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

We demonstrate that Dvl-1, casein kinase Iε (CKIε), 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 CKIε. Coexpression of Dvl-1 and Frat-1 caused accumulation of β-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-(Δ228–250), which lacks the amino acid region 228–250 from Dvl-1, and Frat-1 did not. Dvl-1, but not Dvl-1-(Δ228–250), acted synergistically with CKIε to activate Tcf-4. Depletion of CKIε 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 CKIε reduced the Wnt-3a-induced accumulation of β-catenin, although it did not affect the basal level of β-catenin. These results indicate that CKIε-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.

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 ␤-catenin is controlled by the Wnt signal. In unstimulated cells, cytoplasmic ␤-catenin is destabilized by a multiprotein complex containing Axin (or its homolog Axil/conductin), GSK-3␤, 1 and APC (4 -8).
When Wnt binds to the Frizzled/LRP co-receptor at the cell surface, a cytoplasmic protein, Dvl, antagonizes GSK-3␤-dependent phosphorylation of ␤-catenin. Once the phosphorylation of ␤-catenin is reduced, it dissociates from the Axin complex, and ␤-catenin is no longer degraded, resulting in its accumulation in the cytoplasm. Stabilized ␤-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 ␤-catenin, thereby regulating the expression of various genes. However, the precise molecular mechanism by which the signal is transmitted to stabilize ␤-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)(2)(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 ␤-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-3␤-dependent phosphorylation of ␤-catenin, APC, and Axin (13)(14)(15)(16)(17). Furthermore, Dvl has been shown to bind to CKI⑀ and Frat-1 (18 -21).
CKI comprises a large family of related gene products, namely ␣, ␤, ␥, ␦, and ⑀ (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. CKI⑀ forms a complex with Dvl and Axin, and CKI⑀ and Dvl-1 activate Tcf-4 cooperatively in mammalian cells (18 -20,24). Overexpression of CKI⑀ in Xenopus embryos induces expression of siamois, a Wnt-response gene, and axis duplication (18,19,25). These results suggest that CKI⑀ regulates the Wnt signaling pathway positively. It has also been shown that CKI␣ primes phosphorylation of ␤-catenin by GSK-3␤ and induces the degradation of ␤-catenin (26 -28). Furthermore, disruption of CKI␣ stabilizes ␤-catenin in mammalian HEK-293T cells and Drosophila Schneider cells (26,27). These results suggest that CKI␣ functions as a negative regulator of the Wnt signaling pathway. One possible explanation for these different find-ings 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, CKI⑀, and Frat-1, functionally interact with one another to regulate the Wnt signaling pathway is not known.
Here we demonstrate that CKI⑀ 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 CKI⑀, but not CKI␣, is essential for Wnt-3a-induced ␤-catenin accumulation by the use of ds RNAi.
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-␤catenin antibodies for 1 h. After being washed with PBS, they were further incubated for 1 h with Alexa 546-labeled mouse IgG, Cy5labeled 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.
RNA Interference-Two RNA interferences specific to human CKI␣ (sense) and human CKI⑀ (sense), 5Ј-CCAGGCAUCCCCAGUUGCUT-T-3Ј and 5Ј-UGGCCAAGAAGUACCGGGATT-3Ј, 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 l of PBS containing 25 mM ␤-glycerophosphate, 5 mM sodium orthovanadate, 5 mM NaF, 20 g/ml leupeptin, 20 g/ml aprotinin, and 5 mM phenylmethylsulfonyl fluoride. The homogenates were centrifuged at 100,000 ϫ g for 30 min at 4°C, and the supernatant was used as the cytosolic extract. Aliquots (10 l) of the cytosolic extract were probed with the anti-Dvl, anti-CKI⑀, anti-CKI␣, anti-GSK-3␤, and monoclonal anti-␤-catenin antibodies.

Enhancement of the Interaction of Dvl-1 with Frat-1 by CKI⑀-
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 CKI⑀ forms a complex with and phosphorylates Dvl-1 (20), the physiological significance of the phosphorylation of Dvl-1 by CKI⑀ remained unclear. Because the phosphorylation of Dvl-2 by CKII enhances the interaction of Dvl-2 with ␤-arres-tin1 (35), we tried to identify protein(s) that associate with Dvl-1 phosphorylated by CKI⑀. It has been shown that CKI⑀ stimulates the binding of GBP to Dvl in Xenopus extracts in Functional Interaction of Dvl, CKI, and Frat 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 CKI⑀ enhances the binding of Dvl-1 to Frat-1. HA-Dvl-1 and Myc-Frat-1 were expressed with GFP-CKI⑀ or its kinase negative form, GFP-CKI⑀KN, in COS cells. GFP-CKI⑀, but not GFP-CKI⑀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][3][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-CKI⑀, but not GFP-CKI⑀KN, greatly enhanced the formation of the complex between HA-Dvl-1 and Myc-Frat-1 ( Fig. 2A, lanes 15  and 16).
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-(⌬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-(⌬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.
Synergistic Effects by Dvl-1 and Frat-1 on ␤-Catenin Accumulation and Tcf-4 Activation-Transient overexpression of Dvl-1 and Dvl-1-(⌬228 -250) in L cells induced the nuclear accumulation of ␤-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 ␤-catenin was not observed in L cells stably expressing Dvl-1 (L/Dvl cells) or Dvl-1-(⌬228 -250) (L/ Dvl-(⌬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-(⌬228 -250) in L cells is not sufficient for the stabilization of ␤-catenin. Although expression of Frat-1 in wild-type L cells did not induce the accumulation of ␤-catenin (data not shown), its expression in L/Dvl cells increased the level of ␤-catenin in the nucleus (Fig. 4, E and F,  arrows). However, expression of Frat-1 in L/Dvl-(⌬228 -250) did not induce the nuclear accumulation of ␤-catenin (Fig. 4, G  and H, arrows). Furthermore, expression of CKI⑀ in L/Dvl cells, but not in L/Dvl-(⌬228 -250) cells, resulted in the accumulation of ␤-catenin (Fig. 4, I-L, arrows). Taken together with our previous observations (20), these results suggest that not only CKI⑀ but also Frat-1 act synergistically with Dvl-1 to induce the accumulation of ␤-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 CKI⑀.
Inhibition of the Wnt Signaling Pathway by Depletion of CKI⑀ Expression-It has been shown that CKI␣ is essential for ␤-catenin degradation in mammalian cells (26). Therefore, we depleted the endogenous CKI⑀ in HeLa S3 cells via ds RNAi (36) to ask whether CKI⑀ is involved in the Wnt-dependent accumulation of ␤-catenin. A ds RNAi oligo for CKI⑀ reduced the protein level of CKI⑀ but not that of CKI␣, and, conversely, a ds RNAi oligo for CKI␣ reduced the protein level of CKI␣ but not that of CKI⑀ (Fig. 9A, third and fourth panels from the top). Single-stranded sense oligos for CKI␣ and CKI⑀ did not affect the protein levels of CKI␣ or CKI⑀ (data not shown). These oligos did not alter the protein level of GSK-3␤ (Fig. 9A, bottom  panel).
A decrease of CKI⑀, but not CKI␣, inhibited the Wnt-3adependent 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 CKI⑀ 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 CKI⑀ was depleted  1-7). The same lysates (200 g 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. via ds RNAi (Fig. 9C, f-h). Depletion of CKI␣ 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 CKI⑀ enhances the binding of Dvl-1 and Frat-1.
Furthermore, CKI⑀ ds RNAi did not affect the protein level of ␤-catenin in the absence of Wnt-3a but did inhibit Wnt-3ainduced ␤-catenin accumulation significantly (Fig. 9A, first  panel). A decrease of CKI␣ led to an accumulation of ␤-catenin in the absence of Wnt-3a, consistent with the previously reported observations (26), but did not affect Wnt-3a-induced ␤-catenin accumulation (Fig. 9A, first panel). These results clearly show that CKI⑀, but not CKI␣, is essential for the Wnt-3a-induced accumulation of ␤-catenin in HeLa S3 cells.

DISCUSSION
Complex Formation between Dvl-1 and Frat-1 and Its Enhancement by CKI⑀-In this study, we demonstrated that CKI⑀ 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 CKI⑀ (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 CKI⑀, 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 CKI⑀ have shown that CKI⑀ is required for the Wnt-3a-induced phosphorylation of Dvl-1 in HeLa S3 cells and that CKI⑀ is necessary for the interaction of Dvl-1 with Frat-1. Depletion of CKI␣ 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 CKI⑀ but not by CKI␣, 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-(⌬228 -250) does not act synergistically with either Frat-1 or CKI⑀ to activate the Wnt signaling pathway. Frat-1-(1-185) inhibited the synergistic activation of Tcf-4 by Dvl-1 and CKI⑀ or by Dvl-1 and Wnt-3a. Because Frat-1-(1-185) binds not to GSK-3␤ but to Dvl-1, these results support the idea that the binding of Dvl-1 and Frat-1 induces the accumulation of ␤-catenin and the activation of Tcf-4. The stability of ␤-catenin is regulated in a destruction complex that includes ␤-catenin, Axin, APC, GSK-3␤, Dvl-1, CKI␣, 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 CKI⑀ in HEK-293 cells decreases the association between PP2A and Axin (25). Therefore, the Wnt signal appears to destabilize the ␤-catenin degradation complex. Consistent with these observa- FIG. 9. Inhibition of the Wnt signaling pathway by depletion of CKI⑀ expression. A, inhibition of Wnt-3a-induced accumulation of ␤-catenin by depletion of CKI⑀ expression. HeLa S3 cells were transfected with a ds RNAi oligo specific for human CKI␣ (lanes 2 and 5) or a ds RNAi oligo specific for human CKI⑀ (lanes 3 and 6). Untransfected cells were used as a control (lanes 1 and 4). The cells were treated with Wnt-3a-conditioned medium (ϩ) (lanes 4 -6) or control medium (Ϫ) (lanes 1-3) for 1 h. Aliquots (10 l) of the cytosolic extract were probed with the anti-Dvl-1, anti-CKI⑀, anti-CKI␣, anti-GSK-3␤, or monoclonal anti-␤-catenin antibody. ␣, CKI␣ ds RNAi; ⑀, CKI⑀ 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 M CKI-7. Aliquots (20 l) 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 CKI⑀ expression. HeLa S3 cells were transfected with a single-stranded sense oligo (a-e), a ds RNAi oligo specific for human CKI⑀ (f-h), or a ds RNAi oligo specific for human CKI␣ (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. tions, 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 ␤-catenin by causing the disintegration of its degradation complex remains unclear. CKI⑀dependent interaction of Dvl-1 with Frat-1 may cause a conformational change of the complex that phosphorylates ␤-catenin. Dvl-1 forms a complex with GSK-3␤ via Axin or Frat-1. The level of GSK-3␤ 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-3␤ maintains its association with Dvl-1 after the disintegration of the complex. Because Wnt reduces the phosphorylation of ␤-catenin by GSK-3␤ (26), one possibility is that a conformational change of the degradation complex leads to the dissociation of GSK-3␤ from Axin and recruits it to Frat-1, thereby reducing the phosphorylation of ␤-catenin by GSK-3␤.
Involvement of CKI⑀ as a Positive Regulator in the Wnt Signaling Pathway-Several reports have shown that CKI binds to Dvl and stabilizes ␤-catenin (18 -20,42), whereas other reports have demonstrated that CKI phosphorylates ␤-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 CKI␣, but not of CKI⑀, stabilizes the basal level of ␤-catenin in HEK-293 cells (26). The expression of CKI⑀, but not of CKI␣, induces axis duplication in Xenopus embryos (19). However, arguing against these observations, it has shown that depletion of either CKI␣ or CKI⑀ stabilizes Armadillo in Drosophila cells (27) and that expression of either CKI␣ or CKI⑀ induces axis duplication in Xenopus embryos (42). Furthermore, it has also been demonstrated that Diversin recruits CKI⑀ to the ␤-catenin destruction complex, thereby inducing the down-regulation of ␤-catenin (43). Therefore, we performed a loss of function study using the ds RNAi methods in mammalian cells to clarify the roles of CKI⑀ and CKI␣ in the Wntdependent accumulation of ␤-catenin.
Our results support the hypothesis of CKI-isoform-specific differences. Depletion of CKI⑀ expression by ds RNAi in HeLa S3 cells did not affect the basal level of ␤-catenin but reduced Wnt-3a-induced accumulation of ␤-catenin. Wnt-3a-dependent phosphorylation of Dvl-1 and the interaction of Dvl-1 with Frat-1 were reduced in the cells in which CKI⑀ was depleted. Furthermore, reduction of CKI␣ increased the basal level of ␤-catenin but did not affect Wnt-3a-induced accumulation of ␤-catenin. These results strongly suggest that CKI␣ and CKI⑀ 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 CKI⑀ 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 CKI⑀ in the Wnt signaling pathway.