228-250) was detected as small particles, like HA-Dvl-1, indicating that the amino acid region 228-250 of Dvl-1 is not essential for the localization of Dvl-1. However, HA-Dvl-1-(Delta 228-250) did not affect the distribution of GFP-Frat-1 (Fig. 3, E-G). These results support the findings that the region 228-250 of Dvl-1 is important for its binding to Frat-1 in intact cells.


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Fig. 3.   Subcellular localization of Dvl-1 and Frat-1. GFP-Frat-1 alone (A), HA-Dvl-1 and GFP-Frat-1 (B-D), or HA-Dvl-1-(Delta 228-250) and GFP-Frat-1 (E-G) were expressed in wild-type L cells. Some of the cell cultures were directly viewed with a confocal laser-scanning microscope to detect GFP-Frat-1 (A, C, and F), and the others were stained with the anti-HA antibody to detect HA-Dvl-1 (B) or HA-Dvl-1-(Delta 228-250) (E). Merged images of B and C, and of E and F are shown in D and G, respectively. GFP-Frat-1 and Alexa 546-labeled HA-Dvl or HA-Dvl-1-(Delta 228-250) produced no cross staining. The results shown are representative of three independent experiments.

Synergistic Effects by Dvl-1 and Frat-1 on beta -Catenin Accumulation and Tcf-4 Activation-- Transient overexpression of Dvl-1 and Dvl-1-(Delta 228-250) in L cells induced the nuclear accumulation of beta -catenin (Fig. 4, A-D, arrows), indicating that the amino acid region 228-250 of Dvl-1 is not essential for the ability of Dvl-1 to activate the Wnt signal canonical pathway. However, accumulation of beta -catenin was not observed in L cells stably expressing Dvl-1 (L/Dvl cells) or Dvl-1-(Delta 228-250) (L/Dvl-(Delta 228-250) cells) (Fig. 4, F, H, J, and L, the cells not indicated by arrows). This finding suggests that a low expression level of Dvl-1 or Dvl-1-(Delta 228-250) in L cells is not sufficient for the stabilization of beta -catenin. Although expression of Frat-1 in wild-type L cells did not induce the accumulation of beta -catenin (data not shown), its expression in L/Dvl cells increased the level of beta -catenin in the nucleus (Fig. 4, E and F, arrows). However, expression of Frat-1 in L/Dvl-(Delta 228-250) did not induce the nuclear accumulation of beta -catenin (Fig. 4, G and H, arrows). Furthermore, expression of CKIepsilon in L/Dvl cells, but not in L/Dvl-(Delta 228-250) cells, resulted in the accumulation of beta -catenin (Fig. 4, I-L, arrows). Taken together with our previous observations (20), these results suggest that not only CKIepsilon but also Frat-1 act synergistically with Dvl-1 to induce the accumulation of beta -catenin and that the amino acid region 228-250 of Dvl-1 is important for the functional interaction of Dvl-1 with Frat-1 or CKIepsilon .


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Fig. 4.   Synergistic effects of Dvl-1 and Frat-1 on the accumulation of beta -catenin. HA-Dvl-1 (A and B) or HA-Dvl-1-(Delta 228-250) (C and D) was transiently expressed in wild-type L cells (L/WT). GFP-Frat-1 was transiently expressed in L/Dvl cells (E and F) or L/Dvl-(Delta 228-250) cells (G and H). GFP-CKIepsilon was transiently expressed in L/Dvl cells (I and J) or L/Dvl-(Delta 228-250) cells (K and L). Some of the cells were directly viewed with a confocal laser-scanning microscope to detect GFP-Frat-1 (E and G) and GFP-CKIepsilon (I and K), and others were stained with the anti-HA antibody to detect HA-Dvl-1 and HA-Dvl-1-(Delta 228-250) (A and C) or with the polyclonal anti-beta -catenin antibody to observe endogenous beta -catenin (B, D, F, H, J, and L). Arrows indicate L/WT cells expressing HA-Dvl-1 (A and B) or HA-Dvl-1-(Delta 228-250) (C and D), L/Dvl cells expressing GFP-Frat-1 (E and F) or GFP-CKIepsilon (I and J), or L/Dvl-(Delta 228-250) cells expressing GFP-Frat-1 (G and H) or GFP-CKIepsilon (K and L). The results shown are representative of three independent experiments.

We also examined the effects of the combination of Dvl-1, Frat-1, and CKIepsilon on the activation of Tcf-4 by the use of Top-fos-Luc as a reporter gene (33). Expression of either HA-Dvl-1 or HA-Dvl-1-(Delta 228-250) alone in HEK-293 cells activated Tcf-4 in a dose-dependent manner, although HA-Dvl-1-(Delta 228-250) was slightly less active than HA-Dvl-1 (Fig. 5A). Frat-1 alone increased the activity of Tcf-4 slightly (Fig. 5B). Dvl-1 promoted the ability of Frat-1 to stimulate Tcf-4, whereas Dvl-1-(Delta 228-250) did not affect the ability of Frat-1 to stimulate Tcf-4 (Fig. 5B). CKIepsilon alone increased the activity of Tcf-4 only slightly (Fig. 5C). Although Dvl-1 greatly promoted the ability of CKIepsilon to stimulate Tcf-4, Dvl-1-(Delta 228-250) did not act synergistically with CKIepsilon (Fig. 5C). Dvl-1, Frat-1, or CKIepsilon did not activate Tcf-4 for FOP-fos-Luc, in which the Tcf-4-binding elements are mutated (data not shown). These results suggest that Dvl-1 acts synergistically with Frat-1 or CKIepsilon to activate Tcf-4 and that the amino acid region 228-250 of Dvl-1 is necessary for the synergistic activation of Tcf-4 by Dvl-1 and Frat-1 or CKIepsilon .


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Fig. 5.   Requirement for amino acid region 228-250 of Dvl-1 for the synergistic activation of Tcf-4 by Dvl-1 and Frat-1 or Dvl-1 and CKIepsilon . A, the indicated amounts of pCGN/hDvl-1 or pCGN/hDvl-1-(Delta 228-250) were transfected into HEK-293 cells. , pCGN/hDvl-1; open circle , pCGN/hDvl-1-(Delta 228-250). B, the indicated amounts of pCGN/hFrat-1 were transfected into HEK-293 cells with pCGN/hDvl-1 (0.2 µg) or pCGN/hDvl-1-(Delta 228-250) (0.2 µg). open circle , pCGN/hFrat-1 alone; black-square, pCGN/hFrat-1 and pCGN/hDvl-1; , pCGN/hFrat-1 and pCGN/hDvl-1-(Delta 228-250). C, the indicated amounts of pCGN/hCKIepsilon were transfected into HEK-293 cells with pCGN/hDvl-1 (0.2 µg) or pCGN/hDvl-1-(Delta 228-250) (0.2 µg). open circle , pCGN/hCKIepsilon alone; black-triangle, pCGN/hCKIepsilon and pCGN/hDvl-1; triangle , pCGN/hCKIepsilon and pCGN/hDvl-1-(Delta 228-250). The luciferase activities were assayed and expressed as fold increase compared with the level in cells transfected with TOP-fos-Luc and pEF-BOS-HA/hTcf-4E alone.

To confirm further that the amino acid region 228-250 of Dvl-1 is important in the synergistic effects of Dvl-1 and CKIepsilon , they were expressed in Xenopus embryos. Ventral injection of a high dose (1 ng) of Dvl-1 or Dvl-1-(Delta 228-250) mRNA into embryos resulted in dorsalization of the phenotype, causing effects such as axis duplication, with a similar efficiency (Fig. 6, A and B), but ventral injection of embryos with a low dose (50 pg) of Dvl-1 or Dvl-1-(Delta 228-250) mRNA did not cause significant abnormalities (Fig. 6B). Coinjection of low doses of Dvl-1 and CKIepsilon mRNA markedly induced axis duplication (Fig. 6, A and B). However, coinjection of low doses of Dvl-1-(Delta 228-250) and CKIepsilon mRNA did not result in significant axis duplication (Fig. 6, A and B). These results demonstrate that the amino acid region 228-250 of Dvl-1 is necessary for the synergistic effects of CKIepsilon and Dvl-1 on axis formation in Xenopus embryos, consistent with the results observed in mammalian cells.


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Fig. 6.   Requirement of amino acid region 228-250 of Dvl-1 for the synergistic effects of Dvl-1 and CKIepsilon on axis duplication in Xenopus embryos. A, embryos were injected ventrally with the mRNAs of CKIepsilon and Dvl-1 or Dvl-1-(Delta 228-250). a, Dvl-1 mRNA (1 ng); b, Dvl-1-(Delta 228-250) mRNA (1 ng); c, CKIepsilon mRNA (50 pg); d, CKIepsilon mRNA (50 pg) and Dvl-1 mRNA (50 pg); e, CKIepsilon mRNA (50 pg) and Dvl-1-(Delta 228-250) mRNA (50 pg). WT, wild type; Delta 228-250, Dvl-1-(Delta 228-250). B, the results in A were expressed as the percentage of secondary axis formation. Black bars indicate complete axis duplication, including eyes and cement glands. White bars indicate incomplete axis duplication characterized by a lack of head structures but with a distinct branched axis.

Functional Relationships among Dvl-1, Frat-1, and CKIepsilon -- In Figs. 4-6 we found that Dvl-1 and Frat-1 or Dvl-1 and CKIepsilon activate the Wnt signaling pathway synergistically. CKIepsilon also enhanced the ability of Frat-1 to activate Tcf-4 in HEK-293 cells (Fig. 7A, lanes 3, 5, and 7). Therefore, we next examined the functional relationships among these three proteins, Dvl-1, Frat-1, and CKIepsilon . Frat-1-(1-185) inhibited the Dvl-1-and CKIepsilon -dependent activation of Tcf-4, suggesting that Frat-1-(1-185) functions as a dominant negative form (Fig. 7A, lanes 8 and 9). Although CKIepsilon enhanced the synergistic effects of Dvl-1 and Frat-1 on the activation of Tcf-4, CKIepsilon KN suppressed them (Fig. 7A, lanes 10, 11, and 12).


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Fig. 7.   Functional relationships among Dvl-1, Frat-1, and CKIepsilon . A, dominant negative effects of Frat-1-(1-185) and CKIepsilon KN on the activation of Tcf-4. HEK-293 cells were transfected with pCGN/hDvl-1, pCGN/hFrat-1, pCGN/hFrat-1-(1-185), pCGN/hCKIepsilon , or pCGN/hCKIepsilon (KN) (0.2 µg each) as indicated. The luciferase activities were assayed and expressed as fold increase compared with the level in cells transfected with pTOPFLASH and pEF-BOS-HA/hTcf-4E alone (lane 1). B, inhibition of Dvl-1- and Wnt-3a-dependent Tcf-4 activation by Frat-1-(1-185). HEK-293 cells were transfected with pCGN/hDvl-1, pPGK/Wnt-3a, pCGN/hFrat-1, or pCGN/hFrat-1-(1-185) (0.2 µg each) as indicated. The luciferase activities were assayed and expressed as fold increase compared with the level observed in cells transfected with TOP-fos-Luc and pEF-BOS-HA/hTcf-4E alone (lane 1). The results represent the mean ± S.E. of four independent experiments.

Wnt-3a and Dvl-1 synergistically activated Tcf-4 (Fig. 7B, lanes 2, 3, and 6), and Frat-1 enhanced the synergistic effect of Wnt-3a and Dvl-1, and Frat-1-(1-185) inhibited it (Fig. 7B, lanes 6-8). These results support the idea that the binding of Dvl-1 and Frat-1 is important for the efficient activation of the Wnt signaling pathway and that CKIepsilon modulates their binding.

Inhibition of the Complex Formation between Dvl-1 and Axin by Frat-1-- We examined whether the binding of Dvl-1 and Frat-1 affects the Axin complex in the presence of CKIepsilon . Myc-Dvl-1 and Myc-Dvl-1-(Delta 228-250) formed a complex with FLAG-rAxin with similar efficiencies (Fig. 8, lanes 12 and 13). When HA-Frat-1 was further expressed, the amount of FLAG-rAxin immunoprecipitated with Myc-Dvl-1 was reduced, whereas that with Myc-Dvl-1-(Delta 228-250) was unaffected (Fig. 8, lanes 10 and 11). The level of GSK-3beta in the immune complexes seemed unchanged (Fig. 8, lanes 10 and 11). These results demonstrate that CKIepsilon -dependent binding of Dvl-1 and Frat-1 inhibits the complex formation of Dvl-1 with Axin and suggest that it promotes the disintegration of the beta -catenin destruction complex.


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Fig. 8.   Inhibition of the complex formation between Dvl-1 and Axin by Frat-1. Lysates (20 µg of protein) of COS cells expressing the indicated proteins were probed with the anti-FLAG, anti-Myc, anti-GFP, anti-GSK-3beta , or anti-HA antibody (lanes 1-7). The same lysates (200 µg of protein) were immunoprecipitated with the anti-Myc antibody, and then the immunoprecipitates were probed with the same antibodies (lanes 8-13). The results shown are representative of three independent experiments.

Inhibition of the Wnt Signaling Pathway by Depletion of CKIepsilon Expression-- It has been shown that CKIalpha is essential for beta -catenin degradation in mammalian cells (26). Therefore, we depleted the endogenous CKIepsilon in HeLa S3 cells via ds RNAi (36) to ask whether CKIepsilon is involved in the Wnt-dependent accumulation of beta -catenin. A ds RNAi oligo for CKIepsilon reduced the protein level of CKIepsilon but not that of CKIalpha , and, conversely, a ds RNAi oligo for CKIalpha reduced the protein level of CKIalpha but not that of CKIepsilon (Fig. 9A, third and fourth panels from the top). Single-stranded sense oligos for CKIalpha and CKIepsilon did not affect the protein levels of CKIalpha or CKIepsilon (data not shown). These oligos did not alter the protein level of GSK-3beta (Fig. 9A, bottom panel).


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Fig. 9.   Inhibition of the Wnt signaling pathway by depletion of CKIepsilon expression. A, inhibition of Wnt-3a-induced accumulation of beta -catenin by depletion of CKIepsilon expression. HeLa S3 cells were transfected with a ds RNAi oligo specific for human CKIalpha (lanes 2 and 5) or a ds RNAi oligo specific for human CKIepsilon (lanes 3 and 6). Untransfected cells were used as a control (lanes 1 and 4). The cells were treated with Wnt-3a-conditioned medium (+) (lanes 4-6) or control medium (-) (lanes 1-3) for 1 h. Aliquots (10 µl) of the cytosolic extract were probed with the anti-Dvl-1, anti-CKIepsilon , anti-CKIalpha , anti-GSK-3beta , or monoclonal anti-beta -catenin antibody. alpha , CKIalpha ds RNAi; epsilon , CKIepsilon ds RNAi. B, inhibition of Wnt-3a-dependent phosphorylation of Dvl by CKI-7. HeLa S3 cells were treated with Wnt-3a-conditioned medium (lanes 2 and 3) or control medium (lane 1) in the presence (lane 3) or absence (lanes 1 and 2) of 100 µM CKI-7. Aliquots (20 µl) of the cytosolic extract were probed with the anti-Dvl antibodies. C, interference of the interaction of Dvl-1 with Frat-1 by depletion of CKIepsilon expression. HeLa S3 cells were transfected with a single-stranded sense oligo (a-e), a ds RNAi oligo specific for human CKIepsilon (f-h), or a ds RNAi oligo specific for human CKIalpha (i-k), and 3 days later the cells were further transfected with pCGN/hDvl-1 (a), or pEGFP-C1/hFrat-1 (b), or pCGN/hDvl-1 and pEGFP-C1/hFrat-1 (c-k). GFP-Frat-1 was directly visualized with a confocal laser-scanning microscope (b, d, g, and j), and HA-Dvl-1 was stained with the anti-HA antibody (a, c, f, and i). Merged images of c and d, f and g, and i and j are shown in e, h, and k, respectively. The results shown are representative of three independent experiments.

A decrease of CKIepsilon , but not CKIalpha , inhibited the Wnt-3a-dependent mobility shift of endogenous Dvl (Fig. 9A, second panel from the top). The Wnt-3a-dependent mobility shift of Dvl-1 was inhibited by treatment with CKI-7, a CKI inhibitor (18, 37) (Fig. 9B), or with alkaline phosphatase (data not shown). Therefore, the slowly migrating Dvl band appears to be a phosphorylated form of Dvl. These results indicate that CKIepsilon is necessary for the Wnt-3a-dependent phosphorylation of Dvl-1.

When HA-Dvl-1 or GFP-Frat-1 was expressed alone in HeLa S3 cells, HA-Dvl-1 was observed as small particles, and GFP-Frat-1 was diffusely distributed in the cytoplasm (Fig. 9C, a and b) as well as in L cells (see Fig. 3). When HA-Dvl-1 and GFP-Frat-1 were coexpressed in HeLa S3 cells, the two proteins were colocalized with each other (Fig. 9C, c-e). However, HA-Dvl-1 did not change the subcellular distribution of GFP-Frat-1 in the cells when the expression of CKIepsilon was depleted via ds RNAi (Fig. 9C, f-h). Depletion of CKIalpha did not affect colocalization of HA-Dvl-1 and GFP-Frat-1 (Fig. 9C, i-k). These results are in agreement with the observations that CKIepsilon enhances the binding of Dvl-1 and Frat-1.

Furthermore, CKIepsilon ds RNAi did not affect the protein level of beta -catenin in the absence of Wnt-3a but did inhibit Wnt-3a-induced beta -catenin accumulation significantly (Fig. 9A, first panel). A decrease of CKIalpha led to an accumulation of beta -catenin in the absence of Wnt-3a, consistent with the previously reported observations (26), but did not affect Wnt-3a-induced beta -catenin accumulation (Fig. 9A, first panel). These results clearly show that CKIepsilon , but not CKIalpha , is essential for the Wnt-3a-induced accumulation of beta -catenin in HeLa S3 cells.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Complex Formation between Dvl-1 and Frat-1 and Its Enhancement by CKIepsilon -- In this study, we demonstrated that CKIepsilon enhances the interaction of Dvl-1 with Frat-1 in intact mammalian cells. The amino acid region 228-250 of Dvl-1 was necessary for the binding of Dvl-1 to Frat-1. This region is conserved evolutionarily, supporting the idea that it is important for the functions of Dvl. Although there is GBP, a Frat homolog, in Xenopus, no gene similar to Frat/GBP has been found in Drosophila. Therefore, a different protein may act on this region in Drosophila. Although multiple sites of Dvl-1 were phosphorylated by CKIepsilon (20), the amino acid region 228-250 was not phosphorylated. Because 17% of the 670 amino acids in human Dvl-1 are serines and threonines, it is difficult to precisely map the phosphorylation sites. Although we have not yet identified the sites of phosphorylation of Dvl-1 by CKIepsilon , phosphorylation may result in conformational changes of Dvl-1, rendering the region, including amino acids 228-250, able to associate with Frat-1. Mutations of each of the putative phosphorylation sites by CKII in Drosophila Dsh do not affect the ability of the mutant proteins to rescue dsh mutant animals (38). Therefore, studies of compound phosphorylation mutants may be required to identify the most important phosphorylation sites.

It has been shown that Wnt induces the phosphorylation of Dvl (39). Our results using ds RNAi for CKIepsilon have shown that CKIepsilon is required for the Wnt-3a-induced phosphorylation of Dvl-1 in HeLa S3 cells and that CKIepsilon is necessary for the interaction of Dvl-1 with Frat-1. Depletion of CKIalpha did not affect the Wnt-3a-induced phosphorylation of Dvl-1 and the interaction of Dvl-1 with Frat-1. Taken together, these findings suggest that, when Wnt binds to the Frizzled/LRP co-receptor, Dvl is phosphorylated by CKIepsilon but not by CKIalpha , resulting in enhancement of the binding of Dvl-1 and Frat-1.

Molecular Mechanism by Which Complex Formation between Dvl-1 and Frat-1 Activates the Wnt Signaling Pathway-- We demonstrated that Dvl-1-(Delta 228-250) does not act synergistically with either Frat-1 or CKIepsilon to activate the Wnt signaling pathway. Frat-1-(1-185) inhibited the synergistic activation of Tcf-4 by Dvl-1 and CKIepsilon or by Dvl-1 and Wnt-3a. Because Frat-1-(1-185) binds not to GSK-3beta but to Dvl-1, these results support the idea that the binding of Dvl-1 and Frat-1 induces the accumulation of beta -catenin and the activation of Tcf-4. The stability of beta -catenin is regulated in a destruction complex that includes beta -catenin, Axin, APC, GSK-3beta , Dvl-1, CKIalpha , and PP2A (4, 21, 26, 40, 41). It has been demonstrated that the expression of Wnt-1 in COS cells promotes the disintegration of this complex, resulting in the dissociation of Frat from the Dvl and Axin complex (21) and that the expression of CKIepsilon in HEK-293 cells decreases the association between PP2A and Axin (25). Therefore, the Wnt signal appears to destabilize the beta -catenin degradation complex. Consistent with these observations, we showed that the binding of Dvl-1 and Frat-1 in COS cells decreases the interaction of Dvl-1 with Axin.

How the Wnt signal stabilizes beta -catenin by causing the disintegration of its degradation complex remains unclear. CKIepsilon -dependent interaction of Dvl-1 with Frat-1 may cause a conformational change of the complex that phosphorylates beta -catenin. Dvl-1 forms a complex with GSK-3beta via Axin or Frat-1. The level of GSK-3beta associated with Dvl-1 remained the same in the presence or absence of Axin when Frat-1 was present in the complex. Therefore, it is likely that GSK-3beta maintains its association with Dvl-1 after the disintegration of the complex. Because Wnt reduces the phosphorylation of beta -catenin by GSK-3beta (26), one possibility is that a conformational change of the degradation complex leads to the dissociation of GSK-3beta from Axin and recruits it to Frat-1, thereby reducing the phosphorylation of beta -catenin by GSK-3beta .

Involvement of CKIepsilon as a Positive Regulator in the Wnt Signaling Pathway-- Several reports have shown that CKI binds to Dvl and stabilizes beta -catenin (18-20,42), whereas other reports have demonstrated that CKI phosphorylates beta -catenin and promotes its degradation (26-28). One means by which CKI could play dual positive and negative roles in the Wnt signaling pathway might be that some CKI isoforms perform positive functions while others perform negative functions. Indeed, it has been shown that depletion by ds RNAi of the expression of CKIalpha , but not of CKIepsilon , stabilizes the basal level of beta -catenin in HEK-293 cells (26). The expression of CKIepsilon , but not of CKIalpha , induces axis duplication in Xenopus embryos (19). However, arguing against these observations, it has shown that depletion of either CKIalpha or CKIepsilon stabilizes Armadillo in Drosophila cells (27) and that expression of either CKIalpha or CKIepsilon induces axis duplication in Xenopus embryos (42). Furthermore, it has also been demonstrated that Diversin recruits CKIepsilon to the beta -catenin destruction complex, thereby inducing the down-regulation of beta -catenin (43). Therefore, we performed a loss of function study using the ds RNAi methods in mammalian cells to clarify the roles of CKIepsilon and CKIalpha in the Wnt-dependent accumulation of beta -catenin.

Our results support the hypothesis of CKI-isoform-specific differences. Depletion of CKIepsilon expression by ds RNAi in HeLa S3 cells did not affect the basal level of beta -catenin but reduced Wnt-3a-induced accumulation of beta -catenin. Wnt-3a-dependent phosphorylation of Dvl-1 and the interaction of Dvl-1 with Frat-1 were reduced in the cells in which CKIepsilon was depleted. Furthermore, reduction of CKIalpha increased the basal level of beta -catenin but did not affect Wnt-3a-induced accumulation of beta -catenin. These results strongly suggest that CKIalpha and CKIepsilon have opposite actions on the Wnt signaling pathway in HeLa S3 cells. Of course, we can not rule out the possibility that the positive and negative roles of CKIepsilon in the Wnt signaling pathway may be dependent on the abundance of various substrates and interacting proteins. Further studies will be necessary to clarify the dual roles of CKIepsilon in the Wnt signaling pathway.

    ACKNOWLEDGEMENTS

We are grateful to Drs. D. M. Virshup, H. Clevers, S. Tanaka, K. Miyazono, S. Takada, Y. Matsuura, and K. Matsumoto for donating plasmids, viruses, and cells.

    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 (2001, 2002).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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

Published, JBC Papers in Press, January 28, 2003, DOI 10.1074/jbc.M213265200

    ABBREVIATIONS

The abbreviations used are: GSK-3beta , glycogen synthase kinase-3beta ; GBP, GSK-3-binding protein; APC, adenomatous polyposis coli gene product; LRP, low density lipoprotein-related protein; Tcf, T-cell factor; Dsh, Dishevelled; CKI, casein kinase I; HEK-293, human embryonic kidney; ds RNAi, double-stranded RNA interference; KN, kinase negative; GST, glutathione S-transferase; MBP, maltose-binding protein; PBS, phosphate-buffered saline; HA, hemagglutinin A; GFP, green fluorescent protein; PP2A, protein phosphatase 2A.

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