Another layer of CK regulation in the Hh
Another layer of CK1 regulation in the Hh and Wnt pathways is to employ different CK1 isoforms to phosphorylate distinct pathway components or even distinct sites on the same substrates. In this regard, it has been shown recently that the membrane-associated CK1 isoform CK1γ, but not the cytosolic isoform CK1α or CK1ɛ, is responsible for phosphorylating a membrane-proximal cluster of S/T residues on the Smo C-tail to promote high-level Hh pathway activity (Li et al., 2016). Similarly, CK1γ has been shown to phosphorylate a membrane-proximal site (T1497) on the LRP6 intracellular domain to modulate the activity of Wnt signaling (Davidson et al., 2005), and in Drosophila, CK1γ is the most potent CK1 isoform that phosphorylates Arrow, the Drosophila homolog of LRP6, likely due to its membrane association (Zhang, Jia, et al., 2006). On the other hand, CK1α is the major CK1 isoform responsible for β-catenin phosphorylation and degradation. However, both CK1α and CK1ɛ are involved in the regulation of Ci phosphorylation and degradation in the Hh way (Jia et al., 2005). Interestingly, Hh switches the CK1α/ɛ substrate from Ci to Smo, thus converting CK1α and CK1ɛ from negative to positive regulators of the pathway (Jia et al., 2004, Zhang et al., 2005). This conversion is achieved, at least in part, by regulating kinase/substrate interaction because Hh signaling inhibits the formation of Cos2-Ci-kinase complex but promotes the formation of Cos2–Smo–kinase complexes (Li et al., 2014, Zhang et al., 2005).
Although it is generally thought that CK1 activity is not regulated but rather its substrate accessibility is regulated in the Hh and Wnt pathways, a recent study reveals a novel mechanism of CK1 regulation by DDX3, which belongs to a family of ATP-dependent DEAD-box RNA helicases (Cruciat et al., 2013). DDX3 was identified in a genome-wide siRNA screen for novel Wnt regulators in cultured mammalian Pasireotide (Cruciat et al., 2013). DDX3 directly binds to CK1ɛ in a manner stimulated by Wnt, and DDX3 stimulates CK1ɛ kinase activity and promotes phosphorylation of Dvl2 although the physiological relevance of Dvl2 phosphorylation has not been directly tested (Cruciat et al., 2013). The enzymatic activities of DDX3, i.e., ATP hydrolysis and RNA unwinding, appear to be dispensable for binding to CK1ɛ, suggesting that DDX3 activates CK1ɛ through an allosteric mechanism (Cruciat et al., 2013). Interestingly, mutations in DDX3 were found in the Wnt-subgroup of medulloblastoma, illustrating its physiological role in Wnt/β-catenin signaling in humans (Jones et al., 2012, Pugh et al., 2012). It remains to be determined whether DDX3 regulates other CK1 isoforms in vivo and whether other DDX family helicases are involved in CK1 regulation.
Conclusion Multiple CK1 family members regulate both Hh and Wnt signaling, and in each pathway, CK1 exerts both positive and negative influences depending on the signaling status, the specific CK1 isoforms involved, and which pathway components CK1 phosphorylates. Although many relevant CK1 substrates have been identified and the specific phosphorylation sites on individual pathway components defined, not all the biological relevant sites have been determined. For example, it has shown that CK1 activates the Hh pathway at the level of Smo as well as downstream of Smo by phosphorylating Fu and Ci (Shi et al., 2014, Zhou and Kalderon, 2011); however, the precise role and relevant CK1 sites on Fu remain to be determined. In addition, the precise function of individual CK1 isoforms in the Hh and Wnt pathways remains to be clarified, especially in the case of LRP6 phosphorylation where multiple CK1 isoforms have been implicated (Davidson et al., 2005, Zeng et al., 2005). In the past, loss-of-function study mainly employed dominant negative forms of individual isoforms, which may invoke cross-regulation among different CK1 isoforms and thus may not be exclusively “isoform specific.” In addition, redundancy among different CK1 family members might have underscored the role of individual CK1 isoforms in certain signaling processes, which is particularly problematic for CK1γ because of the presence of three CK1γ family members in mammals. Recent advance in gene editing technology, especially the CRISPR/Cas9 technology, that offers an efficient way to knock out multiple genes in the same cells (Doudna & Charpentier, 2014), will undoubtedly facilitate the loss-of-function study of individual CK1 family members in different cellular and developmental contexts and help elucidating their roles in human diseases.