Casein Kinase Iε Modulates the Signaling Specificities of Dishevelled

The Wnt family of glycoproteins regulate cell growth and cell fate determination in a wide range of metazoan organisms, including Drosophila melanogaster and mammals. Wnts play pivotal roles in diverse developmental processes, such as segmentation in D. melanogaster, control of asymmetric division in Caenorhabditis elegans, and axis formation and patterning of the central nervous system in vertebrates (10, 56). Genetic experiments in D. melanogaster and biochemical studies in Xenopus and mammalian cells have established a framework for the Wnt signaling pathway (15, 39). In the absence of a Wnt signal, cytoplasmic β-catenin is bound to Axin and constitutively phosphorylated at several N-terminal Ser and Thr residues by glycogen synthase kinase 3 (GSK3); phosphorylated β-catenin is then recognized by β-TrCP and degraded by the ubiquitin-proteasome system. Wnt signaling is initiated by the binding of secreted Wnt protein to its receptors, Frizzled and LRP5/6, activating Dishevelled (Dvl) by an unknown mechanism. Dvl induces the dissociation of the Axin-GSK3-β-catenin complex and the stabilization of β-catenin, presumably through the inactivation of GSK3 and failure to phosphorylate β-catenin. Accumulated β-catenin enters the nucleus, binds to members of LEF/TCF family of transcription factors, and alters the transcription of Wnt target genes. Deregulated Wnt signaling contributes to tumorigenesis. Wnt-1, the founding member of the Wnt family, was first identified as a gene activated by insertion of mouse mammary tumor provirus, leading to the formation of mouse mammary tumors (36). Activating mutations of β-catenin and inactivating mutations of adenomatous polyposis coli and Axin, whose products are negative regulators of the Wnt signaling pathway, have been associated with various types of human cancers (41). Some components of the Wnt signaling pathway, including Frizzled and Dvl, are also involved in a β-catenin-independent pathway that determines planar cell polarity (32, 49). The planar cell polarity pathway regulates cytoskeletal organization through small GTPases and the c-Jun N-terminal kinase (JNK). In D. melanogaster, planar cell polarity signaling is essential for proper orientation of hairs and bristles on the thorax, abdomen, wing, and leg as well as correct polarity of ommatidia in the eye. In vertebrates, planar cell polarity is required for cell polarity and movement during gastrulation (52, 55). Analysis of Wingless (Wg) signaling-deficient arm, pan/TCF and zw3/sgg clonal tissue in the wing (6) and eye (9) revealed no polarity-related phenotype, indicating that D. melanogaster Dishevelled (Dsh) is the branching point of the planar cell polarity pathway and the β-catenin pathway. Three conserved domains have been identified in the Dvl protein, including an amino-terminal DIX domain, a central PDZ domain, and the carboxyl-terminal DEP domain (Fig. ​(Fig.1A).1A). A simplified view is that the DIX and the PDZ domains are required for the β-catenin pathway while the PDZ and the DEP domains are required for planar cell polarity/JNK signaling (6, 9, 33, 60). It is possible that Dvl adopts different conformations and forms different protein complexes in these divergent signaling pathways. The involvement of Dvl in one signaling pathway might exclude its participation in the other pathway. However, it is still not clear how Dvl signaling activities are regulated in these two pathways. FIG. 1. Identification of a hyperactive Dvl mutant that stimulates the β-catenin signaling pathway. (A) Schematic representations of mouse Dvl 1 and its mutants. Domains that are conserved among Dvl molecules in different species are highlighted. The ... Casein kinase Iɛ (CKIɛ) has been identified as a positive regulator of the Wnt signaling pathway (40, 45). Overexpression of CKIɛ in Xenopus embryos induces axis duplication and rescues UV-treated embryos. Epistasis experiments in Xenopus embryos suggest that CKIɛ functions between Dvl and GSK3 (40). Associations of CKIɛ, Dvl and Axin have been demonstrated by coimmunoprecipitation (45). Using yeast two-hybrid experiments, CKIɛ was shown to bind directly to the PDZ domain of Dvl (40) and the C-terminal region of Axin (30, 44). However, it was also shown that CKIɛ binds directly to the DEP domain of Dvl and indirectly to Axin (22). Recently, it has been suggested that CKIɛ is recruited to the Axin-GSK3-β-catenin complex by the ankyrin repeat protein Diversin (46). The CKI gene family consists of seven different genes in mammals, CKIα, β, γ1, γ2, γ3, δ, and ɛ (16). The ɛ and δ isoforms are closely related and share a C-terminal domain that is not present in other CKI isoforms. The shared C-terminal region is responsible for the interaction between Dvl and CKIɛ; accordingly, ectopic overexpression of CKIɛ and CKIδ, but not CKIα, induces axis duplication in Xenopus embryos (45). Recent studies suggest that the α isoform of CKI is, in contrast, a negative regulator of Wnt signaling by functioning as a priming kinase for β-catenin and GSK3 (28, 59). In spite of intensive study, the mechanism by which CKIɛ stimulates the Wnt pathway remains elusive. Overexpression of CKIɛ increases the phosphorylation of multiple Wnt pathway components, including Dvl (17, 22, 40), adenomatous polyposis coli (17, 22, 44), Axin (17), TCF (27), and β-catenin (17), but the physiological significance of these modifications is uncertain. Most of the major components in the Wnt pathway are conserved from Drosophila to mammals. Double time (Dbt) is the D. melanogaster homologue of CKIɛ/δ, but it is not clear what role, if any, Dbt plays in Wnt signaling. For example, Dbt-dsRNA induces the stabilization of Armadillo, the D. melanogaster β-catenin homologue, raising the possibility that Dbt functions differently than its vertebrate homologue (59). Here, we demonstrate that CKIɛ functions as a molecular switch to direct Dvl from the planar cell polarity/JNK pathway to the β-catenin pathway. In addition, we observe a correlation between the subcellular localizations of Dvl and its signaling activities in the β-catenin and the JNK pathways. Our work also indicates that the D. melanogaster CKIɛ homologue Dbt has a dual role in Wnt signaling, and it positively regulates Wnt signaling through Dishevelled.