InteractiveFly: GeneBrief

gilgamesh: Biological Overview | References

BIOLOGICAL OVERVIEW

Signalling by Wnt proteins (Wingless in Drosophila) has diverse roles during embryonic development and in adults, and is implicated in human diseases, including cancer. LDL-receptor-related proteins 5 and 6 (LRP5 and LRP6; Arrow in Drosophila) are key receptors required for transmission of Wnt/β-catenin signalling in metazoa. Although the role of these receptors in Wnt signalling is well established, their coupling with the cytoplasmic signalling apparatus remains poorly defined. Using a protein modification screen for regulators of LRP6, the identification of Xenopus Casein kinase 1 γ (CK1γ), a membrane-bound member of the CK1 family and homolog of Drosophila Gilgamish, is described. Gain-of-function and loss-of-function experiments show that CK1γ is both necessary and sufficient to transduce LRP6 signalling in vertebrates and Drosophila cells. In Xenopus embryos, CK1γ is required during anterio-posterior patterning to promote posteriorizing Wnt/β-catenin signalling. CK1γ is associated with LRP6, which has multiple, modular CK1 phosphorylation sites. Wnt treatment induces the rapid CK1γ-mediated phosphorylation of these sites within LRP6, which, in turn, promotes the recruitment of the scaffold protein Axin. These results reveal an evolutionarily conserved mechanism that couples Wnt receptor activation to the cytoplasmic signal transduction apparatus (Davidson, 2005).

In a human cell-culture-based small pool expression screen for proteins able to covalently modify LRP6, CK1γ, a protein which retards ('upshifts') the migration of LRP6 on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was identified in co-transfection assays. There are three closely related isoforms of CK1γ -- CK1γ1, 2 and 3 -- that are unique within the CK1 family in carrying putative palmitoylation sites at the carboxy terminus (TKCCCFFKR), that anchor the proteins in the plasma membrane. This is unlike CK1α, δ and epsilon, which are cytoplasmic or nuclear and are involved in the direct regulation of Wnt pathway components other than LRP5 and LRP6. Indeed, LRP6 upshift is promoted by CK1γ, while CK1epsilon has no effect on the electrophoretic mobility of LRP6. The cytoplasmic domain of LRP6 is the region modified by CK1γ, as an LRP6-δN protein that lacks the amino-terminal extracellular domain (ECD) is still able to produce the higher molecular weight bands indicative of CK1γ-mediated modification, whereas an LRP6-δC protein that lacks the carboxy-terminal intracellular domain (ICD) does not. These results suggest that the C terminus of LRP6 is modified by CK1γ phosphorylation, and this was confirmed by ³²P-incorporation in vivo. Moreover, modification of LRP6 by CK1γ enhances LRP6 function in Wnt reporter assays, in which LRP6 synergizes strongly with CK1γ and more weakly with CK1epsilon, while Dishevelled-1 (Dvl1) shows the inverse behaviour, consistent with the latter being directly regulated by CK1epsilon. Similar gain-of-function results were obtained in Xenopus embryos (Davidson, 2005).

To investigate the requirement of CK1γ in LRP6 signalling, two dominant-negative forms of CK1γ were designed with point mutations (K73R or D164N) in the ATPase domain, previously identified as generating specific inhibitors for CK1epsilon. Both dominant-negative forms of CK1γ inhibit the upshift, and hence phosphorylation, of LRP6 by wild-type CK1γ, but phosphorylation of Dvl1 by CK1epsilon is unaffected. Therefore, both dominant-negative forms of CK1γ specifically inhibit CK1γ. Dominant-negative CK1γ inhibits Wnt signalling in HEK293T cells, and similar loss-of-function results were obtained in Xenopus embryos; wild-type CK1γ rescues this effect in both cases. Unlike Wnt1, Wnt3a and a constitutively active LRP6-δE(1-4) protein, lacking most of the extracellular domain, β-catenin activity is unaffected by dominant-negative forms of CK1γ. This is consistent with CK1γ acting directly on LRP6 (Davidson, 2005).

Wingless (Wg) reporter assays in Drosophila SL2 cells show that the single Drosophila homologue of CK1γ, Gilgamesh (Gish) (Hummel, 2002), strongly synergizes with the Drosophila LRP6 homologue, Arrow. Conversely, using an interfering RNA (RNAi) loss-of-function approach, a gish double-stranded RNA that targets all eight gish transcripts blocks Wg but not Dishevelled (Dsh) signalling. This is unlike armadillo (arm) and dsh dsRNAs, which block signalling by both Wg and Dsh. These results support an evolutionarily conserved role for CK1γ in LRP6 regulation. The absence of obvious Wg phenotypes in Drosophila gish mutants may be because at least two alternatively spliced transcripts within the gene locus are intact (Davidson, 2005).

In Xenopus embryos, maternally derived ck1γ mRNA transcripts are present ubiquitously at gastrulation, and zygotic transcription starts at around the mid-neurula stage. To address the physiological role of CK1γ, Xenopus gain-of-function and loss-of-function experiments were performed. Overexpression of CK1γ in the animal pole by messenger RNA injection leads to headless embryos, whereas injection of either a ck1γ morpholino or dominant-negative ck1γ^K73R mRNA reduces trunk and tail structures, and induces enlarged heads and cement glands. These phenotypes match hyper- and hypo-activation of zygotic Wnt signalling, respectively. It is concluded that CK1γ is required for Wnt-mediated antagonism of Spemann's head organizer (Davidson, 2005).

Following transfection in HEK293T cells, CK1γ tagged with enhanced yellow fluorescent protein (EYFP-CK1γ) localizes to the plasma membrane, consistent with it containing a C-terminal palmitoylation site. Deleting this region (CK1γ-δC) results in the cytoplasmic localization of CK1γ, and abolishes Wnt/β-catenin signalling, although its kinase activity is unaffected. Bioluminescence resonance energy transfer (BRET) assays reveal that CK1γ and LRP6 interact in live cells, and that this interaction requires membrane localization, as it is abolished in CK1γ-δC. Co-immunoprecipitation (CoIP) confirms that LRP6 specifically interacts with CK1γ and not CK1γ-δC, and this is independent of coexpressed Axin or GSK3β. Neither BRET nor CoIP assays show differences in CK1γ-LRP6 interaction on Wnt treatment. These results indicate that CK1γ and LRP6 associate, and that this association requires the membrane localization of CK1γ (Davidson, 2005).

To identify relevant phosphorylation sites in LRP6, serial C-terminal deletions were tested for loss of CK1γ synergy in Wnt reporter assays. The LRP6-δE(1-4) backbone generates a robust Wnt response. A gradual, not abrupt, decrease in reporter activation with progressive deletions is observed, consistent with the presence of multiple activating phosphorylation sites. LRP6-δE(1-4)-δ87, which retains a single PPPSP motif, was identified as the shortest construct still activated by CK1γ (Davidson, 2005).

Deletion of Ser and/or Thr clusters in LRP6-δE(1-4)-δ87 revealed that two regions are required for cooperation with CK1γ. To test if the two regions are also sufficient for Wnt signalling, they were linked alone, or in combination, to LDLR-δN, a heterologous low-density lipoprotein (LDL) mini-receptor structurally analogous to LRP6-δN but inactive in Wnt signalling. Neither region alone (miniA or miniB) activates Wnt signalling, but they do when combined (miniC), and this effect is further enhanced by CK1γ both in HEK293T cells and Xenopus embryos (Davidson, 2005).

The region of LRP6 contained within miniC consists of a PPPSP motif flanked by two evolutionarily conserved Ser/Thr clusters that are potential CK1 phosphorylation sites. The N-terminal CK1 site (TGA(S)₇TKGT) is referred to as cluster 1, and the C-terminal CK1 site (SPATERSHYT) as cluster 2 (abbreviated 1/S/2, with the Ser residue in the PPPSP motif designated by the S). To analyse the function of the two CK1 site clusters in Wnt signalling, inactivating Ala (A) and phospho-mimicking Asp (D) substitutions of all Ser/Thr residues within these clusters (shown above in bold) were tested, using the miniC receptor as a backbone. The wild-type mini-receptor (1/S/2) is constitutively active, unlike A/S/A, indicating that the CK1 sites are required for Wnt signalling. Elimination of either CK1 site alone (A/S/2 or 1/S/A) suppresses Wnt signalling. Conversely, Asp substitutions in individual (D/S/2 or 1/S/D) or both CK1 sites (D/S/D) lead to Wnt hyperactivation. Constitutive Wnt signalling by 1/S/2, D/S/2 and 1/S/D is blocked by dominant-negative CK1γ, indicating that both clusters require CK1γ for Wnt signalling. D/S/D, on the other hand, in which both CK1 sites are artificially activated, signals independently of CK1γ. Any substitution of the PPPSP site (1/A/2 or 1/D/2) inactivates Wnt signalling, indicating that this serine is essential. The results suggest that (1) clusters 1 and 2 are both physiologically relevant CK1γ phosphorylation sites that activate Wnt signalling, and (2) they require an intact PPPSP motif (Davidson, 2005).

To show direct phosphorylation of LRP6 by CK1γ, in vitro kinase assays were performed. A phospho-specific antibody (Tp1479) was raised against a CK1 site within cluster 1, which recognizes the most C-terminal phosphorylated threonine. In vitro kinase assays were performed with CK1γ using the miniC receptor carrying various internal mutations as the substrate. Following the kinase reaction and SDS-PAGE, samples were analysed for ³²P-incorporation by autoradiography, and T1479 phosphorylation by immunoblotting. The wild-type mini-receptor (1/S/2) is phosphorylated by CK1γ, unlike the construct in which all three sites are inactivated (A/A/A). In agreement with cluster 1 being a CK1 phosphorylation site, 1/A/A is robustly phosphorylated. In contrast, A/S/A, containing only the intact PPPSP site, is not phosphorylated by CK1γ. Notably, cluster 2 is only weakly phosphorylated (A/A/2), but this is enhanced either by the presence of an intact PPPSP motif (A/S/2) or mimicking cluster 1 and PPPSP phosphorylation (D/D/2). Taken together, these results indicate that clusters 1 and 2, but not the PPPSP motif, are direct target sites for CK1γ phosphorylation, and that efficient phosphorylation of cluster 2 requires N-terminal priming from the PPPSP site. T1479 is phosphorylated in all constructs containing an unmodified cluster 1, indicating that it is a bona fide target for phosphorylation by CK1γ (Davidson, 2005).

An important consequence of Wnt activation is the sequestration of Axin, a negative regulator of β-catenin, by LRP6. Therefore, it was asked whether CK1γ is required for Axin recruitment. In CoIP assays, dominant-negative CK1γ markedly reduces binding of Axin to LRP6. It has been shown that the PPPSP site of LRP6 is required for Axin binding. To determine if the two CK1 phosphorylation site clusters in LRP6 also play a role in Axin binding, CoIPs were performed using 1/S/2 site substitutions within the LRP6-δE(1-4)-δ87 protein. While only weak interaction is seen with the wild-type construct (1/S/2), mimicking CK1γ phosphorylation of cluster 1 in D/S/2 leads to robust binding of Axin. Increased binding is also observed in 1/S/D, supporting the notion that phosphorylation of cluster 2 is physiologically relevant. However, activation of this cluster by itself (A/S/D) fails to mediate Axin binding. Likewise, D/S/2 shows stronger binding than D/S/A, arguing that both CK1 sites act synergistically to promote Axin binding when they are phosphorylated. Of note, 1/D/D fails to bind Axin, indicating that a phospho-serine at the PPPSP site cannot be mimicked by an Asp, explaining the inactivity of 1/D/2. It is concluded that CK1γ is required for Axin binding, and that both of the identified CK1 phosphorylation site clusters promote this interaction (Davidson, 2005).

Wnt signalling promotes Axin binding to LRP6, and the data indicate that this is mediated by CK1γ phosphorylation at conserved sites in LRP6. An important prediction, therefore, is that Wnt stimulation leads to phosphorylation of LRP6 at sites of CK1γ phosphorylation. Indeed, CK1γ overexpression in HEK293T cells induces phosphorylation at T1479 (in cluster 1), and this can be mimicked by Wnt treatment. This Wnt-induced T1479 phosphorylation requires CK1γ, as it is blocked by the dominant-negative mutant CK1γ^K73R. The PPPSP motifs are also thought to become phosphorylated following Wnt stimulation. However, using a phospho-specific anti-PPPSpP antibody (Sp1490), no significant phosphorylation increase is detected after either CK1γ overexpression or Wnt treatment. Strikingly, as little as 10 min of Wnt stimulation is sufficient to induce robust T1479 phosphorylation of endogenous LRP6 in mouse embryonic fibroblasts (MEFs), P19 and HeLa cells. Unlike T1479, S1490 (in the PPPSP motif) is constitutively phosphorylated in all cell lines tested, and shows either no or weaker Wnt induction. Using a phospho-independent T1479 antibody, which recognizes total LRP6, a progressive mobility upshift is observed, which saturates after 60 min of Wnt treatment. It is concluded that Wnt stimulation results in phosphorylation of T1479 (cluster1) in a CK1γ-dependent manner (Davidson, 2005).

It has been suggested that the ECD of LRP6 exerts an autoinhibitory effect that is relieved upon Wnt stimulation, based on the observation that its deletion leads to constitutive receptor activation. Alternatively, it was suggested that deletion of the ECD enhances transport to the plasma membrane, because co-transfection of the chaperone MESD (for mesoderm development) with full-length LRP6 activates Wnt signalling as effectively as the ECD-deleted form. To distinguish between these two possibilities either full-length LRP6 and MESD or LRP6-δE(1-4) were coexpressed alone in HEK293T cells, and T1479 phosphorylation was analysed. No signal is detected in full-length LRP6 despite overexpressing MESD, while there is a strong signal in LRP6-δE(1-4). A slight increase in S1490 (PPPSP) phosphorylation was also observed in LRP6-δE(1-4) compared to full-length LRP6. These results therefore support the idea of an autoinhibitory role of the LRP6 ECD, and indicate that one of its functions is to prevent phosphorylation by CK1γ (Davidson, 2005).

In summary, this investigation indicates that CK1γ couples the extracellular Wnt signal and the cytoplasmic signal transduction machinery, and suggests a model where the net effect of CK1γ phosphorylation of LRP6 is Axin recruitment. The cytoplasmic domain of LRP6 contains five reiterated PPPSP motifs that are necessary for Wnt/β-catenin signalling. While the PPPSP motifs are phosphorylated, the data demonstrate that this is not by CK1γ, but by an unknown, probably proline-directed, kinase. As Wnt was reported to stimulate PPPSP phosphorylation, it was surprising to find not only that there is constitutive S1490 (PPPSP) phosphorylation in unstimulated cells, but that Wnt stimulates no or only weak induction at this site. Since CK1 members act on proteins previously phosphorylated by other kinases, constitutive S1490 (PPPSP) phosphorylation may prime CK1γ-mediated phosphorylation. This may well apply to all PPPSP sites in LRP6, as all are succeeded by at least one adjacent CK1 site. It is therefore proposed that the LRP6 cytoplasmic domain is modular, and each element consists of a CK1γ phosphorylation site that is primed by a phosphorylated PPPSP motif (Davidson, 2005).

Role for casein kinase 1 in the phosphorylation of Claspin on critical residues necessary for the activation of Chk1

The mediator protein Claspin is critical for the activation of the checkpoint kinase Chk1 during checkpoint responses to stalled replication forks. This function involves the Chk1-activating domain (CKAD) of Claspin, which undergoes phosphorylation on multiple conserved sites. These phosphorylations promote binding of Chk1 to Claspin and ensuing activation of Chk1 by ATR. However, despite the importance of this regulatory process, the kinase responsible for these phosphorylations has remained unknown. By using a multifaceted approach, this study found that casein kinase 1 gamma 1 (CK1γ1; Gilgamesh) carries out this function. CK1γ1 phosphorylates the CKAD of Claspin efficiently in vitro, and depletion of CK1γ1 from human cells by small interfering RNA (siRNA) results in dramatically diminished phosphorylation of Claspin. Consequently, the siRNA-treated cells display impaired activation of Chk1 and resultant checkpoint defects. These results indicate that CK1γ1 is a novel component of checkpoint responses that controls the interaction of a key checkpoint effector kinase with its cognate mediator protein (Meng, 2011).

This study sought to resolve the molecular basis of a key step in the checkpoint-dependent activation of Chk1 in response to genomic stress. The activation of Chk1 involves phosphorylation-dependent docking of Chk1 with its cognate mediator protein (Claspin) and recognition of the resulting Claspin-Chk1 complex by ATR-ATRIP. In particular, phosphorylation of Claspin on multiple residues in its CKAD mediates the binding of Chk1. Direct biochemical evidence has indicated the presence of Claspin in this complex enhances the ability of ATR-ATRIP to phosphorylate Chk1 on key residues necessary for the ultimate activation of Chk1. Thus, the kinase that phosphorylates Claspin on the CKAD plays a key role in this pathway (Meng, 2011).

To address the identification of this kinase(s) systematically, a kinome-wide RNAi screen was initially performed in Drosophila S2 cells. Various RNAi screens in these cells have been used to identify numerous regulators in diverse cellular pathways. For the purposes of this study, the fact that Drosophila contains approximately one-third as many kinases as humans was a considerable advantage. S2 cells appear to possess checkpoint-signaling pathways generally similar to those in other metazoan cells. In particular, the Drosophila Chk1 homologue Grapes exhibits a conserved checkpoint function in S2 cells. It functions downstream of Mei-41 (the Drosophila homologue of ATR) and is critical for a proper checkpoint response to genotoxic stress (Meng, 2011).

Nonetheless, the reagents available for the study of checkpoint responses in Drosophila S2 cells are still relatively limited. To circumvent this problem, the Xenopus version of Chk1 was introduced into the Drosophila cells as a more readily traceable marker to monitor the checkpoint response. It was possible to establish a system in which phosphorylation of this reporter could be induced following treatment with a variety of replication inhibitors. Moreover, this response seems to have molecular features similar to those present in vertebrate cells. For example, RNAi-mediated knockdown of Drosophila homologues of ATR and Claspin abolished checkpoint-dependent phosphorylation of the exogenously introduced Chk1 reporter molecule (Meng, 2011).

The results of the screen indicated that knockdown of several casein kinases led to the reduced phosphorylation of Chk1. Following this lead, a candidate-based cDNA overexpression approach was employed to further pinpoint a specific kinase that can directly phosphorylate the CKAD of Claspin. From these tests, it was eventually found that Drosophila Gish, a homologue of CK1γ, could phosphorylate the CKAD of Claspin quite effectively both in vitro and in the Drosophila tissue culture cells. At this juncture, the studies were extended to human cells. Interestingly, humans possess three different versions of CK1γ, namely, CK1γ1, CK1γ2, and CK1γ3. All three proteins have very similar central kinase domains but are significantly different in their N- and C-terminal extensions. It was observed that all three kinases could phosphorylate the CKAD relatively well in vitro. However, further characterizations established that CK1γ1 appears to be the form primarily responsible for the phosphorylation of CKAD in human cells. Upon expression in human cells, only CK1γ1 could phosphorylate the CKAD of Claspin in vivo. More importantly, siRNA-mediated knockdown of CK1γ1 resulted in greatly reduced phosphorylation of Claspin, whereas knockdowns of CK1γ2 and CK1γ3 did not have a significant effect. As expected from the known function of the phosphorylation of Claspin, cells with diminished levels of CK1γ1 were greatly compromised in their ability to carry out the activation of Chk1 in response to a variety of genotoxic agents, including APH, HU, and UV. Furthermore, these cells had the physiological defects characteristic of cells with impairment of the Chk1-mediated signaling pathway. In particular, these cells showed reduced survival following treatment with genotoxic agents, impaired recovery of stalled replication forks, a defective G2/M checkpoint response, and spontaneous DNA damage in the absence of exogenous stress. Taken together, our results indicated CK1γ1 is an important regulator in Chk1-mediated cellular checkpoint responses (Meng, 2011).

The casein kinase 1 family of serine/threonine kinases is highly conserved and ubiquitously expressed. The functions of CK1 encompass a wide variety of processes, including cell proliferation, cell division, apoptosis, circadian rhythms, and others. In mammals, this family contains at least seven members (α, β, γ, ε, γ1, γ2, and γ3) with multiple splicing variants. All of the CK1 proteins share significant homology in the central kinase domain (53%-98% identical), but differ significantly in the flanking N- and C-terminal sequences, which most likely confer unique properties to the various kinases. In the case of CK1γ1, this study identified and isolated multiple splicing variants of this kinase from human U2OS cells. These isoforms contain distinct C-terminal sequences (ranging from 3 to 106 amino acids), display discrete subcellular localizations, and exhibit differences in their abilities to phosphorylate the CKAD. A similar phenomenon has also been observed in various organisms in the case of CK1α, which contains four types of isoforms with distinct localizations and kinase activities. These findings strongly suggest various isoforms of CK1 may participate in distinct cellular activities due to differences in access to and/or affinity for substrates (Meng, 2011).

Interestingly, it was initially difficult to achieve good rescue of CK1γ1-depleted cells with vectors encoding an siRNA-resistant version of the major published form of CK1γ1. However, there are multiple forms of CK1γ1 in human cells. It was possible to clone and express three additional versions of CK1γ1, which were designated as isoforms B, C, and D to distinguish them from the published form (isoform A). In cellular localization studies, it was found that isoform A had the expected prominent localization to cell membranes. However, isoforms B and C resided in both the nucleus and cytoplasm, whereas isoform D was mainly cytoplasmic. In coexpression studies, it was found that isoforms A, B, and C (but not D) could phosphorylate the CKAD of Claspin, although isoforms B and C were more effective than isoform A in this in vivo assay. Ultimately, it was possible to rescue depletion of CK1γ1 by introducing siRNA-resistant forms of isoforms A, B, and C into the cells. It is straightforward to understand how the nuclear isoforms could regulate Claspin, but it is intriguing that a membrane-bound enzyme is also partially responsible. Conceivably, some fraction of this isoform could be absent from the membrane. It is also possible that Claspin could shuttle between the nucleus and cytoplasm and thus be subject to regulation by enzymes in both locations. In this regard, a recent study has shown that perturbed cell-surface signaling through the Sonic Hedgehog (Shh) pathway inhibits ATR-mediated signaling by disrupting the interaction between Claspin and Chk1 (Meng, 2011).

It will be important to understand the mechanisms that control the phosphorylation of Claspin by CK1γ1. It is known that phosphorylation of the CKAD in Xenopus egg extracts is dependent upon ATR. However, ATR itself is unable to phosphorylate the critical sites in the CKAD directly. One possibility would be that ATR might somehow regulate the activity of CK1γ1. However, it has been possible to detect only a subtle increase in the activity of CK1γ1 in response to genomic stress. Moreover, when the only apparent potential target site for ATR (Ser361) was mutated to Ala in CK1γ1, no change was observable in its kinase activity toward the CKAD upon coexpression in U2OS cells. Another possibility is that ATR might regulate the accessibility of Claspin to CK1γ1. Further studies will be required to understand the dynamics of this phosphorylation (Meng, 2011).

In summary, this study identified a conserved casein kinase, Gish/CK1γ1, from Drosophila and humans as a specific enzyme that controls phosphorylation of the CKAD of Claspin. Functional studies have revealed that this kinase is critical for mediating activation of Chk1 and ensuring a proper checkpoint response under conditions of genotoxic stress. Further studies of its regulation and function should help gain more insight into the molecular basis of checkpoint responses (Meng, 2011).

Genome-wide identification of phospho-regulators of Wnt signaling in Drosophila

Evolutionarily conserved intercellular signaling pathways regulate embryonic development and adult tissue homeostasis in metazoans. The precise control of the state and amplitude of signaling pathways is achieved in part through the kinase- and phosphatase-mediated reversible phosphorylation of proteins. In this study, a genome-wide in vivo RNAi screen was performed for kinases and phosphatases that regulate the Wnt pathway under physiological conditions in the Drosophila wing disc. These analyses have identified 54 high-confidence kinases and phosphatases capable of modulating the Wnt pathway, including 22 novel regulators. These candidates were also assayed for a role in the Notch pathway, and numerous phospho-regulators were identified. Additionally, each regulator of the Wnt pathway was evaluated in the wing disc for its ability to affect the mechanistically similar Hedgehog pathway. 29 dual regulators were identified that have the same effect on the Wnt and Hedgehog pathways. As proof of principle, this study established that Cdc37 and Gilgamesh/CK1gamma inhibit and promote signaling, respectively, by functioning at analogous levels of these pathways in both Drosophila and mammalian cells. The Wnt and Hedgehog pathways function in tandem in multiple developmental contexts, and the identification of several shared phospho-regulators serve as potential nodes of control under conditions of aberrant signaling and disease (Swarup, 2015).

Divergent disease states have been attributed to be a cause or consequence of aberrant protein phosphorylation. Wnt signaling is phosphor-regulated both in its silent and active states, but thus far understanding of kinases, phosphatases and associated factors of the pathway has been limited. In this study, the first genome-wide in vivo screen was performed under physiological conditions in the Drosophila wing disc for phospho-regulators of the Wnt pathway. 54 high-confidence regulators were identified, 22 of which are novel. The results of these analyses do not indicate whether a high-confidence regulator has a direct or indirect effect on signaling. However, as ~60% of the high-confidence regulators identified have been previously validated to have a direct effect on Wnt signaling, it is predictd that at least some of the novel high-confidence regulators identified would also have a direct effect on the pathway. Indeed, subsequent analyses of Myopic revealed a novel role in regulating Wg secretion. Although the mechanism and components of the Wnt pathway are for the most part conserved between Drosophila and humans, there are possibly vertebrate-specific phospho-regulators of signaling that would not have been identified in these analyses. The dataset represents the largest list of putative phospho-regulators of the Wnt pathway identified to date, almost all of which have identified human orthologs and are therefore likely to be functionally conserved (Swarup, 2015).

As part of this study, previously unknown relationships were established between the Wnt and Hh pathways in vivo by identifying 12 novel dual regulators that are proposed to function at analogous levels of signaling. As proof of concept, the roles of Cdc37 and Gish/CK1γ were biochemically characterized to demonstrate that their functions are conserved from Drosophila to mammalian cells. An initial analysis is reported of candidate regulators of Notch signaling during wing disc development. Although these findings are preliminary, they highlight an emerging theme of phospho-regulation of Notch that likely hold parallels in vertebrate biology. The comparison of signaling pathways in vivo and the identification of specific versus shared phospho-regulators facilitate understanding of human development and disease states (Swarup, 2015).

Gilgamesh (Gish)/CK1gamma regulates tissue homeostasis and aging in adult Drosophila midgut

Adult tissues and organs rely on resident stem cells to generate new cells that replenish damaged cells. To maintain homeostasis, stem cell activity needs to be tightly controlled throughout the adult life. This study shows that the membrane-associated kinase Gilgamesh (Gish)/CK1gamma maintains Drosophila adult midgut homeostasis by restricting JNK pathway activity and that Gish is essential for intestinal stem cell (ISC) maintenance under stress conditions. Inactivation of Gish resulted in aberrant JNK pathway activation and excessive production of multiple cytokines and growth factors that drive ISC overproliferation. Mechanistically, Gish restricts JNK activation by phosphorylating and destabilizing a small GTPase, Rho1. Interestingly, this study found that Gish expression is down-regulated in aging guts and that increasing Gish activity in aging guts can restore tissue homeostasis. Hence, this study identifies Gish/CK1gamma as a novel regulator of Rho1 and gatekeeper of tissue homeostasis whose activity is compromised in aging guts (Li, 2020).

Adult stem cells are essential for tissue homeostasis and regeneration, and their activity needs to be tightly controlled in order to maintain the normal cellular architecture and physiological function of adult organs. The JNK pathway plays a pivotal role in the Drosophila midgut homeostasis and injury response. In addition, JNK pathway activity is elevated in aging guts, leading to aberrant stem cell proliferation and loss of tissue homeostasis. How the JNK pathway is kept in check during gut homeostasis and aging is not well understood. This study provides both genetic and biochemical evidence that the membrane-associated kinase Gish/CK1γ restricts JNK pathway activity by phosphorylating and destabilizing Rho1, an upstream activator of the JNK pathway. This regulatory pathway is critical for the maintenance of midgut homeostasis as loss of Gish results in elevated JNK pathway activity and ISC proliferation. Interestingly, Gish expression in the midgut declines with aging, which may contribute to the elevated JNK pathway activity and ISC overproliferation in old guts. In support of this notion, it was found that transgenic expression of Gish in old guts could restore JNK pathway activity and ISC proliferation to homeostatic levels. How Gish expression is down-regulated in aging guts remains an open question, but it appears to occur at the level of transcription, although the possibility cannot be ruled out that posttranscriptional regulation may also occur. The precise mechanism awaits further investigation (Li, 2020).

Prolonged activation of JNK can induce apoptosis in both Drosophila and mammalian cells. As a negative regulator of JNK pathway activity, Gish synergizes with Puc, another JNK pathway inhibitor, to protect ISCs from undergoing apoptosis; thus, it may play a role in stem cell maintenance under stress condition. The synergistic interaction between Gish and Puc is not restricted to ISCs but also occurs in other tissues. For example, in wing imaginal discs, depletion of Gish only resulted in low levels of apoptosis; however, removal of one copy of puc (puc^E96/+) in Gish-depleted wing discs (MS>GishRNAi, puc^E96/+) resulted in massive cell death, leading to the formation of small wings. The increased apoptosis in MS>GishRNAi, puc^E96/+ appeared to mirror the dramatic activation of the JNK pathway. Hence, the cell- and tissue-protective function of Gish could simply be attributed to its role in suppressing JNK signaling. However, it remains possible that other pathways downstream of Rho1 and/or Gish may contribute to the induction of apoptosis in Gish and Puc double-deleted cells in parallel to heightened JNK pathway activity (Li, 2020).

Although mainly regulated by GTP-GDP cycling, Rho GTPases can also be regulated by posttranslational modifications such as phosphorylation that control their subcellular localization, stability, and complex formation. For example, a previous study showed that Erk2-mediated phosphorylation of RhoA targeted it for ubiquitin/proteasome-mediated degradation in cultured epithelial cells. However, the phosphorylation site(s) on RhoA that regulates its stability remained unidentified, and the physiological role of Erk2-mediated phosphorylation of RhoA was not determined. This study provides both genetic and biochemical evidence that Gish phosphorylates a cluster of sites in the N-terminal region of Rho1, which targets Rho1 for ubiquitination, followed by proteasome-mediated degradation. Gish-mediated down-regulation of Rho1 restricts JNK pathway activity, which is critical for adult Drosophila midgut homeostasis. How Rho1 inhibits JNK pathway remains an open question. A previous study revealed that Rho1 physically interacts with Slpr/JNKKK regardless of its GDP/GTP binding state and that Rho1 promotes Slpr cortical localization. Therefore, it is possible that plasma membrane-associated Rho1 promotes JNK pathway activation by increasing the local concentration of Slpr/JNKKK at the plasma membrane. The precise biochemical mechanism by which Rho1 activates Slpr/JNKKK awaits further investigation (Li, 2020).

Rho GTPases shuttle between plasma membrane and cytoplasm with GTP-bound active forms associated with membrane. Gish/CK1γ belongs to the CK1 family kinases. Unlike other family members that are located mainly in the cytoplasm, Gish/CK1γ is associated with plasma membrane through its C-terminal lipid modification. This study has demonstrated that the function of Gish in restricting ISC overproliferation depended on its kinase activity and membrane association, suggesting that Gish may phosphorylate plasma membrane-associated Rho1 to prevent its aberrant accumulation at the plasma membrane where it can activate the JNK pathway. This may explain why the role of Gish/CK1γ cannot be replaced by other CK1 family members. Since the CK1 phosphorylation sites on Rho1 are also found in all members of mammalian Rho GTPase, it is speculated that CK1γ may play a conserved role in the regulation of Rho GTPase activity. Future study is needed to determine whether CK1γ regulates Rho GTPase activity and whether such regulation plays a role in development, tissue homeostasis, and aging in mammals (Li, 2020).

A GFP trap study uncovers the functions of Gilgamesh protein kinase in Drosophila melanogaster spermatogenesis

gilgamesh encodes a casein kinase that functions in Drosophila spermatogenesis. The chimeric Gilgamesh-GFP protein in spermatocytes is cortically located. In the polar and apolar spermatocytes, it concentrates at the terminal ends of the fusome, the organelle that passes through the system of ring canals of the spermatocyte cyst. At the stage of spermatid elongation, the protein associates with the nucleus. A spot of the highest Gilgamesh-GFP concentration in the nucleus co-localizes with γ-tubulin in the basal body. At later stages, Gilgamesh is localized to the individualization complex (IC), leaving the nuclei somewhat before the IC investment cones, as detected by actin binding. The sterile mutation due to the gilgamesh gene leads to the phenotype of scattered nuclei and altered structure of actin cones in the individualizing spermatid cyst. Ultrastructural evidence confirmed defective spermatid individualization due to the mutation. The phylogenetic origin of the protein, and the connection between vesicular trafficking and spermatid individualization, are discussed (Nerusheva, 2009).

The intracellular distribution of the chimeric Gish-GFP protein suggests the cellular structures that can be targets for the Gilgamesh protein, namely the cortical membrane, fusome, basal body, spermatid nucleus and individualization complex. The sterile gish allele shows an abnormality in spermatid individualization appearing as a phenotype of scattered nuclei and altered morphology of the bundle of actin cones. It fits well with the localization of the fused protein within the individualization complex. This result increases knowledge of the function of this protein kinase in the male gonads. Phylogenetic analysis has demonstrated that the earliest event in the evolution of casein kinases 1 was the divergence between the YCK1, YCK2 and YCK3 ancestral isoform and the HRR25 ancestral isoform. Presumably this divergence occurred in unicellular organisms. The structure of the subtree corresponding to the divergence of animal gamma isoforms fits well with the divergence of animal species. The unexpected result of the second earliest event in the evolution of casein kinases 1 was the divergence between the yeast HRR25 ancestral form (and the cognate animal alpha and delta isoforms) and the ancestral animal gamma isoform. From the formal standpoint, this suggests that the animal gamma isoform can share the function with the nuclear-cytoplasmic kinase HRR25, but this interpretation is incorrect. Mammalian gamma casein kinase 1 can substitute the function of YCK1 and YCK3 proteins, restoring the colony growth and morphology in the double mutants yck1 yck2, proving that these proteins have a common function. The mammalian casein kinase delta can restore the growth abnormality of the cells mutant at HRR25, thereby also showing a common function in this case. Thus the nuclear-cytoplasmic protein kinase appeared in the phylogenetic branch leading to the yeast HRR25 and the animal alpha and delta protein kinases. However, the animal gamma isoform retained the cytoplasmic function of its ancestor (Nerusheva, 2009).

Vesicular trafficking and spermatid individualization are processes of membrane remodeling. They are also similar from the standpoint of genetic control. The molecular function is known for 4 mutations affecting individualization of the 9 studied. In 2 cases (dud and cbx), the products are involved in protein ubiquitination, whereas in the other cases (Chc4 and scat), the products are the proteins directly involved in the vesicular trafficking. Monoubiquitination is a marker of internalization of yeast cell receptors; therefore, it is probable that the first 2 genes are also involved in the vesicular trafficking. These data on Gish protein confirm that the protein is involved in spermatid internalization and that it displays a very close relation to the genes of yeast vesicular trafficking (Nerusheva, 2009).

Regulation of wingless signaling by the CKI family in Drosophila limb development

The Wingless (Wg)/Wnt signaling pathway regulates a myriad of developmental processes and its malfunction leads to human disorders including cancer. Recent studies suggest that casein kinase I (CKI) family members play pivotal roles in the Wg/Wnt pathway. However, genetic evidence for the involvement of CKI family members in physiological Wg/Wnt signaling events is lacking. In addition, there are conflicting reports regarding whether a given CKI family member functions as a positive or negative regulator of the pathway. This study examined the roles of seven CKI family members in Wg signaling during Drosophila limb development. Increased CKIepsilon stimulates whereas dominant-negative or a null CKIepsilon mutation inhibits Wg signaling. In contrast, inactivation of CKIalpha by RNA interference (RNAi) leads to ectopic Wg signaling. Interestingly, hypomorphic CKIepsilon mutations synergize with CKIalpha RNAi to induce ectopic Wg signaling, revealing a negative role for CKIepsilon. Conversely, CKIalpha RNAi enhances the loss-of-Wg phenotypes caused by CKIepsilon null mutation, suggesting a positive role for CKIalpha. While none of the other five CKI isoforms can substitute for CKIalpha in its inhibitory role in the Wg pathway, several CKI isoforms including CG12147 exhibit a positive role based on overexpression. Moreover, loss of Gilgamesh (Gish)/CKIgamma attenuates Wg signaling activity. Finally, evidence is provided that several CKI isoforms including CKIalpha and Gish/CKIgamma can phosphorylate the Wg coreceptor Arrow (Arr), which may account, at least in part, for their positive roles in the Wg pathway (Zhang, 2006).

The Wnt family of secreted growth factors controls many key developmental processes, including cell proliferation, cell fate determination, tissue patterning, and planar cell polarity in a wide variety of organisms. Mutations in Wnt signaling components lead to many types of cancers including colon and skin cancers. The Drosophila Wingless (Wg), a founding member of the Wnt family, controls embryonic segmental polarity and patterning of adult appendages such as wing, leg, and eye. Wg exerts its biological influence through the canonical Wnt/β-catenin pathway, which is evolutionarily conserved from invertebrates to vertebrates (Zhang, 2006).

Genetic and biochemical studies in several organisms have suggested a model for Wnt/Wg signal transduction. Binding of Wnt/Wg proteins to their cognate receptors, members of the Frizzled (Fz) family of seven transmembrane proteins, and co-receptors, LRP5/6/Arrow (Arr), activates a cytoplasmic signaling component Dishevelled (Dsh), which counteracts the activity of a destruction complex composed of Axin, APC, and the Ser/Thr kinase GSK3β/Shaggy (Sgg)/Zest White 3 (Zw3), leading to the accumulation and nuclear translocation of the transcriptional effector β-catenin/Armadello (Arm). β-catenin/Arm forms a complex with the DNA binding protein Lef1/TCF to activate Wnt/Wg target genes (Zhang, 2006).

A cohort of studies have provided evidence that CKI family members participate in many aspects of the Wnt/Wg signaling pathway (Price, 2006). CKIε was first identified as a positive regulator of the canonical Wnt pathway. Overexpression of CKIε in Xenopus embryos induced ectopic dorsal axis formation, activated Wnt-responsive genes, and rescued the axial formation of UV treated embryos. Dominant negative forms of CKIε and a pharmacological inhibitor of CKI blocked the responses to ectopic Wnt signaling in Xenopus. Biochemical and epistasis study suggested that CKIε binds Dsh and acts between Dsh and GSK3β. In vivo and In vitro kinase assays showed that CKIε can phosphorylate Dsh and a pharmacological CKI inhibitor can block Wnt induced Dsh phosphorylation, suggesting that Dsh is a target of CKIε. However, the role of CKIε appears to be more complex than it was originally anticipated. For example, it has also been shown that CKIε interacts with Axin, and Axin-bound CKIε phosphorylates APC and modulates its ability to regulate β-catenin. What makes the picture even more complicated is the finding that, in a reconstituted system of Xenopus extracts, CKIε can phosphorylate Tcf3 and enhance Tcf3-β-catenin association and β-catenin stability, implying that CKIε may also exert a positive influence downstream of GSK3β (Zhang, 2006 and references therein).

The potential role of other CKI isoforms in Wnt signaling has also been examined in several systems. In an overexpression study using Xenopus embryonic explants, all other CKI isoforms, including α, β, γ, and δ, can activate Wnt signaling (McKay, 2001). All of these CKI isoforms with the exception of CKIγ can stimulate Dsh phosphorylation in cultured cells. However, subsequent studies provided evidence that CKIα plays a negative role in Wnt/Wg signaling that acts as a priming kinase for GSK3β-mediated phosphorylation of β-catenin/Arm. Purification of the Axin-bound kinases that can prime GSK3β-mediated phosphorylation of β-catenin identified CKIα. RNAi knockdown of CKIα inhibited phosphorylation at Ser45 of β-catenin and subsequent phosphorylation by GSK3β, resulting in β-catenin stabilization. Consistent with the vertebrate results, CKIα RNAi of Drosophila embryos resulted in 'naked cuticle', a phenotype consistent with gain-of-Wg signaling (Liu, 2002). The possible role of CKIε as a priming kinase for β-catenin remained unclear. Overexpression of a dominant negative CKIε inhibited Axin-induced phosphorylation at Ser45 of β-catenin in 293 cells. In addition, RNAi knockdown of CKIε stabilized Arm in Drosophila S2+ cells, although the effect was less dramatic than CKIα RNAi knockdown. In contrast, RNAi knockdown of CKIε in 293T cells had no detectable effect on Ser45 phosphorylation and stability of β-catenin. It remains possible that CKIε plays a minor partially redundant role in β-catenin/Arm phosphorylation and the effect of its inactivation on β-catenin/Arm phosphorylation and degradation could have been masked by CKIα (Zhang, 2006 and references therein).

Although CKIα RNAi in Drosophila embryos resulted in phenotypes consistent with 'gain-of-Wg' function, the recent finding that CKIα is also a negative regulator of the Hh pathway complicated the interpretation. Because Wg and Hh cross-regulate each other during embryonic development, the 'gain-of-Wg' phenotype resulted from CKIα RNAi could be attributed to ectopic Hh signaling. To further investigate the physiological roles of the CKI family members in Wg signaling In vivo, overexpression, dominant-negative, genetic mutations, and RNAi approaches were applied to study the function of CKIε, CKIα and Gish/CKIγ in Drosophila wing development where Wg signaling is independent of Hh. The potential roles of other CKI family members were also assessed (Zhang, 2006).

This study provides the first genetic evidence that DBT/CKIε plays a pivotal positive role in the Wg pathway and provides evidence that DBT/CKIε exerts its positive influence both upstream and downstream of GSK3β. Moreover, the first genetic evidence is provided that DBT/CKIε has a negative role in addition to its predominantly positive role in the Wg pathway. Using RNAi, evidence that CKIα is the major CKI isoform that negatively regulates Wg signaling in Drosophila wing development. In addition, evidence is provided that CKIα may also have an unappreciated positive role and this could be achieved, at least in part, at the level of Arr phosphorylation. Finally, genetic evidence is provided that Gish/CKIγ has a positive role in the Wg pathway. Consistent with this finding, a recent study showed that RNAi knockdown of Gish in cultured cells reduced Wg-stimulated luciferase reporter gene expression (Davidson, 2005). In addition, it was found that Gish/CKIγ, like its vertebrate counterpart, is mainly localized on the cell surface, and can effectively phosphorylate Arr, which may account for its positive role in the Wg pathway (Zhang, 2006).

CKIε was initially identified as a positive regulator in the Wnt pathway based on overexpression studies. Indeed, overexpression of XCKIε in Drosophila limb caused cell autonomous accumulation of Arm and activation of Wg responsive genes, leading to pattern abnormality consistent with ectopic Wg signaling. Although DBT/CKIε shares over 85% amino acid sequence identity with XCKIε in the kinase domain, overexpression of DBT or its kinase domain didn't induce ectopic Wg signaling. Nevertheless, overexpression of DBT induced ectopic Wg signaling in a sensitized genetic background (Zhang, 2006).

Despite the fact that CKIε has been implicated as a positive regulator of the Wnt/Wg pathway, no genetic evidence for such a role has ever been obtained until now. One reason could be that CKIε participates in multiple cellular processes and null or strong mutations cause cell lethality. In contrast, hypomorphic mutations do not significantly perturb Wg signaling, probably because a low dose of CKIε suffices to transduce the Wg signal and/or because other CKI family members can compensate for the partial loss of CKIε. To facilitate the recovery of mutant clones homozygous for dbt null mutation, a combination of several approaches was applied: (1) mitotic clones were generated in the Minute background, which gave mutant cells a growth advantage; (2) P35, a cell death inhibitor, was overexpressed in discs where dco mutant clones were generated to block apoptosis due to loss of CKIε; (3) a wing specific, constitutive source of flipase (MS1096/UAS-flp) was used to induce FRT-mediated mitotic recombination in the wing pouch region. Under these conditions, all wing discs of the appropriate genotype contained dco⁻ clones occupying most of the wing pouch region. These wing discs exhibited diminished levels of Wg target gene expression, demonstrating that DBT/CKIε is a positive regulator of the Wg pathway. The approach described in this study can be applied to study other cell lethal genes (Zhang, 2006).

Although most of the evidence supports a positive role for CKIε in the Wnt/Wg pathway, several observations implied that CKIε also impinged on β-catenin/Arm phosphorylation and degradation. For example, it has been shown that CKIε is associated with Axin and DN-CKIε blocks Axin-induced phosphorylation of β-catenin at Ser45. In addition, RNAi knockdown of DBT/CKIε resulted in stabilization of Arm in S2 cells, albeit to a lesser extent than CKIα knockdown, and increased the basal transcription from a Tcf-luciferase reporter gene. However, one caveat of these studies is that the activities of other CKI isoforms might also be affected by DN-CKIε or DBT/CKIε RNAi. A genetic approach was taken to address whether DBT/CKIε has any negative function in the Wg pathway, and hypomorphic dbt mutations were found to cause ectopic Wg signaling, but only when CKIα activity was partially blocked. Hence, DBT/CKIε is normally dispensable for Arm degradation due to sufficent CKIα; however, DBT/CKIε levels become critical when CKIα activity is reduced. This result is not inconsistent with a previous observation that CKIε RNAi did not affect β-catenin phosphorylation and degradation in cultured cells (Liu, 2002). In that study, RNAi did not completely block CKIε, and the presence of CKIα in the same cells could have masked any effect CKIε RNAi might have had on β-catenin phosphorylation and degradation. It would be interesting to determine if CKIε RNAi could enhance the effect of CKIα RNAi on β-catenin phosphorylation and degradation, which is predicted by the current study (Zhang, 2006).

CKIε binds and phosphorylates Dsh. However, a previous study placed CKIε downstream of Dsh based on the observation that overexpressing XCKIε could rescue Wnt signaling defects caused by a dominant negative form of Dsh (DN-Dsh). In contrast, this study found that the ability of XCKIε to induce Wg pathway activation depends on Dsh, as dsh null mutant clones overexpressing XCKIε fail to activate Wg target genes. Hence genetic epistasis study places CKIε upstream of or parallel to Dsh. It is possible that DN-Dsh might not completely block endogenous Dsh, and overexpressed XCKIε could transduce the Wnt signal through residual Dsh activity. Consistent with the notion that CKIε acts upstream of or parallel to Dsh, it was found that coexpression of Nkd, an inducible Wg pathway inhibitor that acts by binding to Dsh, suppresses the 'gain-of-Wg' phenotypes caused by XCKIε. In addition, DN-GSK3β can reverse the 'loss-of-Wg' phenotypes caused by DN-CKIε. Hence a critical role that CKIε plays is to antagonize the activity of the Arm/β-catenin destruction complex, and antagonism of GSK3β alleviates such a requirement. CKIε could bind Dsh and destabilize the Arm/β-catenin destruction complex. In addition, CKIε could destabilize Axin complex through phosphorylation of Arr (Zhang, 2006).

Epistasis analysis also revealed a role for CKIε downstream of GSK3β phosphorylation. It was found that the levels of ectopic sen in wing discs coexpressing DN-CKIε and DN-GSK3β are significantly lower than those in wing discs expressing DN-GSK3β alone, suggesting that DN-CKIε attenuates Wg signaling activity even when phosphorylation and degradation of Arm is blocked by DN-GSK3β. One likely target for CKIε downstream of GSK3β is Tcf as it has been shown that in Xenopus oocyte extracts, CKIε phosphorylated Tcf3 and stabilized its interaction with β-catenin (Zhang, 2006).

The role of CKIα in the Wnt/Wg pathway has largely been deduced from studies using cell culture systems. Thus, RNAi knockdown of CKIα inhibits β-catenin/Arm phosphorylation and degradation, and induces Tcf/Lef mediated luciferase expression. CKIα RNAi in Drosophila embryos resulted in a 'naked cuticle' phenotype, consistent with ectopic Wg signaling (Liu, 2002; Yanagawa, 2002). However, two recent studies revealed that loss-of-CKIα also results in ectopic Hh signaling. This finding complicated the interpretation of the 'gain-of-Wg' phenotypes resulting from CKIα RNAi as Hh and Wg regulate each other’s expression in Drosophila embryos. To circumvent this problem, this study used Drosophila wing development as a model to address the In vivo function of CKIα since Wg and Hh do not regulate each other in this system. It was found that overexpressing two shorter forms of CKIα RNAi constructs (CRS and CRS2), which are specific for CKIα, led to ectopic Wg signaling in a dose dependent manner: one copy of CRS or CRS2 barely affected Wg target gene expression whereas two copies resulted in ectopic expression of sc and sen. A longer form of CKIα RNAi construct (CRL) was more potent than CRS, as expressing one copy resulted in robust ectopic expression of sc and sen. This is likely due to the fact that CRL knocks down CKIα more effectively than CRS. In addition, CRL may knock down DBT/CKIε to reduce a compensatory effect on loss of CKIα by DBT/CKIε. Intriguingly, expressing CRL at higher levels caused adverse effect on the Wg signaling activity, as manifested by the reduced levels of ectopic sc expression. A likely explanation is that high levels of CRL diminish the level of CKIε to the extent that its positive role in the Wg pathway is compromised. In support of this notion, coexpressing DBT/CKIε with CRL restored high levels of ectopic sc expression (Zhang, 2006).

Despite the predominantly negative role of CKIα in the Wg pathway, a positive role has been underscored in double mutant analysis. It was observed that CKIα knockdown enhanced the 'loss-of-Wg' phenotypes caused by dbt null mutation, as manifested by more complete loss of sen and vg expression in dbt mutant discs expressing CRS2. CKIα may positively regulate Wg signaling by phosphorylating Dsh, as suggested by previous studies. Alternatively, CKIα could exert a positive influence on the Wg pathway by phosphorylating Arr (Zhang, 2006).

Overexpression assays were applied to explore the potential role of the other five CKI isoforms that share over 50% amino acid sequence identity in their kinase domains with CKIα. First, it was asked if any of these CKI isoforms could functionally substitute for CKIα in blocking Wg pathway activation. Unlike CKIα, none of other CKI isoforms including CG7094, CG2577, CG12147, Gish/CKIγ, and CG9962 were able to rescue the 'gain-of-Wg' phenotype caused by CRL, suggesting that these CKI isoforms are unlikely to play any major role in priming GSK3β-mediated phosphorylation and degradation of Arm/β-catenin. In contrast, CG12147 induced ectopic Wg signaling activity when CKIα was partially blocked, albeit to a lesser extent than DBT. Although Gish overexpression failed to induce ectopic Wg signaling activity even when CKIα was partially blocked, loss-of-Gish mutation resulted in a reduction in Wg signaling activity and enhanced the 'loss-of-Wg' phenotypes caused by the dbt null mutation, suggesting that Gish/CKIγ positively regulates the Wg pathway (Zhang, 2006).

It has recently been shown that CKI family members phosphorylate multiple sites in the cytoplasmic domain of LRP6 (Davidson, 2005; Zeng, 2005) and a set of these CKI sites are primed by GSK3β phosphorylation of the PPPSP motif (Zeng, 2005). Overexpressing CKIγ but not CKIε caused phosphorylation of LRP6, whereas dominant negative CKIγ inhibited Wnt3a-induced LRP6 phosphorylation in HEK293T cells (Davidson, 2005), suggesting a specific role for CKIγ in phosphorylating LRP6. In contrast, Zeng showed that a combination of dominant negative CKIα and CKIδ but neither CKIα or CKIδ alone blocked Wnt3a-induced LRP6 phosphorylation in CKIε^−/− MEF cells, suggesting that CKIα and CKIγ/ε act redundantly in phosphorylating LRP6 in response to Wnt (Zeng, 2005). However, dominant negative CKI isoforms may not exhibit absolute specificity, which could account for the discrepancy between these two studies. While it awaits for genetic mutations in individual CKI isoforms to confirm the results obtained with the dominant negative forms of CKI, it is likely that multiple CKI family members could participate in LRP5/6 phosphorylation (Zhang, 2006).

Multiple PPPSP motifs as well as adjacent CKI sites are conserved in the cytoplasmic domain of Drosophila Arr. In Drosophila S2 cells, multiple CKI family members can phosphorylate Arr cytoplasmic domain and this phosphorylation appears to rely on GSK3β primed phosphorylation. Among all the CKI isoforms that can phosphorylate Arr, Gish/CKIγ exhibited the highest potency whereas CKIα and CKIε show weak activity toward Arr, suggesting that Gish/CKIγ is the major CKI isoform that phosphorylates Arr. Consistent with its high potency toward Arr phosphorylation, Gish/CKIγ is primarily associated with plasma membrane, as is the case for its vertebrate counterpart (Davidson, 2005). Phosphorylation of Arr by Gish/CKIγ is likely to account for the positive role that Gish/CKIγ plays in the Wg signaling pathway. It was found that gish^e01759 attenuates but not completely blocks Wg responsive gene expression. The residual Wg signaling activity in gish^e01759 mutant cells could be due to the hypomorphic nature of this mutation. Alternatively, other CKI isoforms could partially substitute for Gish/CKIγ in phosphorylating Arr (Zhang, 2006).

CG12147 and CG9962 phosphorylate Arr more effectively than CKIα or CKIε, although they are less potent than Gish/CKIγ. Consistent with their ability to phosphorylate Arr, overexpressing CG12147 or CG9962 resulted in ectopic Wg signaling in a genetic sensitized background. However, phosphorylation of Arr alone might be insufficient to account for their positive roles as overexpressing Gish/CKIγ did not have the same magnitude of effect on Wg signaling as CG12147 and CG9962. It is possible that CG12147 and CG9962 can phosphorylate other targets in the Wg pathway. Future loss of function study and biochemical analysis should probe the precise roles of these CKI isoforms in the Wg pathway (Zhang, 2006).

Temporal control of glial cell migration in the Drosophila eye requires gilgamesh, hedgehog, and eye specification genes

In the Drosophila visual system, photoreceptor neurons (R cells) extend axons towards glial cells located at the posterior edge of the eye disc. In gilgamesh (gish) mutants, glial cells invade anterior regions of the eye disc prior to R cell differentiation and R cell axons extend anteriorly along these cells. gish encodes casein kinase Igamma. gish, sine oculis, eyeless, and hedgehog (hh) act in the posterior region of the eye disc to prevent precocious glial cell migration. Targeted expression of Hh in this region rescues the gish phenotype, though the glial cells do not require the canonical Hh signaling pathway to respond. It is proposed that the spatiotemporal control of glial cell migration plays a critical role in determining the directionality of R cell axon outgrowth (Hummel, 2002).

In the developing Drosophila visual system, photoreceptor cell (R cell) axons contact numerous glial cell types along the pathway to the target and within the target itself. These glial cells originate from different regions of the developing visual system and migrate to their final destinations where they associate with axons. The first glial cells encountered by extending R cell axons are retinal basal glial (RBG) cells located along the basal surface of the eye disc epithelium. As R cells differentiate, they extend growth cones basally where they contact RBG cells and turn posteriorly toward the optic stalk. These glial cells originate in the optic stalk and migrate into the eye disc. Migration is temporally and spatially linked to R cell development. The extension of axons from R cell clusters occurs in a sequential fashion that reflects the highly ordered pattern of R cell differentiation in the eye disc. R cells in the posterior region differentiate first and additional R cell clusters form more anteriorly as a wave of differentiation sweeps across the eye disc. The leading edge of this wave is marked by a depression in the apical region of the disc epithelium called the morphogenetic furrow (MF) (Hummel, 2002).

Glial cells start to migrate into the eye disc as R cell differentiation at the posterior margin is initiated. During MF progression, RBG cells migrate along the basal surface up to but not past the youngest R cell axons. RBGs appear to migrate along R cell axons thereby making a guidance role for them unlikely. Glial cells can migrate out of the optic stalk into the eye disc in the absence of axon contact; instead, glial cells are essential for R cell axons to enter the optic stalk. These observations suggest that the precise coordination of glial cell development in the optic stalk and R cell differentiation in the eye disc is important for normal visual system development. In the course of a genetic screen for axon guidance mutants, a loss-of-function mutation was identified that temporally uncoupled glial cell migration from R cell differentiation. Mutations in the gilgamesh locus lead to precocious glial cell migration from the optic stalk into the eye disc prior to R cell differentiation. As a result, glial cells are misplaced anteriorly in the eye disc as R cell axons extend. R cell axons frequently project along pathways demarcated by these ectopic glial cells, providing strong evidence that the positioning of glial cells plays an instructive role in regulating the directionality of R cell axon outgrowth in the eye disc. The gish locus acts in conjunction with the eye specification genes, eyeless and sine oculis, to prevent precocious entry of glial cells into the eye disc. Loss-of-function hh mutants exhibit precocious glial cell migration and targeted expression of Hh in the posterior region of the eye disc suppresses the ectopic glial cell migration phenotype in gish mutants (Hummel, 2002).

A set of genes encoding nuclear proteins [e.g., eyeless (ey), eyes absent (eya), sine oculis (so) and secreted factors such as hedgehog (hh)] regulates the initiation of neuronal differentiation in the posterior region of the eye disc. The effect of loss-of-function mutations in these genes on glial cell migration was tested. As in gish mutants, glial cells migrate precociously out of the optic stalk in a hh temperature-sensitive mutation incubated at the nonpermissive temperature during first and second instar. This is an early function of hh, since ectopic glial cells are not observed in hh¹; in this allele, the posterior eye field develops normally, but anterior progression of the MF is inhibited. A similar early onset glial cell migration defect is observed in eye-specific alleles of so and ey. In contrast, glial cells did not migrate out from the optic stalk in an eye-specific allele of eya, raising the possibility that eya is required to activate glial cell migration. Since glial cells migrate out of the stalk precociously in eya/gish double mutants, the production of an eya-dependent signal is not necessary to promote anterior migration. Hence, in contrast to their role in R cell development, eye specification genes ey and so seem to function independent of eya to control the onset of glial cell migration (Hummel, 2002).

These observations raise the possibility that gish also contributes to the genetic circuitry regulating eye specification. Indeed, while ey-FLP-induced clones of gish lead to only minor defects in MF progression during third instar stage, gish mutant adult eyes are smaller and frequently contain a reduced number of ommatidia in the anterior region. These phenotypes are frequently seen in weak alleles of eye specification genes. Furthermore, double mutants of ey¹ and gish¹ , as well as so¹ and gish¹, reveal strong synergistic effects in R cell development. The glial cell migration phenotypes in double mutants is similar in severity to the single mutants. In summary, these data argue that gish acts in conjunction with eye specification genes to coordinate neuronal development and glial cell migration in the eye disc (Hummel, 2002).

Since Hh is expressed in the right place and time to function as the glial cell repellent, and since glial cells migrate precociously in hh mutants, it became important to assess whether Hh directly regulates glial cell migration. In support of this view, targeted expression of Hh at the posterior region of the second instar eye disc rescues precocious cell migration in gish mutants. In about 80% of the gish mutant larvae carrying Dpp-Gal4 driving UAS-hh, premature glial cell migration was prevented in second instar, and glial cells remain posterior to the MF at third instar (Hummel, 2002).

Does Hh directly regulate glial cell migration? To address this question, whether the canonical Hh signaling pathway is activated in glial cells was assessed and whether it is required to prevent precocious migration. patched (ptc) expression, an indicator of reception of the Hh signal, is not elevated in glial cells prior to migration into the eye disc. It is, however, induced in epithelial cells located at the most posterior edge of the eye disc immediately juxtaposed to the pre-migratory glial cells in the optic stalk. In support of the importance of the posterior margin in signaling glial cells, a significant reduction in the level of ptc-lacZ expression was observed in this region of the second instar eye in gish mutants. Alternatively, this reduction may simply provide a sensitive indicator that the level of Hh is reduced in gish mutants. Since the level of Hh protein is only slightly above background in wild-type, the level of the Hh signal in these mutants could not be critically assess with the available reagents. Further evidence that the canonical Hh pathway is not required in glial cells came from the analysis of mutations in smoothened (smo) and Cubitis interruptus (Ci) genes encoding a Hh receptor component and a downstream transcription factor, respectively. Mosaic clones of smo mutant glial cells do not migrate prematurely at second instar and were found exclusively posterior to the MF. Similarly, targeted expression of a dominant-negative version of Ci in glial cells does not alter early migratory behavior of eye disc glial cells. These findings argue that Hh acts either indirectly to control glial cell migration or acts directly upon glial cells through a novel pathway independent of ptc, smo, and Ci (Hummel, 2002).

Thus, the timing of glial cell migration plays an essential role in regulating axon guidance in the fly visual system. Glial cells in the posterior region of the eye disc epithelium provide an intermediate target for R cell axons as they project from the eye to the brain. In gish mutants, glial cells migrate out of the optic stalk to more anterior regions of the eye disc prior to R cell differentiation, and as a consequence, R cell axons frequently extend anteriorly in gish mutants along the surface of these ectopically located glial cells. gish acts in combination with eye specification genes, ey and so, and the extracellular signaling protein Hh to control glial cell migration. Since these genes, including gish, also regulate neuronal development in the eye disc, it is proposed that they define a signaling center in the posterior region of the eye disc that controls both neurogenesis and glial cell migration to ensure normal patterns of R cell axon outgrowth (Hummel, 2002).

Through genetic mosaic analysis and transgene rescue experiments, it has been established that gish acts within the eye disc epithelium to inhibit glial cell migration. In principle, gish could regulate the production of a repellent preventing migration of glial cells out of the optic stalk, an attractant that promotes their close association to the posterior edge or alternatively an antagonist to an attractive signal produced by cells in the disc. Gish belongs to the casein kinase I family of highly conserved and widely expressed enzymes. These enzymes contain small but varied amino termini and large, highly diverse carboxy-terminal domains. Gish is most similar to mammalian CKIgamma3. CKIgamma3, like gish, is alternatively spliced -- this can result in kinases with different biochemical properties and functions. CKIs act on proteins previously phosphorylated by other kinases. CKIs have been shown to phosphorylate a large number of proteins in vitro. Regrettably, little evidence exists to establish a link between phosphorylation by CKI and specific developmental pathways. Recent studies have shown that casein kinase Iepsilon (CKIepsilon) can regulate ß-catenin in the Wnt pathway in both worms and frogs. Loss- and gain-of-function manipulation of Wg signaling components, however, did not disrupt glial cell migration into the eye disc. This is not surprising given that CKIepsilon and CKIgamma differ significantly in their C-terminal regions and deletion analysis has revealed that the unique C-terminal domain is important for the interaction of CKIepsilon with the axin signaling complex. Since gish does not encode a secreted molecule, it must act indirectly to affect signaling from the epithelium to the glial cells (Hummel, 2002).

Several lines of evidence support a model in which gish regulates Hh signaling: (1) like gish, hh is required in the posterior region of the second instar eye disc to inhibit anterior migration of glial cells; (2) gish mutants display defects in morphogenetic furrow progression similar to those seen for hypomorphic alleles of hh, and (3) the expression of Hh target genes ptc and Ci in the eye disc epithelium is reduced in gish mutants. These data suggest that gish acts upstream of (or in parallel to) hh. Although genetic evidence suggests that the gish mutants studied here are strong loss-of-function alleles, since low levels of gish mRNA can still be detected in homozygous gish¹ larvae, the epistatic relationship between gish and hh must be qualified. Indeed, Gish may function downstream from Hh and limiting levels of activity in loss-of-function alleles may be compensated by increasing the level of Hh upstream (Hummel, 2002).

The location of a specific subtype of glial cells is not only necessary for posterior-directed outgrowth of R cell axons, but also sufficient. Indeed, some 50% of gish mutant eye discs at an early stage of development contain R cell fibers that project anteriorly along the surface of misplaced glial cells. During normal development, however, more anterior R cells differentiate as a continuous wave, and glial cells migrate to a region just posterior to differentiating R cells. As a consequence, as these axons extend to the basal surface, they contact glial cells that lie posterior to them and follow them into the optic stalk. Axon-glial cell interactions in the fly eye show interesting parallels to intra-retinal pathfinding in the vertebrate eye. Retinal ganglion cell axons project radially toward the optic disk, their first intermediate target in the center of the eye. Here, axons receive contact-dependent signals from glial cells to exit the retina and enter the optic nerve. In addition, in vertebrates, a barrier at the junction between the optic nerve and the retina has been proposed to inhibit glial cell precursors from migrating into the retina. It will be interesting to assess whether the molecular strategies coordinating axon outgrowth and glial cell migration have been conserved between vertebrate and invertebrate visual systems (Hummel, 2002 and references therein).

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Davidson, G., Wu, W., Shen, J., Bilic, J., Fenger, U., Stannek, P., Glinka, A. and Niehrs, C. (2005). Casein kinase 1 gamma couples Wnt receptor activation to cytoplasmic signal transduction. Nature 438(7069): 867-72. PubMed ID: 16341016

Hummel, T., Attix, S., Gunning, D. and Zipursky, S. L. (2002). Temporal control of glial cell migration in the Drosophila eye requires gilgamesh, hedgehog, and eye specification genes. Neuron 33: 193-203. PubMed ID: 11804568

Li, S., Tian, A., Li, S., Han, Y., Wang, B. and Jiang, J. (2020). Gilgamesh (Gish)/CK1gamma regulates tissue homeostasis and aging in adult Drosophila midgut. J Cell Biol 219(4). PubMed ID: 32328627

Liu, C., et al. (2002). Control of beta-catenin phosphorylation/degradation by a dual-kinase mechanism. Cell 108: 837-47. PubMed ID: 11955436

McKay, R. M., Peters, J. M. and Graff, J. M. (2001). The casein kinase I family in Wnt signaling. Dev. Biol. 235: 388-96. PubMed ID: 11437445

Meng, Z., Capalbo, L., Glover, D. M. and Dunphy, W. G. (2011). Role for casein kinase 1 in the phosphorylation of Claspin on critical residues necessary for the activation of Chk1. Mol Biol Cell 22(16): 2834-2847. PubMed ID: 21680713

Nerusheva, O. O., et al. (2009). A GFP trap study uncovers the functions of Gilgamesh protein kinase in Drosophila melanogaster spermatogenesis. Cell Biol. Int. 33(5): 586-93. PubMed ID: 19269340

Price, M. A. (2006). CKI, there'’s more than one: casein kinase I family members in Wnt and Hedgehog signaling. Genes Dev. 20: 399-410. PubMed ID: 16481469

Swarup, S., Pradhan-Sundd, T. and Verheyen, E. M. (2015). Genome-wide identification of phospho-regulators of Wnt signaling in Drosophila. Development 142(8): 1502-1515. PubMed ID: 25852200

Yanagawa, S., et al. (2002). Casein kinase I phosphorylates the Armadillo protein and induces its degradation in Drosophila. Embo J. 21: 1733-42. PubMed ID: 11927557

Zeng, X., et al. (2005). A dual-kinase mechanism for Wnt co-receptor phosphorylation and activation. Nature 438: 873-7. PubMed ID: 16341017

Zhang, L., et al. (2006). Regulation of wingless signaling by the CKI family in Drosophila limb development. Dev. Biol. 299(1): 221-37. PubMed ID: 16987508

date revised: 15 December 2020

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