discs overgrown


REGULATION

Protein Interactions

Because Dbt and Per appear to be expressed in the same cells in the Drosophila brain and eyes, and patterns of Per phosphorylation and accumulation are altered in dbt mutants, it was asked whether functional interactions between Per and Dbt might include a physical association of these proteins. Two independent methods were employed to test and confirm such a physical interaction: in vitro binding studies, and coimmunoprecipitation of Dbt and Per from cultured Drosophila cells (S2) programmed to express both proteins. In vitro translation of Dbt from dbt cDNA reveals a protein of ~46 kDa as predicted from sequence analysis. GST (glutathione-S-transferase) fusions, involving varying segments of Per, were tested for evidence of affinity for this Dbt protein. The GST-Per fusions were immobilized on glutathione agarose beads and subsequently incubated with in vitro translated, 35S-labeled DBT. After extensive washing to remove nonspecifically bound proteins, SDS-PAGE analysis of labeled Dbt proteins bound to the beads showed that Dbt binds to Per 1-640 and Per 1-365, but the protein does not bind to Per 530-640 or GST alone. These results show that Dbt and Per can physically associate in vitro and that Dbt interacts directly with an N-terminal region of Per (Kloss, 1998).

The clock gene double-time (dbt) encodes an ortholog of casein kinase Iepsilon that promotes phosphorylation and turnover of the Period protein. Whereas the period, timeless, and Clock genes of Drosophila each contribute cycling mRNA and protein to a circadian clock, dbt RNA and Dbt protein are constitutively expressed. Robust circadian changes in Dbt subcellular localization are nevertheless observed in clock-containing cells of the fly head. These localization rhythms accompany formation of protein complexes that include Per, Tim, and Dbt, and reflect periodic redistribution between the nucleus and the cytoplasm. Nuclear phosphorylation of Per is strongly enhanced when Tim is removed from Per/Tim/Ddt complexes. The varying associations of Per, Ddt and Tim appear to determine the onset and duration of nuclear Per function within the Drosophila clock (Kloss, 2001).

Dbt RNA levels are constant throughout the day. In this respect, the same is true for Dbt protein levels, since there was no detectable circadian oscillation of Dbt accumulation in timed head extracts. Furthermore, a variety of mutations disrupting the circadian clock and molecular oscillations have no effect on the level of Dbt protein. Thus, production of Dbt protein is not under the control of clock genes. In contrast, the subcellular localization of Dbt in the lateral neurons and photoreceptor cells changes over the course of a daily cycle. Dbt is consistently detected in the nucleus. However, at the end of the day and in the early part of the night, a substantial increase is found in cytoplasmic Dbt, coincident with the cytoplasmic accumulation of Per proteins and Per/Tim complexes. Furthermore, when Per/Tim complexes translocate to the nucleus at ~ZT18, and early during the day when Per remains in the nucleus in absence of Tim, a substantial nuclear accumulation of Dbt is observed. These changes in subcellular location of Dbt appear to be influenced exclusively by the locus of Per accumulation (in the presence or absence of Tim). Tim protein has little or no effect on the localization of Dbt because Dbt is always detected in the nucleus in per01 flies, which lack Per and have a substantial amount of Tim in the cytoplasm. Consequently, there is circadian regulation of Dbt proteins, in the form of a changing subcellular distribution. The fact that the movement of Per and Tim from the cytoplasm to the nucleus predicts the distribution of Dbt implies a close correspondence between maximum levels of Per/Tim complex and cytoplasmic levels of Dbt. Such a relationship could indicate that Tim associates with cytoplasmic Per once the latter protein has effected cytoplasmic localization of most cellular Dbt (Kloss, 2001).

Because Dbt preferentially accumulates in nuclei in the absence of Per, cytoplasmic Per proteins must affect this default localization at certain times of day in wild-type flies. Although the half-life of Dbt has not been determined, Dbt RNA and proteins are constantly synthesized. Therefore, the subcellular fate of newly translated Dbt may simply depend on whether cytoplasmic Per is available to associate with Dbt and retard its nuclear translocation. Alternatively, accumulation of Dbt may involve mechanisms promoting both nuclear import and export, with the predominant localization of Dbt governed by the presence or absence of cytoplasmic Per. Regardless of the specific mechanism, since Dbt has also been implicated in vital developmental and cellular functions that are not mediated through Per, an important product of any device generating cycling subcellular localization of this kinase could be temporal regulation of its access to alternative substrates (Kloss, 2001).

Dbt has been shown to be a component of the cytoplasmic activity that destabilizes Per. Evidence was also found that Dbt influences the stability of nuclear Per proteins. However, it has been unclear whether Dbt acts in both subcellular compartments, or whether nuclear stability of Per is affected by a Dbt-dependent phosphorylation in the cytoplasm, with delayed effects once Per translocates into the nucleus. This study shows that Dbt proteins are found both in the cytoplasm and in the nucleus. Coupled with the finding that Per proteins are always found associated with Dbt, this suggests that Dbt is required both in the nucleus and in the cytoplasm for Per phosphorylations (Kloss, 2001).

The simultaneous changes in subcellular localization of Per, Tim, and Dbt make it likely that direct physical associations among these proteins cause the cycling Dbt localizations. Per and Dbt proteins can associate in vitro and in cultured cells. Per/Dbt complexes can be recovered at all times during the day from head extracts, regardless of whether the majority of these proteins are localized in the cytoplasm or in the nucleus. Thus, Per proteins are associated with Dbt proteins in vivo when Per is in a Per/Tim complex and when Per proteins are free from Tim (Kloss, 2001).

Conversely, while Dbt binds to Per and Per/Tim complexes, no evidence has been found that Tim protein, free from Per, associates with Dbt in vivo. This finding is in line with the conclusion that Dbt's effects on the circadian clock are primarily mediated through Per (Kloss, 2001).

Extensive efforts have failed to obtain a functional assay for bacterially produced, recombinant Dbt in vitro. The putative kinase domains of Dbt and its mammalian ortholog CKIepsilon are very closely related (86% aa identity), so it was surprising to find that recombinant, mammalian CKIepsilon readily phosphorylates Drosophila Per and human Per in vitro. These observations suggest that Dbt function might be tightly regulated in the fly. It has been established that truncation of mammalian CKIepsilon substantially increases its activity in vitro, and truncated forms of the enzyme were used in the above mentioned Per and hPer assays. Although a corresponding truncation of Dbt failed to generate activity, such studies of mammalian CKIepsilon also indicate more complex regulation for this kinase in vivo (Kloss, 2001).

Without direct kinetic measurements of the activity of Dbt at different times of day, it cannot be determine whether Dbt function is under circadian control. However, it can be asked whether Per phosphorylation in vivo is (1) dependent upon the presence of Dbt and (2) influenced by Tim. In timUL flies entrained to LD 12:12, where Per remains complexed with TimUL for a prolonged interval in the nucleus, Per remains hypophosphorylated during the dark phase. Because wild-type flies begin to phosphorylate their Per proteins during the dark phase of such LD cycles, the results with timUL suggest that Tim influences the timing of light-independent Per phosphorylation (Kloss, 2001).

Light-triggered removal of TimUL protein is correlated with a rapid and progressive increase in the level of Per phosphorylation. Because a similar, cytoplasmic association of Per and Dbt in tim01 flies results in cytoplasmic Per degradation, and such Per degradation requires Dbt, the most parsimonious explanation of these results should be that nuclear association of Per with TimUL protects Per from phosphorylation and, secondarily, from turnover. It has been shown that light eliminates Tim, but will not promote Per phosphorylation in a hypomorphic mutant of Dbt (dbtP). Thus, Per phosphorylation appears to be influenced by the formation of Per/Tim complexes, and only when Per is free from Tim is it subject to phosphorylation by a Dbt-dependent mechanism. While this view is favored, it is also possible that light directly activates elements of a Dbt-dependent mechanism to promote some Per phosphorylations, or that additional factors associate with Per (or Dbt) after Tim is removed by light. Such factors would then be essential for Dbt-regulated phosphorylation of Per (Kloss, 2001).

The following is a model for the accumulation, phosphorylation, and degradation of Per: Dbt-dependent phosphorylation of Per in the cytoplasm is thought to delay the accumulation of Per proteins until lights off. Increasing Tim levels result in stable Per/Tim/Dbt complexes containing hypophosphorylated Per. These complexes are translocated to nuclei, where continued physical association of Tim with Per prolongs the cycle. Subsequently, the formation of Per free from Tim allows the clock to advance by Dbt-dependent phosphorylation of nuclear Per. This phosphorylation could be indirectly controlled by Dbt. The cycle restarts after degradation of phosphorylated nuclear Per proteins. According to this model, Dbt would have opposing effects on the cycle at different times of day and in different subcellular compartments. This regulation would determine the onset and duration of Per's activity in the nucleus, and should therefore be required to establish rhythmicity and set the period of Drosophila's circadian clock (Kloss, 2001).

The secreted signaling molecule Hedgehog regulates gene expression in target cells in part by preventing proteolysis of the full-length Cubitus interruptus (Ci-155) transcriptional activator to the Ci-75 repressor form. Ci-155 proteolysis depends on phosphorylation at three sites by Protein Kinase A (PKA). These phosphoserines prime further phosphorylation at adjacent Glycogen synthase kinase 3 (GSK3) and Casein kinase I (CK1) sites. Alteration of the GSK3 or CK1 sites prevents Ci-155 proteolysis and activates Ci in the absence of Hedgehog. Ci-155 proteolysis is also inhibited if cells lack activity of the Drosophila GSK3. Conversely, Ci-155 levels are reduced in Hedgehog-responding cells by overexpression of PKA and the Drosophila CK1, Double-time, a regulator of circadian rhythms. Thus Shaggy/GSK3 is implicated in the functioning of the Hedgehog pathway, in addition to its well known role in the Wingless pathway (Price, 2002).

Phosphorylation of Ci at three defined PKA sites primes further phosphorylation at adjacent GSK3 and CK1 sites. This PKA-primed phosphorylation could be catalyzed by purified mammalian GSK3ß and CK1delta enzymes or by activities in Drosophila embryo extracts. Changing the target serines of either GSK3 or CK1 consensus sites to alanines prevents proteolysis of Ci-155 to Ci-75 in flies. This result was demonstrated both by Western blots of embryo extracts and by assaying for the activity of Ci-75 as a transcriptional repressor in wing imaginal discs. It is argued that the resistance of these altered Ci molecules to proteolysis results from altered phosphorylation rather than a change in amino acid identity per se, because elimination of Sgg GSK3 activity produces a similar result and because the PKA sites required for priming further phosphorylation must themselves be intact in order for Ci-155 to be proteolyzed to Ci-75 (Price, 2002).

The basic arrangement of PKA sites flanked by PKA-primed GSK3 and CK1 sites is conserved in Gli2 and Gli3 for each of the three PKA sites in Ci, with an additional fourth motif between PKA sites 2 and 3 of Ci. The identity of amino acids in each cluster extends beyond the consensus SGSK3RRXSPKAXXSCK1. For instance, PKA site 1 has an adjacent CK1 site followed by a second CK1-primed site (RRXSPKAXXSCK1XXSCK1), but there are no GSK3 sites. PKA sites 2 and 3 are flanked by GSK3 sites and CK1 sites (SGSK3RRXSPKAXXSCK1), but only site 3 includes a second GSK3 site (SGSK3XXXSGSK3RRXSPKA). Ignoring the possibility of additional interstitial phosphorylations in this region due to GSK3 priming of CK1 sites and vice versa, Ci contains a total of eight PKA-primed GSK3 or CK1 phosphorylation sites, whereas Gli2 and Gli3 contain eleven and nine, respectively, in this region of less than 80 amino acids. Gli1 has only two PKA sites in this region with three associated CK1 sites and only one GSK3 site. Commensurate with sequence conservation, both Gli2 and Gli3 appear to be proteolyzed when expressed in Drosophila, whereas Gli1 remains full-length. Processing of Gli proteins in flies appears to correspond, at least approximately, to their fate in their normal environment. These data are consistent with the proposal that a conserved mechanism of PKA-dependent proteolysis of Ci/Gli proteins depends on creating highly phosphorylated clusters of regularly spaced phosphoserine residues (Price, 2002).

How do multiple phosphorylations of Ci target it for degradation? Paired GSK3 phosphorylation sites are crucial for recognition of ß-catenin by Slimb/ß-TrCP, but they fall within a more specific consensus sequence DS(P)GXXS(P) that is conserved in IkappaB. None of the GSK3 or, of course, the CK1 or PKA sites in Ci conform to this consensus. It is possible that Slimb/ß-TrCP recognizes more epitopes than currently appreciated or that the presence of multiple weak binding sites collectively contributes to association with Slimb. The latter mechanism has been demonstrated for the recognition of yeast Sic1, which is phosphorylated within multiple suboptimal binding sequences, by the F box protein Cdc4. At least six such sites in Sic1 must be phosphorylated to exceed a physiological threshold for recognition (Price, 2002).

Only one of the eight putative CK1 genes in Drosophila has been extensively investigated genetically. Weak alleles of this gene were named double-time because they alter circadian rhythms. Stronger alleles affect imaginal disc growth and patterning in a variety of ways, but relating these phenotypes to specific cellular processes or signaling pathways has been hampered by the limited growth and viability of cells homozygous for null and strong alleles. This property of dbt/dco also limits these investigations to showing that overexpression of Dbt can enhance the reductions of Ci-155 levels at the A/P border of wing discs due to PKA hyperactivity. This observation is consistent with the idea that increased PKA-primed phosphorylation of Ci by Dbt can promote Ci-155 proteolysis even in cells exposed to Hh, but it was not directly demonstrated that proteolysis is responsible for the reduced Ci-155 levels observed, nor does this result show that Dbt is normally involved in Ci phosphorylation. Dbt remains a good candidate for the CK1 homolog that phosphorylates Ci. It is a member of the CK1 delta/epsilon family, which has been implicated in Wnt signaling in Xenopus and in mammalian tissue culture cells (Price, 2002).

The identification of GSK3 and CK1 as components of the Hh signaling pathway extends previously noted similarities with the Wnt signaling pathway. In addition to these kinases, the F box protein Slimb is shared between the pathways, and both pathways include a component with similarity to the G protein-coupled receptors Smo on the Hh pathway and Frizzled, the Wg receptor. Finally, both pathways share the feature of constitutive phosphorylation-dependent degradation of a key effector that is reversed by ligand signaling. These shared components and other similarities invite speculations about 'crosstalk' and about conserved mechanisms (Price, 2002).

Casein kinase I (CKI) is a positive regulator of Wnt signaling in vertebrates and Caenorhabditis elegans. To elucidate the function of Drosophila CKI in the wingless pathway, CKI was disrupted by double-stranded RNA-mediated interference (RNAi). While previous findings were mainly based on CKI overexpression, this is the first convincing loss-of-function analysis of CKI. Surprisingly, CKIalpha- or CKIepsilon-RNAi markedly elevates Armadillo (Arm) protein levels in Drosophila Schneider S2R+ cells, without affecting Arm mRNA levels. Pulse-chase analysis showed that CKI-RNAi stabilizes Arm protein. Moreover, Drosophila embryos injected with CKIalpha double-stranded RNA showed a naked cuticle phenotype, which is associated with activation of Wg signaling. These results indicate that CKI functions as a negative regulator of Wg/Arm signaling. Overexpression of CKIalpha induces hyper-phosphorylation of both Arm and Dishevelled in S2R+ cells and, conversely, CKIalpha-RNAi reduces the amount of hyper-modified forms. His-tagged Arm is phosphorylated by CKIalpha in vitro on a set of serine and threonine residues that are also phosphorylated by Zeste-white 3. Thus, it is proposed that CKI phosphorylates Arm and stimulates its degradation (Yanagawa, 2002).

Since loss-of-function studies are the key to revealing the actual function of Drosophila CKI in the Wg pathway, RNAi was used to disrupt the CKI gene expression in Drosophila Schneider S2R+ cells. S2R+ cells were cultured in the presence of double-stranded (ds)RNA for CKIalpha, CKIepsilon, alpha-Catenin, casein kinase II catalytic (alpha) subunit (CKII-alpha) or LacZ for 3 days and then the protein levels in the cell lysates were analyzed by Western blotting. Addition of dsRNA for CKIepsilon, alpha-Catenin and CKII-alpha causes a selective decrease in the corresponding proteins. While previous studies with Xenopus, Caenorhabditis elegans and mammalian systems have reported that CKI is a positive regulator of Wnt signaling, both CKIalpha- and CKIepsilon-RNAi markedly elevate Arm protein levels, suggesting that CKI functions as a negative regulator of Arm protein in Drosophila. Since CKIalpha-RNAi induces higher levels of Arm protein accumulation than CKIepsilon-RNAi, CKIalpha was mainly used for subsequent analyses (Yanagawa, 2002).

To search for the sequence in Arm that responds to CKIalpha-RNAi, stable S2R+ cell lines expressing wild-type and various mutant forms of myc-tagged Arm were established and the effects of CKIalpha-RNAi on accumulation of these Arm mutant proteins were examined by Western blotting. Similar to endogenous Arm, wild-type Arm with the myc-tag is markedly stabilized by CKIalpha-RNAi. Since phosphorylation of Arm at the N-terminus is known to determine its stability, Arm mutants lacking the N-terminal 58 or 138 amino acids were analyzed. These two mutants, which are more stable than the wild-type, no longer respond to CKIalpha-RNAi, indicating that the target sequence for CKIalpha-RNAi resides in the N-terminal 58 amino acids. Therefore, a series of N-terminal mutants was made. In the serine/threonine to alanine mutant, the Ser and Thr residues originally identified as phosphorylation target sites for ZW3 (S at codon 44, 48, 56 and T at 52) were changed to Ala. In S56A and S58A, the Ser at 56 and 58, respectively, was changed to Ala. In the ED to QN mutant, a stretch of acidic amino acids (E and D) was replaced with Q and N (E at 61, 63, 64, 66 to Q and D at 62 to N). This mutant was produced because CKI is known to phosphorylate a Ser or Thr residue close to the acidic residues and this stretch of acidic amino acids is also conserved in ß-catenin and plakoglobin (Yanagawa, 2002).

Analyses with this series of Arm mutants has revealed that protein levels of the S58A mutant are somewhat elevated even without CKI-RNAi, but this mutant responds to CKI-RNAi similarly to the wild-type Arm, while, the S56A mutant responds slightly less than the wild-type Arm. The S/T to A mutant no longer responds to CKIalpha-RNAi, while the ED to QN mutant response is much weaker than that of the wild-type Arm. These results suggest that CKIalpha directly or indirectly stimulates phosphorylation of Ser44, 48 and 56, as well as Thr52, thereby destabilizing Arm and that the stretch of acidic amino acids may facilitate this process. If so, the ED to QN mutant would be expected to be more stable than the wild-type Arm. Hence, the stabilities of the wild-type, S/T to A, S56A and ED to QN forms of Arm were compared. The S/T to A mutant is the most stable, with the S56A mutant second. The ED to QN mutant is more stable than the wild-type Arm, but less stable than the S/T to A mutant (Yanagawa, 2002).

Next to be examined was whether CKIalpha directly phosphorylates a set of Ser and Thr residues in the N-terminal region of Arm phosphorylated by ZW3. The results indicate that phosphorylation sites for CKIalpha are Ser44, 48 and 56, as well as Thr52 residues (among these, S56 seems to be the major phosphorylation site, whose phosphorylation affects those of the other three sites). A cluster of acidic amino acids is also required for this phosphorylation. The cluster of acidic amino acids described above is conserved in ß-catenin (amino acid sequence from 53 to 58: EEEDVD). Notably, mutations in this region have been reported in tumors. Of 37 independent anaplastic thyroid carcinoma samples, four had mutations. One hepatoblastoma has been reported that had a 42 base pair deletion in ß-catenin exon 3, which leads to deletion of amino acids from S45 to D58. Clearly, CKI mutations in certain tumors remain to be explored (Yanagawa, 2002).

Protein phosphorylation has a key role in modulating the stabilities of circadian clock proteins in a manner specific to the time of day. A conserved feature of animal clocks is that Period (Per) proteins undergo daily rhythms in phosphorylation and levels, events that are crucial for normal clock progression. Casein kinase Iepsilon (CKIepsilon) has a prominent role in regulating the phosphorylation and abundance of Per proteins in animals. This was first shown in Drosophila with the characterization of Doubletime (Dbt), a homolog of vertebrate casein kinase Iepsilon. However, it has not been clear how Dbt regulates the levels of Per. Using a cell culture system, this study shows that Dbt promotes the progressive phosphorylation of Per, leading to the rapid degradation of hyperphosphorylated isoforms by the ubiquitin–proteasome pathway. Slimb, an F-box/WD40-repeat protein functioning in the ubiquitin–proteasome pathway interacts preferentially with phosphorylated Per and stimulates its degradation. Overexpression of slimb or expression in clock cells of a dominant-negative version of slimb disrupts normal rhythmic activity in flies. These findings suggest that hyperphosphorylated Per is targeted to the proteasome by interactions with Slimb (Ko, 2002).

Phosphorylation by double-time/CKIepsilon and CKIalpha targets cubitus interruptus for Slimb/beta-TRCP-mediated proteolytic processing

Hedgehog (Hh) proteins govern animal development by regulating the Gli/Ci family of transcription factors. In Drosophila, Hh signaling blocks proteolytic processing of full-length Ci to generate a truncated repressor form. Ci processing requires sequential phosphorylation by PKA, GSK3, and a casein kinase I (CKI) family member(s). This study shows that Double-time (DBT)/CKIε and CKIα act in conjunction to promote Ci processing. CKI phosphorylates Ci at three clusters of serine residues primed by PKA and GSK3 phosphorylation of other residues. CKI phosphorylation of Ci confers binding to the F-box protein Slimb/β-TRCP, the substrate recognition component of the SCFSlimb/β-TRCP ubiquitin ligase required for Ci processing. CKI phosphorylation sites act cooperatively to promote Ci processing in vivo. Substitution of Ci phosphorylation clusters with a canonical Slimb/β-TRCP recognition motif found in β-catenin renders Slimb/β-TRCP binding and Ci processing independent of CKI. It is proposed that phosphorylation of Ci by CKI creates multiple Slimb/β-TRCP binding sites that act cooperatively to recruit SCFSlimb/β-TRCP (Jia, 2005).

Regulation of Ci/Gli processing is a key regulatory step in the Hh signal transduction pathway; however, the underlying mechanism is still not fully understood. This study provides evidence that two CKI isoforms, DBT/CKIε and CKIα, act additively to promote Ci processing. It was found that CKI phosphorylates multiple Ser residues arranged in three clusters in the C-terminal half of Ci, and that CKI can phosphorylate sites primed by PKA or GSK3 phosphorylation. In addition, DBT/CKIε and CKIα are required for Ci phosphorylation in vivo. CKI sites in different phosphorylation clusters act cooperatively to promote Ci processing in vivo. More importantly, Slimb/β-TRCP was shown to directly bind CKI-phosphorylated Ci through its WD40 repeats. Finally, substitution of multiple CKI sites with a Slimb/β-TRCP binding motif found in β-catenin renders Ci processing independent of CKI. Based on these and other observations, it is proposed that PKA- and GSK3-primed CKI phosphorylation of Ci creates docking sites for Slimb/β-TRCP that recruit SCFSlimb/β-TRCP to regulate Ci processing (Jia, 2005).

This study employed dominant-negative kinase, genetic mutations, and heritable RNAi knockdown to investigate the role of two CKI isoforms in Ci processing in vivo. Overexpression of a dominant-negative DBT/CKIε (DN-DBT) caused cell-autonomous accumulation of Ci155 and ectopic dpp expression, suggesting that interference with DBT/CKIε activity impairs Ci processing. As a further support, it was found that A compartment dbt/dco mutant cells accumulate high levels of Ci155. The phenotypes associated with dbt/dco mutations differ depending on the alleles used. The hypomorphic allele, dco3, does not seem to affect Ci processing, although it does affect cell growth and proliferation. By contrast, more severe alleles, including dcoP103 and dcole88, affect Ci processing. The lack of Hh-related phenotypes associated with the weak allele of dbt/dco is likely due to compensation by other CKI isoforms. This may explain why RNAi knockdown of DBT/CKIε does not affect Hh signaling in cultured cells, since RNAi knockdown usually does not completely eliminate the function of the targeted genes, and hence often resembles hypomorphic genetic mutations. Alternatively, other CKI isoforms might be expressed in cultured cells at higher levels than in imaginal discs, so that they can compensate for the complete loss of DBT/CKIε in cultured cells (Jia, 2005).

To investigate the role of CKIα in Ci processing, the heritable RNAi approach was used, and two CKIα RNAi constructs were generated: CRS and CRL. CRL knocks down CKIα more effectively than CRS, likely due to its larger targeting sequence; however, it also knocks down DBT/CKIε. In contrast, CRS appears to be more specific for CKIα. Expressing CRL in wing discs induces high levels of Ci155 accumulation and ectopic dpp expression. In contrast, expressing CRS resulted only in a modest increase in Ci155 without inducing ectopic dpp expression. However, expressing CRS in DBT/CKIε hypomorphic (dco3/dcole88) wing discs completely blocked Ci processing, as evident by the accumulation of high levels of Ci155 and ectopic dpp expression in these discs. These data suggest that CKIα and CKIε play partially redundant roles in Ci processing, and that they act additively to provide optimal CKI kinase activity required for efficient Ci phosphorylation and processing. Consistent with this notion, CKIα and CKIε bind equally well to Cos2. This is in contrast to what has been proposed for the Wnt pathway, where CKIε and CKIα appear to play opposing roles and act on distinct protein substrates. Since CKI sites are conserved in Gli proteins, it awaits to be determined whether CKIε or CKIα or both are involved in Gli regulation (Jia, 2005).

Using an in vitro kinase assay, two types of CKI phosphorylation events were uncovered: one primed by PKA and the other by GSK3 phosphorylation. CKI phosphorylation sites are arranged in three clusters. Whereas cluster 1 contains only PKA-primed CKI sites, both cluster 2 and 3 contain PKA- and GSK3-primed CKI sites. Using an in vivo functional assay, it was demonstrated that both PKA- and GSK3-primed CKI sites are involved in Ci processing. For example, the two types of CKI sites in cluster 2 appear to have overlapping function; mutations in either one only partially blocked Ci processing, whereas mutations in both completely blocked Ci processing (Jia, 2005).

CKI sites in different phosphorylation clusters appear to act cooperatively to promote Ci processing. Strikingly, mutating the two CKI sites in cluster 1 (CiSA12) completely abolishes Ci processing. Similarly, mutating all the CKI sites in cluster 2 also abolishes Ci processing. A dosage-sensitive interaction was observed between two phosphorylation clusters. For example, partial loss of function of both cluster 2 and cluster 3 nearly abolish Ci processing. Based on these and other observations, it is proposed that each phosphorylation cluster acts as a functional module, and Ci processing requires cooperative action among the three modules (Jia, 2005).

Ci lacks the canonical Slimb/β-TRCP binding motif (DSGXXS) found in other SCFSlimb/β-TRCP substrates such as β-catenin and Iκ-B, inviting speculation that Ci phosphorylation could recruit a protein(s) other than Slimb/β-TRCP and that the involvement of SCFSlimb/β-TRCP in Ci processing could be indirect. This study assessed whether hyperphosphorylation of Ci directly recruits Slimb/β-TRCP. It was found that a GST-Ci fusion protein binds Slimb/β-TRCP efficiently after it is phosphorylated by CKI, following primed phosphorylation by the other kinases. In addition, binding of GST-Ci to Slimb is compromised when a subset of CKI sites was mutated to Ala. These observations support the hypothesis that phosphorylation of Ci at CKI sites confers Slimb/β-TRCP binding. The in vivo relevance of Slimb/β-TRCP binding was demonstrated by the finding that a single canonical Slimb/β-TRCP binding site can substitute for the three phosphorylation clusters to promote Ci processing. Strikingly, Ci variants bearing the DSGXXS motif can undergo processing even when CKI activity is blocked. These observations suggest that the major function of CKI in Ci processing is to recruit SCFSlimb/β-TRCP by phosphorylating Ci at multiple Ser residues that function as docking sites for Slimb/β-TRCP (Jia, 2005).

The recently solved crystal structure of the β-TRCP/β-catenin phospho-peptide complex reveals that the two phospho-Ser and the aspartate residues in the DSGXXS motif make critical contacts with several basic residues from the WD40 repeats of β-TRCP that form a single substrate binding pocket. Although none of the three phosphorylation clusters in Ci contains a DSGXXS motif, they all contain related sequences. For example, cluster 1 contains DSQNSTAS, cluster 2 contains SSQSS and SSQVSS, and cluster 3 contains SSQMS. It is proposed that these phospho-Ser motifs represent low-affinity or suboptimal sites for Slimb/β-TRCP recognition, and optimal binding of Slimb/β-TRCP to Ci is achieved by cooperative binding among multiple low-affinity sites. The high local concentration of binding sites greatly increases the probability of interaction so that Ci is unable to diffuse away from Slimb/β-TRCP before rebinding occurs. Hence, Ci becomes kinetically trapped in close proximity to Slimb/β-TRCP once the binding is engaged. Alternatively, phosphorylation of Ci could recruit a cofactor that binds cooperatively with Slimb/β-TRCP to hyperphosphorylated Ci. Both models can explain the observed high cooperativity among multiple phosphorylation clusters in Ci processing (Jia, 2005).

The ability to bind a single high-affinity site or multiple low-affinity sites appears to be a general feature for the SCF family of ubiquitin ligases. Another well-characterized SCF complex, SCFCDC4, can bind certain substrates such as Cyclin E through a single high-affinity site and other substrates such as Sic1 through multiple low-affinity sites. In the case of Sic1, phosphorylation at multiple sites appears to set a threshold for kinase activity that converts a smooth temporal gradient of kinase activity into a switch-like response for degradation of Sic1 and onset of S phase. In the case of Ci/Gli regulation, first, the requirement for hyperphosphorylation may render Ci processing highly dependent on the activity of individual kinases and hence highly sensitive to Hh, since low levels of Hh suffice to block Ci processing although such levels of Hh may only cause a small reduction in Ci phosphorylation levels. Second, cooperativity among multiple phosphorylation sites may convert a smooth spatial Hh activity gradient into a sharp response for Ci processing, since a small drop in the level of Ci phosphorylation could result in a dramatic reduction in Ci processing and hence the level of Ci75. Third, employing multiple phosphorylation events may allow the levels of Ci phosphorylation to be fine-tuned by different thresholds of Hh signaling activity, leading to differential regulation of Ci processing and activity, as the activity of Ci155 appears to be regulated by phosphorylation independent of its processing. Finally, employing multiple kinases to regulate Ci/Gli may provide opportunities for crosstalk between the Hh and other signaling pathways in certain developmental contexts (Jia, 2005).

PER-dependent rhythms in CLK phosphorylation and E-box binding regulate circadian transcription

Transcriptional activation by Clock-Cycle (Clk-Cyc) heterodimers and repression by Period-Timeless (Per-Tim) heterodimers are essential for circadian oscillator function in Drosophila. Per-Tim has been found to interact with Clk-Cyc to repress transcription, and this interaction is shown to inhibit binding of Clk-Cyc to E-box regulatory elements in vivo. Coincident with the interaction between Per-Tim and Clk-Cyc is the hyperphosphorylation of Clk. This hyperphosphorylation occurs in parallel with the Per-dependent entry of Double-time (Dbt) kinase into a complex with Clk-Cyc, where Dbt destabilizes both Clk and Per. Once Per and Clk are degraded, a novel hypophosphorylated form of Clk accumulates in parallel with E-box binding and transcriptional activation. These studies suggest that Per-dependent rhythms in Clk phosphorylation control rhythms in E-box-dependent transcription and Clk stability, thus linking Per and Clk function during the circadian cycle and distinguishing the transcriptional feedback mechanism in flies from that in mammals (Yu, 2006).

ChIP studies demonstrate that Clk-Cyc is only bound to E-boxes when target genes are being actively transcribed. Since Per-Dbt/Per-Tim-Dbt complexes interact with Clk-Cyc to inhibit transcription (Darlington, 1998; Lee, 1998), these data imply that these Per-containing complexes inhibit transcription by removing Clk-Cyc from E-boxes. It is also possible that binding of these Per-containing complexes to Clk-Cyc effectively blocks Clk and Cyc antibody access, in which case Per complexes would inhibit transcription while Clk-Cyc is bound to E-boxes. Given that the polyclonal Clk and Cyc antibodies used in this study were raised against full-length proteins and have been used to immunoprecipitate Per-containing complexes (Lee, 1998), it is highly unlikely that all Clk and Cyc epitopes are fully blocked by Per complex binding. Thus, it is concluded that Clk-Cyc rhythmically binds E-boxes in concert with target gene activation (Yu, 2006).

Per complex binding could remove Clk-Cyc from E-boxes by directly altering their conformation or by promoting Clk phosphorylation. The region of Per that inhibits Clk-Cyc transcription, called the Clk-Cyc inhibitory domain or CCID, is near the C terminus. The CCID can act independently of the N terminus of Per, where the Dbt-binding domain resides. This observation argues that Per does not inhibit Clk-Cyc binding to E-boxes by promoting Dbt-dependent Clk phosphorylation. Dbt- and CK2-dependent phosphorylation nevertheless enhances transcriptional repression in S2 cells by potentiating Per inhibition or by inhibiting Clk activity directly. Unfortunately, these disparate results from S2 cells do not allow distinguishing between the different effects of Per complex binding to inhibit transcription outlined above (Yu, 2006).

In mammals, mCry complexes bind to CLOCK-BMAL1 and repress transcription without removing CLOCK-BMAL1 from E-boxes. This contrasts with the situation in flies, where Per complexes inhibit transcription by inhibiting Clk-Cyc E-box binding, and suggests that these Per and mCry complexes repress transcription via different mechanisms. Although mCry complexes do not remove CLOCK-BMAL1 from E-boxes, they repress transcription by inhibiting the CLOCK-BMAL1-induced acetylation of histones by blocking p300 histone acetyl transferase function or introducing a histone deacetylase. Even though Per complexes repress transcription by inhibiting Clk-Cyc binding to E-boxes, this does not exclude the possibility that rhythms in histone acetylation are also involved in regulating rhythmic transcription in flies. Since chromatin remodeling is generally accepted as a prerequisite for transcription initiation, it would be surprising if rhythms in transcription were not accompanied by rhythms in histone acetylation or some other form of chromatin remodeling (Yu, 2006).

A rhythm in Clk phosphorylation has been defined in which hyperphosphorylated Clk predominates during times of transcriptional repression and hypophosphorylated Clk predominates during times of transcriptional activation. This rhythm occurs in parallel to the rhythm in Per phosphorylation; hyperphosphorylated Per and Clk accumulate in nuclei during the late night and early morning, then these forms are degraded and hypophosphorylated forms of Per and Clk accumulate in the cytoplasm and nucleus, respectively, during the late day and early evening. The rhythm in Clk and Per phosphorylation are not merely coincidental; the accumulation of hyperphosphorylated Clk is Per dependent. Although Per is not itself a kinase, it is bound by Dbt kinase. Per brings Dbt into the nucleus, where Per-Dbt or Per-Tim-Dbt complexes bind Clk-Cyc to inhibit transcription (Yu, 2006 and references therein).

Since Dbt enters a complex containing Clk-Cyc at times when Clk becomes hyperphosphorylated, Dbt may also act to phosphorylate Clk. However, an in vitro assay for Dbt phosphorylation is not available, thus it iw not known whether Dbt directly phosphorylates Clk. Dbt acts to reduce Clk levels in S2 cells even though Per levels are very low. It is therefore possible that Dbt can act to destabilize Clk in a Per-independent manner, although it is believed this is unlikely to be the case since Clk hyperphosphorylation and complex formation with Dbt are both Per dependent (Yu, 2006).

Clk is phosphorylated to some extent in the absence of Per and is hyperphosphorylated in the absence of functional Dbt, indicating that other kinases act to phosphorylate Clk. The accumulation of hyperphosphorylated Clk in dbtAR/dbtP flies suggests that Dbt triggers Clk degradation subsequent to Clk hyperphosphorylation. A similar situation is seen for Per, where hyperphosphorylated Per accumulates in the absence of functional Dbt, and phosphorylation by CK2 precedes Dbt-dependent phosphorylation and Per destabilization. In addition, rhythmically expressed phosphatases may also contribute to Clk phosphorylation rhythms (Yu, 2006 and references therein).

Rhythms in Clk phosphorylation may function to modulate Clk stability, subcellular localization, and/or activity. Clk levels do not change appreciably throughout the daily cycle despite approximately fivefold higher levels of Clk mRNA at dawn than at dusk. If a less stable hyperphosphorylated form of Clk accumulates when Clk mRNA is high and a more stable hypophosphorylated form of Clk accumulates when Clk mRNA is low, they would tend to equalize total Clk levels over the daily cycle. This possibility is supported by results in ARK flies, which express Clk mRNA in the opposite circadian phase (i.e., Clk mRNA peak at dusk rather than dawn). The overall level of Clk cycles in ARK flies with a peak in (hypophosphorylated) Clk around dusk, consistent with hypophosphorylated Clk being more stable than hyperphosphorylated Clk. This possibility is also supported by Dbt-dependent destabilization of Clk in S2 cells since Dbt associates with Clk as hyperphosphorylated Clk accumulates in wild-type flies. If hypophosphorylated Clk is relatively stable, higher levels of Clk might be expected to accumulate in per01 flies. However, constant low levels of Clk mRNA likely limit Clk accumulation in per01 flies. Clock phosphorylation is coupled to its degradation in cultured mammalian cells, yet degradation of phosphorylated Clock does not lead to a rhythm in Clock abundance even though Clock mRNA levels are constant (Yu, 2006).

Studies in cultured mammalian cells also demonstrate that Clock phosphorylation promotes Clock-BMAL1 nuclear localization, although the significance of this nuclear localization is not clear given that Clock-BMAL1 binding to E-boxes is either constant or more robust during transcriptional repression in vivo. In contrast, Clk is nuclear throughout the daily cycle in flies (Yu, 2006).

The coincidence between Clock phosphorylation and transcriptional repression in mice supports the possibility that phosphorylation inhibits Clock-BMAL1 activity, perhaps by promoting HDAC binding or inhibiting HAT binding. Likewise, hypophosphorylated and hyperphosphorylated Clk accumulate in parallel with target gene activation and repression, respectively, in flies. This relationship suggests that the state of Clk phosphorylation may alter its ability to activate target genes. Given that target gene activation occurs when Clk-Cyc is bound to E-boxes and that E-box binding coincides with the accumulation of hypophosphorylated Clk, it is possible that Clk hyperphosphorylation compromises Clk-Cyc binding to E-boxes and, consequently, target gene transcription is repressed. Precedent for such a regulatory mechanism is seen in the Neurospora clock, where limiting levels of FREQUENCY (FRQ) promote phosphorylation of WHITE COLLAR 1 (WC1) and WHITE COLLAR 2 (WC2), thereby inhibiting WC1-WC2 binding to C-box regulatory elements and repressing transcription. In contrast to FRQ in Neurospora, Per is considerably more abundant than Clk in Drosophila and forms stable complexes with Clk-Cyc. In addition, Per/Per-Tim can release Clk-Cyc from E-boxes in vitro, thus demonstrating that Per/Per-Tim binding is sufficient to release Clk-Cyc from E-boxes independent of Clk phosphorylation. Taken together with the in vitro E-box binding results, the high levels of Per relative to Clk and the formation of stable Per-Tim-Clk-Cyc complexes in flies argue that Per/Per-Tim binding may also be sufficient to inhibit E-box binding by Clk-Cyc in vivo, although they do not rule out a role for Clk hyperphosphorylation in inhibiting Clk-Cyc E-box binding. For instance, Per/Per-Tim binding could function to initially remove Clk-Cyc from E-boxes, and subsequent Clk phosphorylation could maintain Clk-Cyc in a form that is incapable of binding E-boxes (Yu, 2006).

The constant levels and rhythmic phosphorylation of Clk defined in this study are similar to those previously characterized for mammalian Clock. This similarity extends beyond metazoans to fungi, where positive elements of the Neurospora circadian feedback loop; i.e., WC1 and WC2, are also rhythmically phosphorylated. In each of these organisms, phosphorylation of positive factors increases when they interact with their respective negative feedback regulators, and decreases when they activate target gene transcription in the absence of these feedback inhibitors. This remarkable similarity suggests that phosphorylation controls one or more critical aspects of positive element function, and consequently, the rhythm in the positive element phosphorylation has become a conserved feature of circadian feedback loops in eukaryotes (Yu, 2006 and references therein).

Per-dependent regulation of Clk-Cyc binding to E-boxes, Per-dependent formation of Per-Dbt and/or Per-Dbt-Tim complexes with Clk-Cyc, and Per-dependent rhythms in Clk phosphorylation suggest a model for the regulation of rhythmic transcription. During the late day and early evening, hypophosphorylated Clk-Cyc binds E-boxes to activate transcription of per, tim, and other genes within and downstream of the transcriptional feedback loop. Accumulating levels of per mRNA peak during the early evening, but Per accumulation is delayed due to Dbt-dependent (and possibly CK2-dependent) phosphorylation, which destabilizes Per. Per is subsequently stabilized via Tim binding, which inhibits further phosphorylation of Per by Dbt. Phosphorylation of Tim by SGG then promotes the translocation of Tim-Per-Dbt complexes into the nucleus, where they bind (hypophosphorylated) Clk-Cyc and repress transcription by inhibiting E-box binding and promoting Clk hyperphosphorylation and degradation. These transcriptional repression mechanisms are not mutually exclusive; Clk hyperphosphorylation may inhibit E-box binding as well as promote Clk degradation. Dbt is able to enter the nucleus in per01 flies, but does not associate with Clk in the absence of Per. This suggests that Per is required to either bring Dbt into a complex with Clk-Cyc, enable phosphorylation of Clk after Dbt enters the complex, or both. Once the Tim-Per-Dbt-Clk-Cyc complex has formed, hyperphosphorylated Per and Clk levels decline in a coordinated fashion by mid-day. Tim is eliminated prior to hyperphosphorylated Per and Clk via separate light-dependent and light-independent mechanisms. As hyperphosphorylated Clk and Clk mRNA decline during the day, hypophosphorylated Clk accumulates. This hypophosphorylated Clk forms complexes with Cyc and binds E-boxes in the absence of nuclear Tim-Per-Dbt complexes, thus initiating the next cycle of transcription (Yu, 2006 and references therein).

The contributions of protein kinase A and smoothened phosphorylation to hedgehog signal transduction in Drosophila; Doubletime targets Smoothened

Protein kinase A (PKA) silences the Hedgehog (Hh) pathway in Drosophila in the absence of ligand by phosphorylating the pathway's transcriptional effector, Cubitus interruptus (Ci). Smoothened (Smo) is essential for Hh signal transduction but loses activity if three specific PKA sites or adjacent PKA-primed casein kinase 1 (CK1) sites are replaced by alanine residues. Conversely, Smo becomes constitutively active if acidic residues replace those phosphorylation sites. These observations suggest an essential positive role for PKA in responding to Hh. However, direct manipulation of PKA activity has not provided strong evidence for positive effects of PKA, with the notable exception of a robust induction of Hh target genes by PKA hyperactivity in embryos. This study shows that the latter response is mediated principally by regulatory elements other than Ci binding sites and not by altered Smo phosphorylation. Also, the failure of PKA hyperactivity to induce Hh target genes strongly through Smo phosphorylation cannot be attributed to the coincident phosphorylation of PKA sites on Ci. Finally, it has been shown that Smo containing acidic residues at PKA and CK1 sites can be stimulated further by Hh and acts through Hh pathways that both stabilize Ci-155 and use Fused kinase activity to increase the specific activity of Ci-155 (Zhou, 2006; full text of article).

When the role of PKA in Hh signaling was first discovered it appeared that PKA acted simply to silence the pathway in the absence of Hh. This aspect of PKA function has been studied further, revealing that it is conserved in vertebrate Hh signaling and can be explained adequately by the phosphorylation of three clustered consensus PKA sites on Ci-155. Loss of these sites, loss of PKA activity, and even the consequences of excessive PKA activity in wing discs all lead to a coherent picture of how PKA silences Ci and the Hh signaling pathway in the absence of Hh. This role of PKA had disguised recognition of any potential positive role for PKA in transduction of an Hh signal on the basis of simply manipulating PKA activity. Indeed, a positive role for PKA in Hh signaling was clearly revealed only by altering PKA (and PKA-primed CK1) phosphorylation sites in Smo; changes to alanine residues eliminated activity and changes to acidic residues endowed some constitutive activity. A number of significant questions remain. Are the consensus PKA sites on Smo actually phosphorylated by PKA and only by PKA, and is phosphorylation of Smo by PKA required to transmit an Hh signal? Does Smo with acidic residues at PKA and CK1 sites mimic the consequences of phosphorylation at those sites, and does it elicit the normal process of Hh pathway activation (Zhou, 2006 and references therein)?

Smo absolutely requires PKA sites for activity. Furthermore, those sites can be phosphorylated by PKA in vitro to prime phosphorylation of adjacent CK1 sites, and those CK1 sites are also essential for Smo activity. Hence, Smo PKA sites must be critical in their phosphorylated form and elimination of the relevant protein kinase activity should prevent all responses to Hh. Expression of a dominant-negative PKA regulatory subunit (R*) in embryos does substantially reduce Fu phosphorylation induced by endogenous or ectopically expressed Hh, consistent with the idea that PKA is the major protein kinase that phosphorylates Smo on PKA sites in embryos. However, PKA inhibition with R* in embryos does not prevent all Hh-stimulated phosphorylation of Fu or Hh-dependent maintenance of wg expression. Since PKA inhibition by R* is likely incomplete it is not possible to distinguish whether these residual responses to Hh result from phosphorylation of Smo by residual PKA activity or by another protein kinase, but it should be noted that PKA inhibition by R* is sufficient to produce very high levels of Ci-155, indicative of a complete block in Ci-155 processing (Zhou, 2006).

In wing discs PKA-C1 activity can be eliminated cleanly in large clones using null alleles. PKA-C1 (formerly named DC0) is the major PKA catalytic subunit in flies and the only PKA catalytic subunit with demonstrated developmental functions, even though at least one other gene encodes an equivalent biochemical activity. Loss of PKA-C1 activity in wing disc clones does reduce Hh signaling, as revealed most clearly by strongly reduced or absent expression of En at the AP border. This deficit of PKA-C1 mutant clones at the AP border can be complemented by expressing SmoD1-3 in place of wild-type Smo. This supports the idea that PKA-C1 must phosphorylate Smo for Hh to elicit maximal pathway activity, which is required for strong induction of En. It is not so straightforward to determine whether Hh requires PKA-C1 activity to induce target genes such as collier (col) or ptc, which require lower levels of Hh pathway activity. This is because loss of PKA-C1 by itself induces strong ectopic ptc and col expression. Nevertheless, when induction of col in PKA-C1 mutant clones was largely suppressed by reducing the dose of ci, it was clear that Hh still induced high levels of col in PKA-C1 mutant clones at the AP border and that this induction required Smo activity. Thus, Smo retains some but not maximal activity in response to Hh when PKA-C1 activity is lost, implying that another kinase can phosphorylate Smo at PKA sites in wing discs. This inference is also supported by the observations that Smo is stabilized in anterior cells when its PKA sites are substituted by alanine residues but not when PKA-C1 activity is eliminated (Zhou, 2006).

In contrast to the limited effects of eliminating PKA-C1 activity on Smo activity and protein levels, the same manipulations of PKA-C1 completely block processing of Ci-155 to Ci-75 and strongly activate Ci-155 in wing discs. Why might Smo and Ci-155 show different sensitivities to PKA-C1? One possibility is that scaffolding molecules may allow special access of PKA-C1 to Ci-155 that is not available to other kinases that might otherwise phosphorylate PKA sites. Indeed, Cos2 does appear to ensure efficient phosphorylation of Ci-155 by PKA-C1 by binding to both components. However, Cos2 also binds to Smo and therefore presumably also provides similarly enhanced access for PKA-C1. A more likely explanation of the different responses of Smo and Ci-155 to PKA-C1 manipulation concerns the stoichiometry of phosphorylation. A key functional consequence of Ci-155 phosphorylation is the binding of Slimb, and this requires extensive phosphorylation of Ci-155 primed by each of the three relevant PKA sites. Thus, any significant reduction in the rate of phosphorylation of these sites might be translated into strong stabilization of Ci-155. Conversely, since Smo retains considerable activity in the absence of PKA-C1 it is speculated that a low rate of phosphorylation of Smo at PKA sites may suffice for it to be active (Zhou, 2006).

The discovery that substitution of multiple PKA and CK1 site Serines of Smo with acidic residues conferred constitutive activity provoked the simple hypothesis that activation of Smo by Hh can be attributed largely to an Hh-stimulated increase in phosphorylation at these sites. Investigations of the properties of Smo with acidic residues at PKA and CK1 sites (SmoD1-3) and of the consequences of forced phosphorylation of Smo do not support this simple hypothesis (Zhou, 2006).

It was found that Hh can increase pathway activity in cells expressing SmoD1-3. This effect is small in wing discs, where (overexpressed) SmoD1-3 has strong constitutive activity. However, in embryos SmoD1-3 exhibited no clear constitutive activity but transduced a normal response to Hh. Thus, Hh must elicit changes in Smo activity other than phosphorylation at PKA and CK1 sites that are sufficiently important to convert pathway activity from a silent state to being fully active in embryos. It is speculated that these (unknown) changes are conserved elements of all Hh signaling pathways and that phosphorylation of Drosophila Smo at PKA and CK1 sites, which are not conserved in vertebrate Smo proteins, is a prerequisite for Drosophila Smo to undergo these Hh-dependent changes (Zhou, 2006).

It was also found that excess PKA activity and CK1 activity cannot reproduce the ectopic activation of Hh target genes induced by expression of SmoD1-3. This was true despite attempts to sensitize Hh target gene induction by eliminating Su(fu) or by providing additional processing-resistant Ci-155. An analogous difference in the potency of SmoD1-3 and excess PKA and CK1 activity was observed when using Fu phosphorylation as a measure of Hh pathway activity in wing discs (Zhou, 2006).

Why are excess PKA and CK1 activities not sufficient to activate Smo? One possibility is that overexpression of PKA or CK1 did not effectively stimulate Smo phosphorylation. This explanation is not favored because both of the protein kinases used are thought to associate with Cos2 and therefore should have good access to Smo, and analogous overexpression studies show that each can lower Ci-155 levels at the AP border, implying that they induce significant changes in Ci-155 phosphorylation (Zhou, 2006).

Another possibility is that PKA or CK1 may have targets other than Smo that reduce Hh signaling pathway activity, obscuring the effects of any potential activation mediated by Smo phosphorylation. Ci-155 is certainly one such target but this confounding influence was excluded by coexpression of a Ci mutant lacking all known regulatory PKA sites and also by measuring Fu phosphorylation in addition to Hh target gene activation. It is conceivable that there are additional inhibitory targets for PKA in the Hh pathway because it was observed that the induction of ptc-lacZ in posterior wing disc cells by a PKA-resistant Ci variant (Ci-H5m) was, surprisingly, reduced by excess PKA activity (Zhou, 2006).

Finally, the favored explanation is that Smo with acidic residues at PKA and CK1 sites behaves significantly differently from Smo that is phosphorylated at those sites. It has been argued that phosphorylation is essential for the activity of Smo in the presence of Hh but also targets Smo for degradation in the absence of Hh. It is further speculated that Hh might normally stabilize the phosphorylated state of Smo rather than actively promoting Smo phosphorylation and that acidic residues might mimic Smo activation by phosphorylation without simultaneously promoting Smo degradation in the absence of Hh. In this scenario SmoD1-3 would accumulate and exhibit constitutive activity, especially when overexpressed, but it would not be possible to accumulate activated Smo very effectively in the absence of Hh by increasing only its rate of phosphorylation at PKA and CK1 sites. The hypothesis that Hh stabilizes phosphorylated Smo rather than promoting Smo phosphorylation is also consistent with the earlier conjecture that Smo activation by Hh requires only a low rate of phosphorylation at PKA sites (Zhou, 2006).

A significant question for the future is how phosphorylation of Smo contributes to its activity. Some clues have been made available from examining the properties of SmoD1-3 in wing discs. SmoD1-3 stabilizes Ci-155, induces phosphorylation of Fu, shows substantial dependence on Fu kinase activity for induction of Hh target genes and can suffice for strong induction of anterior En expression in wing discs. These results suggest that SmoD1-3 activates two genetically separable aspects of Hh signaling (Ci-155 stabilization and the Fu kinase signaling pathway) that are sometimes hypothesized to correspond to two biochemically distinct pathways. The nonphysiological circumstances of using high levels of expression and acidic residues in place of phosphorylation may contribute to one or the other of the apparent dual attributes of SmoD1-3 in Hh signaling. Nevertheless, it appears that phosphorylation of Smo at PKA and CK1 sites at least makes Smo competent to activate each known aspect of the Hh signaling pathway. This fits with the idea that Smo phosphorylation may be constitutive but necessary to make Smo competent to respond to Hh (Zhou, 2006).

It was found that strong ectopic activation of the Hh target genes, wg and ptc, by excess PKA activity in embryos is the consequence of two distinguishable responses. First, PKA does appear to induce target genes through Ci binding sites, consistent with enhancing Smo activity through phosphorylation. However, this response alone would result in only a very small induction of Hh target genes. The salient evidence is that PKA hyperactivity induces (1) detectable, but very limited, ectopic expression of a reporter gene that essentially contains only Ci binding sites, (2) clear ectopic expression of a wg reporter gene that depends on the presence of Ci binding sites, and (3) a small increase in Fu phosphorylation. Second, PKA hyperactivity induces wg and ptc transcription principally through regulatory elements other than Ci binding sites and through a mechanism that does not require a change in phosphorylation at Smo PKA sites. The salient evidence is that the response to excess PKA is greatly enhanced if regulatory elements from the wg and ptc genes other than just Ci binding sites are present and that wg and ptc are strongly induced by excess PKA activity even when the only Smo protein present has acidic residue substituents at PKA and CK1 sites (Zhou, 2006).

The dual consequences of excess PKA described above clarify a potential misconception in the literature that PKA can strongly activate the Hh pathway through Smo and substantiate the idea that excess PKA produces only a small activation of the Hh pathway through phosphorylation of Smo, whether assayed in wing discs or embryos. These results also raise the question of the nature and physiological significance of the pathway that connects excess PKA activity to induction of wg and ptc through enhancer elements other than Ci binding sites (Zhou, 2006).

PKA is known to phosphorylate many proteins that can influence transcription and thus its ability to activate wg and ptc through sites other than Ci binding sites when hyperactive may simply be an artifact of this nonphysiological condition An alternative possibility is that this consequence of excess PKA activity exposes a regulatory mechanism that is relevant to target gene activation by Hh in embryos. There is some evidence for transcription factors other than Ci contributing to induction of Hh target genes in embryos. Furthermore, it is clear that there must be interactions between Ci and other gene-specific transcription factors that underlie both the different sensitivity of genes with equivalent Ci binding sites to activation by Ci-155 and repression by Ci-75 and the tissue-specific responses of most genes to Hh. Whether Hh signaling affects the activity or interactions of transcription factors that collaborate with Ci is not presently known (Zhou, 2006).

An intriguing aspect of the ectopic induction of wg and ptc by excess PKA through sites other than Ci binding sites is its dependence on concomitant activation through Ci binding sites. Thus, induction of wg and ptc by excess PKA requires both Smo and Ci activities and requires functional Ci binding sites within the Deltawg-lacZ reporter gene. Even the PKA sites on Smo are required for wg to respond to excess PKA, consistent with the idea that some activation of Smo is required. There is as yet no indication that Hh signaling normally involves the PKA-responsive regions of wg and ptc enhancers that can collaborate with Ci binding sites. Indeed, both Ci-Grh-lacZ and FE-lacZ reporters, which lack key regulatory regions required for a strong response to excess PKA activity, are clearly induced by Hh. There are, however, caveats to this evidence; induction of Ci-Grh-lacZ depends on the synthetic Grh binding sites as well as its Ci binding sites and the FE-lacZ reporter is induced only poorly by Hh in comparison to the ptc-lacZ reporter that includes PKA-responsive elements. Thus, it remains possible that the Hh signal is transmitted largely through Ci and supplemented by contributions from enhancer elements other than Ci binding sites, including those that are responsive to PKA. One pathway that is known to supplement Hh-induced wg expression in embryos is the Wg autoregulation pathway. However, this does not appear to be relevant to the PKA-responsive elements under discussion here because PKA hyperactivity did not substitute for the requirement for Wg activity to maintain stripes of wg expression and PKA hyperactivity also induces ectopic ptc expression, which does not depend on Wg activity for its expression. In the future, the clearest way to test the significance for Hh signaling of regulatory elements responsive to excess PKA will be to define and then alter those regulatory elements (Zhou, 2006).

A small conserved domain of Drosophila PERIOD is important for circadian phosphorylation, nuclear localization, and transcriptional repressor activity

A 27-amino-acid motif has been identified that is conserved between the Drosophila Period protein (Per) and the three mammalian PERs. Characterization of Per lacking this motif (Per Delta) shows that it is important for phosphorylation of Drosophila Per by casein kinase I epsilon (CKI epsilon; Doubletime protein or DBT) and CKII. S2 cell assays indicate that the domain also contributes significantly to Per nuclear localization as well as to Per transcriptional repressor activity. These two phenomena appear linked, since Per Delta transcriptional repressor activity in S2 cells is restored when nuclear localization is facilitated. Two less direct assays of Per Delta activity in flies can be interpreted similarly. The separate assay of nuclear import and export suggests that the domain functions in part to facilitate Per phosphorylation within the cytoplasm, which in turn promotes nuclear entry. As there is evidence that the kinases also function within the nucleus to promote transcriptional repression, it is suggested that there is a subsequent collaboration between phosphorylated Per and the kinases to repress Clk-Cyc activity, probably through the phosphorylation of Clk. This is then followed by additional Per phosphorylation, which occurs within the nucleus and leads to Per degradation (Nawathean, 2007; full text of article).

Activating PER repressor through a DBT-directed phosphorylation switch

Protein phosphorylation plays an essential role in the generation of circadian rhythms, regulating the stability, activity, and subcellular localization of certain proteins that constitute the biological clock. This study examines the role of the protein kinase Doubletime (DBT), a Drosophila ortholog of human casein kinase I (CKI) epsilon/delta. An enzymatically active DBT protein is shown to directly phosphorylate the Drosophila clock protein Period (PER). DBT-dependent phosphorylation sites are identified within PER, and their functional significance is assessed in a cultured cell system and in vivo. The perS mutation, which is associated with short-period (19-h) circadian rhythms, alters a key phosphorylation target within PER. Inspection of this and neighboring sequence variants indicates that several DBT-directed phosphorylations regulate PER activity in an integrated fashion: Alternative phosphorylations of two adjoining sequence motifs appear to be associated with switch-like changes in PER stability and repressor function (Kivimae, 2008).

Initial studies of dbt mutations suggested that Per is the primary target of Dbt in the Drosophila circadian clock. Loss of dbt expression led to hypophosphorylation and overaccumulation of Per in vivo. Mutations of dbt that shorten or lengthen behavioral rhythms led to correspondingly earlier or later degradation of Per in fly heads. The sequence of dbt suggested that it encodes an ortholog of the CKI family of protein kinases in mammals. Direct evidence for Dbt kinase activity and Per phosphorylation has been hampered by the absence of specific enzymatic activity in bacterially produced recombinant Dbt preparations, even though highly homologous mammalian CKIɛ (86% primary sequence identity in the kinase domain) has been recovered in an active form from bacterial expression systems. It is possible that for Drosophila Dbt, protein folding is more sensitive to the cellular environment than the mammalian enzyme. Alternatively, Dbt may require posttranslational modification(s) and/or the presence of a stimulating activity or cofactor(s) to produce an active conformation that is not available in bacterial cells. Nevertheless, this report has shown that recombinant Dbt can be expressed and recovered in an enzymatically active state from an insect cell line. However, even this insect-derived activity is short-lived (Kivimae, 2008).

Recombinant Dbt in vitro preferentially phosphorylates Per protein. No significant phosphorylation was observed when Tim, Sgg, and Cyc were used as a substrate in vitro. Although no direct Dbt phosphorylation of the N-terminal half of Clk was observed, recent studies by others have shown that Dbt is required for the generation of a highly phosphorylated and unstable form of Clk in vivo. Furthermore, to achieve this highly phosphorylated state of Clk, it has been suggested that Per must be present and function as a bridge to physically deliver Dbt to Clk. Clk appears to be present in flies in multiple isoforms, including hypophosphorylated, intermediate-, and highly phosphorylated states. The last form is only observed during the late part of the Clk accumulation cycle and may promote Clk degradation. The lack of in vitro phosphorylation of Clk by Dbt in the current studies may reflect a Dbt phosphorylation target in the C-terminal half of Clk, or a requirement for additional kinases to 'prime' a Dbt target, or a dependence on Per for Dbt-directed phosphorylation of Clk (Kivimae, 2008).

Casein and Per are phosphorylated by enzymatically active recombinant Dbt, and mutations of dbt that are known to affect the behavioral rhythm also have an effect on kinase activity. The short- and long-period mutations dbtS and dbtL both reduce the kinase activity of Dbt. Importantly, when the dbtS mutation was introduced specifically into the Drosophila kinase, the effect was far less severe than a previously reported orthologous substitution; using casein as a substrate, the mutation produced a 15% decrease in activity when composing the fly kinase, but decreased enzyme activity by approximately 55% when introduced into Xenopus CKIδ ;. Vertebrate CKI and Dbt include divergent C-terminal protein segments, and the former enzyme cannot restore Dbt function in transgenic Drosophila. These results suggest that any analysis of Dbt mutations in a heterologous system should be applied with caution. The comparatively subtle effect of dbtS on Dbt activity versus its strong effect on period length could reflect a qualitative change in the mutant enzyme's activity. An assessment of potential differences of this sort might come from further mapping studies of the target specificities of DbtS versus Dbt on Per substrates in vitro (Kivimae, 2008).

Three distinct regions of Per are preferentially phosphorylated by Dbt in vitro. In the C-terminal region of Per, two phosphorylation targets, serine 1,134 and threonine 1,219, have been identified. No prior genetic or biochemical studies have suggested a specific role for this Per interval, and the cultured cell assays indicate that both phosphorylation targets can be removed without a detectable effect on Per's function as a Clk repressor. Near the N-terminus of Per, a region of 20 amino acids (residues 149-169) contains five serines that are candidate phosphorylation sites for Dbt. This interval is located upstream of the PAS domain of Per. A region important for Dbt/Per binding in vitro has been mapped to the first 300 amino acids of Per. However, it has not yet been determined whether there is a closer physical correspondence of the binding and phosphorylation target sequences, or whether elimination of potential Dbt targets in this Per interval affects Per/Dbt binding. As in the case of cultured cell studies of serine 1,134 and threonine 1,219, elimination of all phosphorylation targets between aa 149-169, produced no detectable effect on Per-dependent repression in cultured cells (Kivimae, 2008).

Interestingly, two of the putative CKI sites, serine 151 and serine 153, are substrates of casein kinase 2. Transgenic flies carrying S151A and/or S153A mutations showed period lengthening and delayed Per nuclear localization. It will be important in future studies to distinguish CK2 and Dbt phosphorylation targets in this region of Per and to determine their individual and/or interactive effects on Per function in vivo (Kivimae, 2008).

Of most interest in this study are phosphorylation sites identified in the third Dbt target region of Per, between residues 580-645. This is a genetically well-studied interval previously implicated in the regulation of behavioral rhythms. Mutations of a portion of this region, the per-short domain, are predominantly associated with short-period behavior. The first mutation to be mapped to this region, perS, eliminates a candidate phosphorylation site at serine 589, and S2 cell assays indicate that blocking phosphorylation of this site enhances Per's activity as a repressor of Clk. Earlier work has also shown that perS leads to premature nuclear degradation of Per, and the current studies of transgenic Drosophila producing a Per protein truncated by 17 amino acids including serine 589 (perΔS), similarly indicate a role for this sequence in regulating Per stability and function as a repressor. Thus, the wild-type per-short domain appears to promote Per stability while reducing its activity as a transcriptional repressor (Kivimae, 2008).

It is noted that earlier work has also suggested that the mutation perS enhances Dbt activity. If serine 589 is no longer an available Dbt target in perS, by what mechanism might Dbt function be elevated? Although mutating serine 589 enhances Per's activity as a repressor, this response is partially suppressed by a further mutation of some phosphorylation sites in the perSD region. As well, little or no repressor activity is detected if all phosphorylation targets are removed from perSD, even in the presence of the perS mutation. Together, these results suggest that the phosphorylation state of serine 589 may influence Dbt activity on downstream targets within perSD that are required for Per's function as a Clk repressor. In such a model (see Activity of the per-Short and perSD Domains Regulated by DBT Phosphorylation), dephosphorylation of the per-short domain would promote Dbt-directed phosphorylation of perSD, enhancing Per's activity as a repressor and also destabilizing the protein. Reciprocally, phosphorylation of serine 589 would depress activity of Dbt with respect to perSD, providing a more stable, but less active, Per protein. In this view, a fully dephosphorylated state would provide the most stable protein, but would provide an inactive form of Per, consistent with findings in cultured cells and transgenic flies, and with studies of Dbt-deficient Drosophila. Further in vivo evidence comes from transgenic flies expressing Per that contains a deletion of a conserved segment (aa 516-568) downstream of the PAS domain. This mutant Per protein displays increased stability and low-amplitude oscillations. More importantly, high levels of a hypophosphorylated form of Per are produced by these truncated proteins, which constitutively accumulate in the nucleus. This protein appears to have little or no activity as a transcriptional repressor (Kivimae, 2008).

Earlier studies of Per repression in S2 cells identified a region of Per, distinct from those reported in this study, that is also required for Per's function as a repressor of Clk-mediated transcription (Clk-CYC inhibition domain [CCID]], aa 764-1034). A novel, bipartite nuclear localization signal (NLS) is found in this region that influences nuclear localization of Per in S2 cells. Recent mapping studies of CCID led to the identification of a smaller, Per-Dbt binding domain (PDBD; aa 755-809) that upon deletion increases Per stability, decreases Per phosphorylation, and severely impairs Per function as a transcriptional repressor (Kim, 2007). A related deletion of Per (aa 768-792) produced similar defects, and impaired nuclear accumulation of Per in cultured cells (Nawathean, 2007). Studies of the latter deletion in transgenic flies showed that a hypophosphorylated form of Per is constitutively expressed at high levels and is a very poor repressor. However, repressor function was restored by NLS addition, suggesting that dysfunction of the protein was likely due to its inability to localize to the nucleus (Kivimae, 2008).

The arrangement of four of the phosphorylation targets included within the perSD domain (serines 604, 607, and 613, and threonine 610) resembles a CKI phosphorylation motif found in human Per2 that has been implicated in familial advanced sleep phase syndrome (FASPS). In the human Per protein, there are five serines spaced in an arrangement such that every third amino acid residue is a serine. These five serines are thought to be progressively phosphorylated by CKIɛ/δ, and in certain FASPS subjects, the first serine is mutated to glycine. It has been proposed that this mutation inhibits the phosphorylation of the remaining serines, thus leading to altered hPer2 levels or activity in FASPS subjects. In fact, more-recent studies of this mutation indicate that it may alter Per2′s repressor function because lower levels of mper2 RNA are found in transgenic mice expressing the similarly mutated mPer2 protein. Cultured cell studies have also suggested that the mutation affects mPer2 stability. Accordingly, a role for CKI-directed phosphorylation in the regulation of Per's stability and activity as a transcriptional repressor may be conserved from flies to mammals (Kivimae, 2008).

The phospho-occupancy of an atypical Slimb-binding site on Period that is phosphorylated by Doubletime controls the pace of the clock

A common feature of animal circadian clocks is the progressive phosphorylation of Period (Per) proteins, which is highly dependent on casein kinase Idelta/epsilon (CKIdelta/epsilon, termed Doubletime [Dbt] in Drosophila), and ultimately leads to the rapid degradation of hyperphosphorylated isoforms via a mechanism involving the F-box protein, beta-TrCP (Slimb in Drosophila). This study use the Drosophila model system shows that a key step in controlling the speed of the clock is phosphorylation of an N-terminal Ser (S47) by DBT, which collaborates with other nearby phosphorylated residues to generate a high-affinity atypical Slimb-binding site on Per. Dbt-dependent increases in the phospho-occupancy of S47 are temporally gated, dependent on the centrally located Dbt docking site on Per and partially counterbalanced by protein phosphatase activity. It is proposed that the gradual Dbt-mediated phosphorylation of a nonconsensus Slimb-binding site establishes a temporal threshold for when in a daily cycle the majority of Per proteins are tagged for rapid degradation. Surprisingly, most of the hyperphosphorylation is unrelated to direct effects on Per stability. This study also used mass spectrometry to map phosphorylation sites on Per, leading to the identification of a number of 'phospho-clusters' that explain several of the classic per mutants (Chiu, 2008).

To better understand the physiological role of phosphorylation in regulating PER stability, Drosophila was used as a model system. Using a range of strategies, including mutational analysis, mass spectrometry and phospho-specific antibodies S47 was identified as a key phospho-determinant regulating the efficiency of SLIMB binding to dPER. By evaluating the behavior of dPER mutants whereby amino acid 47 is constitutively 'nonphosphorylated' (S47A) or 'phosphorylated' (S47D), it was shown that the phospho-occupancy of S47 is a key biochemical throttle adjusting the pace of the clock. However, phosphorylation of S47 occurs within an atypical SLIMB-binding site. Additional DBT-dependent phosphorylated residues, which likely include one or more nearby Ser residues at amino acid 44/45 and possibly others within the first 100 amino acids, collaborate with pS47 to generate a high-affinity SLIMB-binding region on dPER. As such, the affinity of SLIMB for dPER is proportional to the degree of phospho-occupancy within an extended phosphorylation network centered on S47 that as a unit yields a graded response in the affinity of SLIMB-dPER interactions. Attaining a high proportion of dPER molecules that are phosphorylated at S47 and other key sites mediating SLIMB binding is progressive and occurs several hours after DBT stably interacts with dPER via the centrally located dPDBD, likely because DBT 'activity' is counterbalanced by TIM and protein phosphatases. It is proposed that the relatively slow assembly of a high-affinity SLIMB-binding site on dPER is at least partly 'designed' to extend the time that dPER acts as a transcriptional repressor, critical in generating transcriptional feedback loops with daily time frames. Finally, this mass spectrometry analysis identify 'hot spots' for phosphorylation, indicates that the majority of dPER phosphorylation is unrelated to direct effects on stability and sheds new insights into the underpinnings of several previously characterized mutants, including the classic perS allele (Chiu, 2008).

Early studies identified DpSGPhiX1+npS (pS, phosphorylated Ser; Phi, any hydrophobic amino acid; X, any amino acid) as the consensus motif for recognition by the β-TrCP/SLIMB F-box protein (Fuchs. 2004). Phosphorylation at both sites in this six amino acid consensus generally leads to a high-affinity β-TrCP/SLIMB-binding site. Indeed, the three negatively charged residues (Asp/Glu and two phospho-S/T residues) are important binding contacts underlying β-TrCP/substrate interactions (Wu, 2003). Furthermore, it is thought that the presence of an Asp/Glu at position 2 of the canonical binding domain can circumvent the need for a phospho-S/T at that position, as is the case for Wee1A (Watanabe, 2004). However, accumulating evidence indicates that β-TrCP-binding sites can deviate from this consensus. For example, recent work on the Ci/Gli family of transcription factors suggests a novel class of degenerate and weaker β-TrCP-binding sites that extend beyond the standard six-amino-acid binding motif, especially for those missing a Gly at the third position (Smelkinson, 2007). It was suggested that for these extended β-TrCP/SLIMB-binding motifs, significant contributions are made by the local presence of nonpolar residues, such as those found in the motifs for Ci, Gli, and Wee1A. Additional phosphorylation events at nearby regions are also thought to enhance the inherently weak binding affinities of extended β-TrCP-binding sites, enabling a more graded response compared with the standard sequence (Chiu, 2008).

The major SLIMB-dependent phospho-degron identified in this study [44p*Sp*SGpSSGYGG52; where p* = possible phosphorylation] seems to include signature elements found in both the standard and extended β-TrCP-binding motifs. A rather unique feature of the SLIMB-binding domain on dPER is that it includes two SSG repeats. S47 is phosphorylated, and based on mutational analysis and mass spectrometry, it is almost certain that either S44 and/or S45 are phosphorylated. A physiological role for S45 is further indicated by the perSLIH mutant (S45Y) that exhibits long periods, which based on the findings is likely due to reduced dPER-SLIMB interactions. Although changing S48 to Ala phenocopied the S47A mutation, the S48D mutation did not enhance binding to SLIMB, as was the case for S47D. Together with results showing that S48A did not modulate phosphorylation of S47, the data strongly suggest that S48 has a non-phosphorylation-dependent role as a crucial structural element. Mass spectrometry identified two phospho-residues in a dPER peptide from amino acids 40-48. Thus, there might only be two negatively charged residues in the major SLIMB-binding site on dPER. It is possible that the presence of a SSG tandem and a Tyr at position 50 can compensate for the lack of a third acidic residue normally found in β-TrCP-binding sites. It is also highly probable that other, yet to be identified, DBT-dependent phosphorylation sites besides those within the atypical SLIMB-binding site identified in this study contribute to enhancing SLIMB-dPER interactions (Chiu, 2008).

The presence of numerous suboptimal phospho-determinants is thought to generate a graded response in the binding efficiencies of F-box proteins to substrates. The general molecular framework is that progressive increases in the phospho-occupancy of multiple phosphorylated residues eventually reaches a threshold value that drives sufficient F-box protein/substrate interactions to yield desired outcomes. As such, regulating the kinetics of phosphorylation within the phospho-network mediating F-box recognition is a key determinant in the timing of substrate degradation. In the case of animal PER proteins, they undergo progressive increases in global phosphorylation that occur over an ~10-h time frame, whereby highly phosphorylated isoforms are associated with a rapid decline in levels (Chiu, 2008).

What accounts for the hours-long kinetics underlying the gradual increases in phosphorylation of S47 and likely many other DBT-dependent sites on dPER? Based on the in vitro ability of DBT to phosphorylate S47 despite phosphatase treatment of dPER, it is not believed that hierarchical phosphorylation based on prior priming is a major component in regulating the timing of when S47 is phosphorylated in vivo. Rather, the findings strongly suggest that the gradual build-up in the phospho-occupancy of S47 and other sites is at least partly based on a dynamic balance between DBT-mediated phosphorylation and the opposing activities of TIM and protein phosphatases. In agreement, blocking phosphatase activity strongly enhanced the abundance of phosphorylated S47. Recent evidence suggests that the ability of TIM to stabilize dPER might be by acting as a bridge that facilitates the targeting of protein phosphatase activity toward dPER (Fang, 2007). Indeed, it is likely that the strong protective function of TIM on dPER partially overrides the destabilizing effects of the S47D mutant as it attains peak levels comparable with those of wild-type dPER. Following this line of reasoning, it is suggested that a major reason for the advanced dper RNA and protein cycles in the S47D mutant is that as TIM levels decline in the late night the 'released' dPER(S47D) protein is no longer protected (or less so) and undergoes accelerated nuclear clearance, leading to an earlier disengagement from transcriptional repression, which advances the subsequent dper RNA and protein cycles. Likewise, while this manuscript was under review a recent report showed that the CKIepsilon tau mutation, which shortens rhythms in mice, has an 'asymmetrical' effect on PER protein stability, preferentially accelerating nuclear clearance and hence advancing the molecular oscillations underpinning the clockworks (Meng, 2008). Thus, although differential phosphorylation plays a major role in setting the intrinsic stabilites of PER proteins, additional variables, such as phase-specific protein-protein interactions, are critical in the 'readout' from these phospho-signals (Chiu, 2008).

Many of the DBT-dependent phosphorylation sites that were identified using mass spectrometry do not lie within optimal CKI sites, suggesting that inefficient phosphorylation by DBT might also contribute to the overall rate of progressive increases in dPER phosphorylation. It is also possible that the strong binding of DBT to the centrally located dPDBD, while increasing the local concentration of DBT, could function as a slow 'time-release capsule' whereby the disengagement of DBT is first required prior to phosphorylation of dPER residues at more distantly located sites (Chiu, 2008).

Although the phosphorylation requirements and in vivo significance of regions on mPER1 and mPER2 that interact with β-TrCP are not known, it is likely to also be based on noncanonical β-TrCP-binding sites. In addition, hyperphosphorylation of mammalian PER proteins requires a centrally located CKI-binding site. Therefore, mammalian PER proteins, especially mPER1 and mPER2, are likely to be targeted by β-TrCP to the 26S proteasome in a manner similar to that described here for dPER. This type of mechanism might also apply to other clock proteins such as Frequency (Frq) in Neurospora that undergoes daily changes in phosphorylation and stability that are remarkably similar to those observed for PER proteins. In addition, the phosphorylated state of FRQ is regulated by casein kinases, protein phosphatases, and the rapid degradation of highly phosphorylated isoforms is mediated by the F-box protein FWD1, a homolog of β-TrCP (Chiu, 2008).

An interesting feature of the distribution in phosphorylation sites on dPER that were identified using mass spectrometry is that they seem to concentrate in clusters, suggesting the presence of'phospho-modules' with different functions. Most of these clusters appear anchored by proline-directed phosphorylation sites, which are phosphorylated by endogenous kinases expressed in S2 cells. Of note, one such cluster is located in the dPER 'short domain' (T585-T610). Mutations in this region result in animals with short periods. In fact, the mutated residues of two classic per mutants that have short periods, perS (S589N, 19-h period) and perT (G593D, 16-h period), are right in the heart of this cluster. S589 is phosphorylated in a DBT-dependent manner; and G593, when mutated, may affect phosphorylation at nearby S589 and/or S596. Although the perS mutants was isolated more than 35 years ago, the current results provide the first biochemical understanding for the short period phenotype, suggesting that phosphorylation events in the 'short domain,' some of which are DBT-dependent, may collaboratively function to slow down the clock. It is now becoming apparent that phosphorylation at different sites on PER proteins can result in differential effects on the pace of the clockworks, whereby some lead to faster clocks while others slow it down. The presence of phosphorylated residues with opposing outcomes on the speed of the clock can explain why mutations in CKIepsilon/delta/DBT can yield a variety of period-altering phenotypes from short to long, despite the fact that overall enzymatic activity is generally reduced (Chiu, 2008).

A rather unanticipated finding is that the majority of dPER phosphorylation is unrelated to direct effects on stability. This is supported by the lack of detectable SLIMB binding to a dPER fragment only missing the first 100 amino acids despite extensive phosphorylation as inferred from being the region underlying the majority of phosphorylation-dependent electrophoretic mobility shifts and confirmed by mass spectrometric analysis. Other lines of evidence also imply that a significant amount of multiphosphorylation is not linked to direct effects on PER stability. For example, abolishing phosphorylation at many centrally located sites on mPER3 does not attenuate CKI-mediated in vitro interactions with β-TrCP. Also, a trans-dominant version of CKII reduced global hyperphosphorylation of dPER without major effects on its levels (Chiu, 2008).

Thus, there are likely to be at least two functionally distinct DBT-dependent phosphorylation programs regulating different aspects of PER metabolism and activity: one that controls β-TrCP/SLIMB binding, and another that integrates with other kinases, such as CKII, to modulate nuclear entry/accumulation and/or ability to function as a transcriptional repressor. Indeed, mass spectrometric analysis of dPER identified numerous phosphorylation sites in a putative nuclear localization site and within the CCID mediating dPER inhibition of CLK-mediated transcription. Variants of dPER missing the major DBT docking site are hypophosphorylated and weak repressors. However, the relationship between hyperphosphorylation and repressor potency is not clear, since the DBT docking site on dPER also functions as a molecular scaffold for DBT and perhaps CKII-mediated inhibition of CLK-dependent transcription. Nonetheless, it is clear that the DBT docking site is a critical nexus for coordinating multiple phosphorylation programs. A challenge is to examine the functions of the newly identified phosphorylation sites and dissect the mechanisms by which they regulate dPER metabolism and activity (Chiu, 2008).

Drosophila and vertebrate casein kinase Idelta exhibits evolutionary conservation of circadian function

Mutations lowering the kinase activity of Drosophila Doubletime(DBT) and vertebrate casein kinase Iepsilon/delta (CKIepsilon/delta) produce long-period, short-period, and arrhythmic circadian rhythms. Since most ckIshort-period mutants have been isolated in mammals, while the long-period mutants have been found mostly in Drosophila, lowered kinase activity may have opposite consequences in flies and vertebrates, because of differences between the kinases or their circadian mechanisms. However, the results of this article establish that the Drosophila dbt mutations have similar effects on period(PER) protein phosphorylation by the fly and vertebrate enzymes in vitro and that Drosophila DBT has an inhibitory C-terminal domain and exhibits autophosphorylation, as does vertebrate CKIepsilon/delta. Moreover, expression of either Drosophila DBT or the vertebrate CKIdelta kinase carrying the Drosophila dbtS or vertebrate tau mutations in all circadian cells leads to short-period circadian rhythms. By contrast, vertebrate CKIdelta carrying the dbtL mutation does not lengthen circadian rhythms, while Drosophila DBTL does. Different effects of the dbtS and tau mutations on the oscillations of PER phosphorylation suggest that the mutations shorten the circadian period differently. The results demonstrate a high degree of evolutionary conservation of fly and vertebrate CKIdelta and of the functions affected by their period-shortening mutations (Fan, 2009).

Dbt is essential for circadian molecular oscillations because it introduces time delays into the negative feedback exerted by Per. These phosphorylation-dependent delays are thought to be mediated via several mechanisms. For instance, Dbt is thought to destabilize Per in the cytoplasm, while Tim (Per's dimerization partner) is thought to prevent this destabilization and trigger the movement of both proteins to the nucleus, perhaps indirectly. The destabilization of Per in the cytoplasm delays its nuclear accumulation, while the stabilization of Per in the nucleus by Tim delays the Dbt-dependent decrease of nuclear Per. Because Dbt controls the timing of Per's nuclear accumulation so that it does not occur until after the per/tim mRNA levels have peaked, molecular rhythms of per and tim mRNA are possible. Additional regulation has been proposed to occur at the level of Per's capacity to negatively regulate its transcription factor target, Clk/Cyc (Fan, 2009).

Although Dbt's orthologs CKIδ and CKIɛ are involved in the mammalian circadian clock, the extent of evolutionary conservation of their circadian mechanisms has not been clear. Cellular and biochemical analysis argues for a significant degree of conservation. It has been shown that both the original long-period mutation (dbtL) and the short-period dbt mutation (dbtS) reduce the enzymatic activity of Drosophila Dbt and a Xenopus CKI ortholog of Dbt on casein (Preuss, 2004), and this study has shown that Drosophila DbtS and DbtL also exhibit reduced activity on Per. In the current work, deletion of the Dbt C terminus was shown to increase the kinase activity of Dbt and its capacity to target Per for degradation. The latter result is consistent with interaction studies, as it is the N-terminal catalytic domain rather than the C-terminal domain that interacts with Per (Preuss, 2004). Finally, it was shown in this study that in the presence of a general phosphatase inhibitor Dbt produces forms that migrate more slowly on SDS-PAGE, in a manner that requires its kinase activity. These results suggest that Dbt is autophosphorylated. Inhibitory autophosphorylation of the C-terminal domain is a common feature of both vertebrate CKIɛ and -δ. All of these biochemical results demonstrate a high degree of evolutionary conservation between Drosophila Dbt and vertebrate CKIɛ/δ (Fan, 2009).

in vivo analysis herein of vertebrate CKIδ and Drosophila Dbt further establishes the evolutionary conservation of these kinases and the clock mechanisms in which they participate. Mutations that shorten the circadian period in the context of Dbt produced corresponding shortening in the context of CKI in flies, and the tau mutation produced almost identical period shortening in both the mammalian and the Drosophila clocks, in the context of either the fly or the vertebrate enzyme. The data for the tau mutation are particularly complete for analysis of evolutionary conservation, as this mutation has been tested in all possible combinations of organism (fly or mammal) and kinase (Dbt or CKI) except one (DbtTau in mammals). These results argue against the possibility that reduced kinase activity produces only long periods in flies and short periods in mammals. In contrast, the CKIδ FASPS mutation, which has a small effect on period, produces opposite effects on period in flies and mammals. The mutations analyzed in this study have much stronger effects on period and clearly show similar effects in both vertebrates and flies. These findings strongly support the proposed evolutionary conservation of CKIδ and Dbt protein kinases and of at least some of the circadian processes in which they are involved (Fan, 2009).

The reproduction of the period alteration caused by a mutation in CKIɛ when introduced into CKIδ or Drosophila Dbt and tested in a transgenic fly is strong evidence for the high amount of functional conservation between ckIɛ and ckIδ, both of which are proposed regulators of the vertebrate clocks. However, CKIδ and -ɛ are not completely interchangeable, since similar overexpression experiments with CKIɛ in flies have produced a very different set of results from the ones presented in this study for CKIδ. A catalytically active CKIɛ produces a dominant negative effect on circadian rhythms in Drosophila, with relatively constant expression of hypophosphorylated Per at all times of day, while overexpression of a catalytically inactive form of CKIɛ produces only a mild lengthening of circadian period (Sekine, 2008). Taken together with the current results, this result suggests that vertebrate CKIδ is better able to interact with fly clock proteins than is CKIɛ, and in a manner more comparable to Drosophila Dbt in the same expression protocol. The difference between the finding of Sekine (2008) and the current results, as well as those of Xu, (2005) (who also employed overexpression of CKIδ in flies and did not observe dominant negative phenotypes), may be due to the divergent C-terminal domains of CKIδ and -ɛ (Fan, 2009).

This study has shown that flies expressing the dominant negative form of Dbt have very long periods or are arrhythmic, and this result argues that general reductions in Dbt's kinase activity produce long-period rhythms that grade to arrhythmicity. While the lower activity of DbtL is predicted to (and in fact does) lengthen circadian period, the short-period Per oscillation produced by DbtS and tau mutations is not readily explained by their lower kinase activity in vitro. One possible explanation is that the short-period mutants affect something besides kinase activity - an interaction with a regulator, for example (Fan, 2009).

Another possible explanation is that reduced activity of short-period CKI enzymes in vitro may not translate into lower phosphorylation of Per in vivo because other kinases provide compensatory phosphorylation. In fact, progressive phosphorylation of Per, at least as assessed by a reduction in Per's electrophoretic mobility, occurs more rapidly in dbtS flies or cells than in wild-type or dbtL flies or cells, as is also shown in this study for tim-GAL4>UAS-DbtS flies, so the in vivo phosphorylation profile indicates more rapid phosphorylation in the dbtS mutant. Likewise, in the tau mutant, the phosphorylation of Per is only slightly delayed and ultimately appears to be as complete as in wild-type hamsters, despite the strong reduction in kinase activity caused by the tau mutation in vitro. In mammals, at least two kinases (CKIɛ and CKIδ) associate with Per and phosphorylate it, while in flies casein kinase II phosphorylates Per together with Dbt (Fan, 2009).

While the phosphorylation profiles of Per in tim-GAL4>UAS-DbtS, -CKIS, and -DbtL resemble the phosphorylation profiles of the original dbt mutants, the Per phosphorylation profiles of tim-GAL4>UAS-DbtTau and -CKITau do not resemble the profile that has been previously reported for the endogenous mutant; in particular, the oscillation of Per phosphorylation is notably blunted by overexpression of the DbtTau mutant kinases. It is possible the overexpression of the DbtTau has a stronger effect on the phosphorylation profile of Per in flies than in mammals because the tau mutant effects are partially masked in mammals by compensatory activity of CKIδ, with which CKIɛ may be partially redundant (Meng, 2008). Nevertheless, the period of the circadian clock is dramatically shortened by overexpression of both DbtS and DbtTau mutant kinases, and the circadian period of the tau mutation in flies is similar to that for the hamster tau mutant, suggesting that the alterations in the Dbt/CKI kinase are still altering period as they do in the original mutants (Fan, 2009).

Another possible reason for the phenotypes of the short-period mutants is that phosphorylation of specific sites in Per affects multiple, specific aspects of its regulation with opposite effects on period length. Phosphorylation of Drosophila Per at multiple sites, only some of which affect stability, has recently been demonstrated (Chiu, 2008; Kivimae, 2008). Along this line of thinking, it has been proposed that the ckIɛTau mutation is a gain-of-function mutation that enhances phosphorylation of Per at specific sites—an enhancement that is missed in global analysis of multisite substrates like Per. Other explanations have been offered for the tau mutant that include lowered phosphorylation at all sites, but with cytoplasmic destabilization produced by phosphorylation at some sites and increased nuclear retention (and stabilization) produced by phosphorylation at other sites. If these hypotheses for site-specific effects are correct, the dispersed phosphorylation profile detected for Drosophila Per in CKITau- and DbtTau-expressing flies is indicative of general changes in phosphorylation, most of which are not relevant to the period shortening, gain-of-function phenotype produced at a subset of sites. While both the dbtS and tau mutations produce short circadian periods, their effects on the phosphorylation program of Per are different, as reflected in their different effects on circadian changes in Per electrophoretic mobility (Fan, 2009).

Why does CKIL overexpression not lengthen the period of the Drosophila rhythm, while DbtL overexpression does? It is likely that the dbtL mutation compromises CKIδ function and reduces its ability to compete with endogenous wild-type Dbt for interactions with Per. In fact, CKIδ may have generally less ability to compete with endogenous Dbt than transgenic Dbt, as overexpression of CKIS also has weaker effects than overexpression of the corresponding DbtS protein. Not all mutations that reduce the kinase activity of vertebrate CKI produce short periods, or no effect like CKIL. This study shows that the D/N mutation, which like the K/R mutation is predicted to have a very specific effect on the catalytic properties of the enzyme and not other aspects of its function, produced variable period lengthening. The variability is most likely a consequence of chromosomal position effects at the P-element insertion site on the transgene expression levels. The stronger effects of period-lengthening mutations in the context of fly Dbt than in the vertebrate CKI suggest that there may be differences in the way they affect the fly and vertebrate CKI orthologs, with a consequence that fly Dbt can be mutated more readily to produce a long period. These differences argue against the idea that lack of long periods is produced by a difference in circadian targets, as the lack of long periods correlates with the vertebrate enzyme rather than the species in which the enzyme is expressed ( i.e., DbtL can produce long periods in flies, while CKIL cannot) (Fan, 2009).

The involvement of protein kinases with circadian clocks spans a phylogeny that is larger than the one separating vertebrates and fruit flies. Casein kinases I and II are also involved in the bread mold (Neurospora) clock and target both the FRQ transcriptional repressor and the WCC transcriptional activator in a mechanism reminiscent of the one involving Dbt. Recently, a kinase involved in DNA replication control was also shown to target Neurospora FRQ, with implications for the interplay between circadian rhythms and cell cycle control in higher eukaryotes. The core circadian oscillator mechanism in cyanobacteria involves rhythmic phosphorylation and dephosphorylation of the KaiC protein. The evolutionary conservation of kinase function has led to a synergy between research in different organisms—for instance, with work in Drosophila identifying a circadian role for CKI that has now been shown in diverse phyla and work in Neurospora showing a kinase-targeting role for FRQ that was subsequently shown for Per as well. Further elucidation of the evolutionarily conserved processes regulated by phosphorylation will reveal general mechanisms at the core of the circadian mechanism (Fan, 2009).

Processing and phosphorylation of the Fat receptor

The Drosophila tumor suppressors fat and discs overgrown (dco) function within an intercellular signaling pathway that controls growth and polarity. fat encodes a transmembrane receptor, but post-translational regulation of Fat has not been described. This study shows that Fat is subject to a constitutive proteolytic processing, such that most or all cell surface Fat comprises a heterodimer of stably associated N- and C-terminal fragments. The cytoplasmic domain of Fat is phosphorylated, and this phosphorylation is promoted by the Fat ligand Dachsous. dco encodes a kinase that influences Fat signaling, and Dco is able to promote the phosphorylation of the Fat intracellular domain in cultured cells and in vivo. Evaluation of dco mutants indicates that they affect Fat's influence on growth and gene expression but not its influence on planar cell polarity. These observations identify processing and phosphorylation as post-translational modifications of Fat, correlate the phosphorylation of Fat with its activation by Dachsous in the Fat-Warts pathway, and enhance understanding of the requirement for Dco in Fat signaling (Feng, 2009).

Activation of transmembrane receptors often involves post-translational modifications, such as phosphorylation or cleavage. To investigate potential modifications, Fat was examined by Western blot analysis. In lysates of wing discs, antisera raised against the Fat intracellular domain (anti-Fat ICD) detected a prominent band with a mobility of ~95 kDa (Ft-95), and a faint band with a mobility corresponding to a much larger polypeptide (Ft-565). fat is predicted to encode a 5,147 amino acid protein, with a calculated mass of 565 kDa. Thus, Ft-95 is too small to correspond to full length Fat. Nonetheless, examination of lysates from fat mutant discs confirmed that both Ft-95 and Ft-565 are fat-dependent (Feng, 2009).

To investigate this apparent cleavage of Fat, a C-terminally tagged Fat protein (Fat:FVH) was created. When Fat:FVH was transfected into cultured Drosophila S2 cells, a band with a high apparent molecular weight, consistent with full length Fat, was observed. However, most Fat was detected in lower molecular weight bands. One correlates with the 95-kDa fragment of endogenous Fat (after accounting for the C-terminal tags), but the other appears smaller, ~70 kDa (Ft-70). Although Ft-70 was not detected when endogenous Fat was examined in imaginal discs, it could be detected in discs when Fat:FVH was overexpressed from UAS transgenes. Expression of Fat:FVH under tub-Gal4 control also confirmed that Fat:FVH is functional, because it rescued fat mutant animals. The detection of Ft-95 and Ft-70 with C-terminal epitope tags supports the conclusion that Fat is proteolytically processed. Based on their mobility, the cleavage leading to Ft-95 occurs in or near the 2 extracellular laminin G-like domains, whereas the cleavage leading to Ft-70 occurs near the transmembrane domain. A Fat construct that excludes the cadherin and EGF domains but includes most of the laminin G domain region appears to be processed to the same cleavage products as is full-length Fat, whereas a smaller Fat construct that also lacks the laminin G domains (Fat-STI-4:FVH) yields a single major band, suggesting that it is not processed (Feng, 2009).

To further characterize Fat processing, an N-terminally tagged Fat (V5:Fat) was constructed. Examination of V5:Fat by Western blotting lysates of S2 cells identified 2 bands of high apparent molecular weight, and did not detect Ft-70 or Ft-95. Although the resolving power of the gel and the lack of suitable markers precluded precise determination of the size of these large bands, their mobility is consistent with the expected detection of both full-length Fat (Ft-565) and an approximate 470-kDa N-terminal product of proteolytic processing in the Laminin G domain region (Ft-470). Double staining V5:Fat with anti-Fat ICD and anti-V5 supported the conclusion that slowest mobility isoform is full-length Fat, whereas Ft-470 lacks the Fat ICD. To characterize cleavage of V5:Fat in vivo at endogenous expression levels, the V5 tag was incorporated into a fat+ genomic clone, and then phiC31-mediated recombination was used to insert this into the Drosophila genome. This genomic V5:fat+ construct rescued fat mutants. Western blotting lysates of imaginal discs revealed that Ft-470 is more abundant than Ft-565. Because these proteins are similar in size, this differential detection is unlikely to be due to differences in blotting transfer efficiency. Hence, it is concluded that the majority of Fat protein in vivo is processed (Feng, 2009).

To investigate the nature of Fat displayed on the cell surface, biochemical experiments were performed on cultured cells. S2 cells expressing V5:Fat were incubated with anti-V5 in the absence of detergent, and then cell surface Fat bound by anti-V5 antibodies was immunoprecipitated. As a control, Fat:FVH, which includes a cytoplasmic V5 tag that should not be accessible in intact cells, was expressed. Western blot analysis of the immunoprecipitated material with anti-Fat ICD antibodies confirmed that cell surface V5:Fat is processed. In addition, these experiments demonstrate that Ft-470 and Ft-95 remain stably associated after processing. By contrast, Ft-70 was not detected, indicating that it is not associated with Ft-470. Because coimmunoprecipitation of Ft-470 and Ft-95 could be observed under reducing conditions, the association between them does not require disulfide bonds (Feng, 2009).

Because Fat processing can occur in S2 cells, which do not express detectable levels of Ds and grow as isolated cells, and processing can occur on a truncated Fat polypeptide that lacks the cadherin and EGF domains (Fat-STI:FVH), it appears that Fat processing is part of its normal maturation, rather than a regulated event. In this regard, it appears analogous to the S1 cleavage that is involved in maturation of the Notch receptor, or to the apparent processing of the Starry night/Flamingo cadherin (Feng, 2009).

Under optimal conditions, Ft-95 from wing discs runs as doublet, with a prominent lower band, a weaker upper band, and a faint smear in between. Treatment of lysates with calf intestinal alkaline phosphatase (CIP) resulted in a single sharp band ~95 kDa, with a mobility similar to the fastest of the 95-kDa mobility isoforms in untreated samples. Thus, a fraction of Ft-95 in vivo is phosphorylated. Because Ft-95 is too C-terminal to include the cadherin domains, the phosphorylation detected presumably reflects a phosphorylation of the intracellular domain, rather than Fj-mediated phosphorylation of cadherin domains. To investigate the relationship between Ft-95 phosphorylation and Fat signaling, Fat was examined in lysates of wing imaginal discs in which its putative ligand, ds, was either mutant or overexpressed. Proteolytic processing of Fat was not Ds-dependent, because Ft-95 was observed at similar levels in all cases. Mutation of ds results in enlarged wings and wing discs, and lower levels of Wts protein, a phenotype similar to, although weaker than, that of fat. Western blot analysis of Fat from ds mutant wing discs revealed that levels of the faster mobility Ft-95 band are elevated, whereas the slower mobility band (Ft-95-P) is reduced. Ds overexpression reduces wing size. When Ds was overexpressed under tub-Gal4 control, quantitative Western blot analysis of wing disc lysates identified an average increase in Ds levels of 10-fold. Strikingly, this overexpression of Ds increased the relative amount of Ft-95-P. These observations imply that the presence or absence of Ds modulates Fat phosphorylation. This was confirmed by the observation that phosphatase treatment of lysates from Ds-expressing discs collapsed the Ft-95 doublets into a single band. The visual impression that the presence of the slower mobility (Ft-95-P) isoform(s) was promoted by Ds was confirmed by quantitative line scanning of Western blot analyses (Feng, 2009).

Both mutation of fj and fj overexpression are associated with modest reductions in wing and leg size. When fj was overexpressed under tub-Gal4 control, quantitative Western blot analysis of wing disc lysates identified an average increase in Fj levels of 100-fold. This overexpression of fj was associated with an increase in the relative amount of phosphorylated Fat, and when coexpressed with ds, the increase in phosphorylated Fat appeared even greater, consistent with the reductions in wing size. Mutation of fj had only subtle affects (Feng, 2009).

Altogether, these observations identify a correlation between the presence of the Fat ligand Ds, the level of signaling through Fat to regulate Warts levels and wing growth, and the phosphorylation of the Fat cytoplasmic domain. Thus, they suggest that activation of Fat by its ligand Ds is associated with Fat phosphorylation. From the relative levels of different mobility isoforms if is inferred that in the absence of Ds overexpression, a majority of Fat is in a hypophosphorylated form, whereas overexpression of Ds promotes the production of a hyperphosphorylated form. This identification of a posttranslational modification of Fat that is promoted by Ds is consistent with the hypothesis that Fat and Ds act as receptor and ligand in a signal transduction pathway, and identifies a molecular process that appears correlated with Fat activation. Constructs that lack most of the extracellular domain, and presumably can not interact with Ds, can rescue fat mutants. However, this rescue is only partial, and has only been observed when intracellular domain constructs are overexpressed. One possibility is that interaction with ligand triggers clustering of Fat, and that overexpression of the intracellular domain allows ligand-independent clustering. This could be analogous to other signaling pathways (e.g., TGF-β, receptor tyrosine kinase), in which ligand-mediated clustering promotes phosphorylation of the cytoplasmic domain of the receptor, and for which the requirement for ligand can sometimes be bypassed by receptor overexpression (Feng, 2009).

In considering kinases that might contribute to the Ds-promoted phosphorylation of Fat, the CKIδ/ε family member Dco was a logical candidate. Genetic epistasis tests positioned dco within the Fat pathway, upstream of dachs. At the same time, dco3 exerts cell-autonomous affects on the expression of Fat target genes, which implies that it acts within receiving cells. These observations suggested Dachs or Fat as potential substrates. Initial assessment of the ability of Dco to phosphorylate them was conducted by assaying for mobility shifts in S2 cells. Dco had no effect on Dachs. By contrast, when Dco was cotransfected together with Fat, a shift in the mobility of the C-terminal cleavage products was observed. A Dco-dependent mobility shift was also observed for both the Fat-STI:FVH and Fat-STI-4:FVH constructs. Confirmation that this mobility shift was due to phosphorylation of Fat was provided by the observation that it could be reversed by phosphatase. Overexpression of a Dco construct under UAS-Gal4 control could also increase phosphorylation of endogenous Fat in vivo (Feng, 2009).

If phosphorylation of Fat by Dco is relevant to the participation of Dco in Fat signaling, then the dco3 mutation, which causes loss of Fat signaling, should impair Fat phosphorylation. Sequencing of dco3 identified 2 distinct amino acid substitutions; these were introduced into a Dco:V5 expression construct. Dco3:V5 resulted in much less shift in the mobility of Fat in S2 cells than did wild-type Dco:V5. Thus, the same amino acid changes that cause overgrowth in vivo impair Dco-dependent phosphorylation of Fat in cultured cells. To investigate whether endogenous phosphorylation of Fat could also be influenced by mutation of dco, the mobility of Fat was examined in lysates from dco3 mutant wing discs. Unphosphorylated Fat (Ft-95) appeared slightly elevated, and a distinct Ft-95-P band was no longer visible, but rather a faint smear was detected. This change in Fat mobility was confirmed by line scanning. Thus, dco3 reduces levels of phosphorylated Fat in vivo (Feng, 2009).

To explore the relationship between the Ds-promoted phosphorylation of Fat, and the Dco-dependent phosphorylation of Fat, the mobility of Fat isolated from discs simultaneously overexpressing Ds and mutant for dco3 was examined. Direct examination of Western blots, as well as line scanning, revealed that Fat mobility in these lysates was similar to that in dco3 mutants. Thus, Ds-mediated phosphorylation can be influenced by Dco. dco3 mutant clones have no obvious effect on Fat protein staining in wing imaginal discs, suggesting that they do not affect its overall levels or distribution. Nor did dco3 noticeably affect processing of Fat (Feng, 2009).

The simplest explanation for Dco-promoted Fat phosphorylation, and for dco-dependent effects on Fat signaling, would be that Dco directly phosphorylates Fat. A purified mammalian homologue of Dco (CKIδ) phosphorylated the Fat intracellular domain in vitro, but with reduced specificity, because even greater mobility shifts than those observed in vivo could be induced. CKI's are Ser/Thr kinases, and the 538 amino acid Fat ICD includes 109 Ser or Thr residues. Three different kinase site prediction programs individually predict 7, 15, or 36 CKI sites, and cumulatively identify 46 potential CKI sites. This variation emphasizes the limited accuracy of kinase site predictions. It is also noted that distinct CKI sites could act redundantly, and that among the many potential CKI sites within the Fat ICD, phosphorylation sites responsible for the evident mobility shift on SDS-PAGE gels could be distinct from sites responsible for the influence of ds or dco3 on Fat activity. Thus, the identification of specific phosphorylation sites within the Fat ICD that are required for its biological activity will ultimately be essential for confirming the importance of Dco- and Ds-promoted phosphorylation of Fat to Fat signaling (Feng, 2009).

In contrast to the overgrowth associated with dco3 mutants, dco null mutants lack discs, and dco null mutant clones grow poorly. This could reflect the participation of dco in other processes. However, targets of Fat signaling, including Wingless (WG) in the proximal wing, and Diap1, are up-regulated in dco3 mutant clones, but not in dco null (dcole88) mutant clones. The apparent absence of fat phenotypes in dco null alleles suggests that dco3 is an unusual allele (Feng, 2009).

Dco is also known as double time, because viable alleles were independently isolated as circadian rhythm mutants. This circadian phenotype reflects a role for Dco in phosphorylating, and thereby promoting the turnover, of the circadian protein Period. This activity of Dco can be reproduced in S2 cells. Notably, Dco3:V5 was as effective as wild-type Dco:V5 at promoting Period turnover in S2 cells, whereas a circadian rhythm mutant isoform, DcoDbt-AR, was less effective. Thus, dco3 is impaired in promoting Fat phosphorylation, but active on another substrate (Feng, 2009).

Analysis of the Dco-Period interaction revealed that Dco and Period can be stably associated, as assayed by their ability to be coprecipitated from cultured cells. Similarly, Dco and the Fat-ICD can be coprecipitated, and this association was not impaired by the Dco3 mutations. Because Dco3 can associate with Fat, but does not efficiently phosphorylate it, Dco3 might act as an antimorphic (dominant-negative) protein by competing with wild-type kinase. Indeed, although dco3 is recessive at endogenous expression levels, when dco3 was overexpressed, aspects of the dco3 phenotype, including wing overgrowth and the induction of a Fat pathway target gene could be reproduced. By contrast, overexpression of wild-type forms of Dco does not cause detectable overgrowth phenotypes. Instead overexpression of Dco modestly decreased wing growth and slightly reduced transcription of diap1, suggesting that Fat pathway activity might be increased (Feng, 2009).

In addition to having a CKIδ/ε homologue, Drosophila also have a CKIα homologue, and in some contexts they can act partially redundantly. A partial shift in Fat ICD mobility could be detected when CKIα was expressed in S2 cells or in wing discs. Thus, CKIα can promote phosphorylation of Fat, although it appears less effective than Dco. This observation, together with the dco3 phenotypes observed when Dco3 is overexpressed, and the observation that although dco3 is defective in Fat phosphorylation, dco null mutant cells do not appear to be impaired for Fat signaling, suggest that dco3 might act as an antimorphic, or dominant negative, mutation, failing to effectively phosphorylate Fat and at the same time interfering with an ability of CKIα to phosphorylate Fat. By contrast, it is hypothesized that in dco-null mutant cells, CKIα or other kinases could phosphorylate Fat without interference. Although dco3 could not be rescued with a UAS-CKIα transgene, different CKI transgenes are inserted in different chromosomal locations, and their specific activities on Fat might be distinct. Thus, it remains possible that Dco and CKIα could be partially redundantly for Fat signaling (Feng, 2009).

Dco also participates in other pathways and processes. To determine whether the tumor suppressor phenotype of dco3 can be accounted for solely by its influence on Fat signaling, advantage was taken of the observation that overexpression of Wts under the control of a heterologous promoter (tub-Gal4 UAS-Myc:Wts) could rescue the lethality and tumor suppressor phenotype of fat mutants. The lethality and overgrowth phenotypes of dco3 were also rescued by Wts overexpression (tub-Gal4 UAS-Myc:Wts), resulting in animals that, aside from some mild wing vein phenotypes, are indistinguishable from wild-type animals overexpressing Wts. Because they are rescued simply by elevating Wts expression, dco3 mutant animals are specifically defective in Fat signaling; other essential processes that Dco participates in are not impaired (Feng, 2009).

Although Wts overexpression rescued the overgrowth and lethality of fat mutants, these animals have obvious PCP phenotypes in multiple tissues, consistent with the conclusion that Wts functions specifically in a Fat tumor suppressor pathway, and not in a Fat PCP pathway. By contrast, Wts-rescued dco3 mutants appear to have normal PCP. The absence of an obvious PCP phenotype also indicates that the influence of Dco and CKIα on PCP through phosphorylation of Dishevelled is not affected by dco3 (Feng, 2009).

To confirm the lack of influence of dco3 on PCP, dco3 mutant clones were examined. fat mutant clones in the abdomen exhibit obvious disruptions in the normal posterior orientation of hairs and bristles, but dco3 mutant clones had no effect. In addition to affecting the canonical PCP pathway, studies of the relationship between Fat and its downstream effector Dachs revealed a form of PCP in which Fat signaling causes a polarized distribution of Dachs, which can be visualized by mosaic expression of a tagged form of Dachs, Dachs:V5. In the developing wing, Dachs:V5 is present on distal cell membranes, but not on proximal cell membranes. In clones of cells mutant for fat, Dachs:V5 is equally distributed on proximal and distal membranes. In clones of cells mutants for dco3, Dachs:V5 localization is still polarized. Thus, the regulation of Dachs localization by Fat does not appear to be affected by dco3, although a weak effect on Dachs localization cannot be excluded. The absence of visible Dachs relocalization in dco3 clones appears to conflict with the hypothesis that the influence of Fat signaling on Warts depends on its ability to polarize Dachs, and further studies will be required to resolve this (Feng, 2009).

The atypical cadherin Fat is a transmembrane receptor for pathways that control PCP and transcription. This study has identified 2 posttranslational modifications of Fat. First, Fat is proteolytically processed, resulting in the production of stably associated N- and C-terminal polypeptides. The functional significance of this processing is not known, but its discovery is a necessary precursor to further experiments aimed at this question. Processing appears to be constitutive rather than regulated. Nonetheless, processing may facilitate subsequent events that regulate Fat (Feng, 2009).

Phosphorylation of the Fat cytoplasmic domain was also discovered. Phosphorylation is promoted by the Fat ligand Ds, is influenced by the Fat pathway kinase Dco, and correlates with Fat pathway activity in ds or dco3 mutant animals, or when Ds or Fj are overexpressed. These observations suggest that phosphorylation of Fat is a key step in Fat receptor activation. When Dco or CKIα are overexpressed, the phenotypic effects appear mild compared with the evident increase in phosphorylation. However, because there could be multiple CKI sites within the Fat ICD, it is possible that the phosphorylation-dependent mobility shift of Fat is a general marker of the extent of Fat phosphorylation, rather than a precise marker of phosphorylation at a site or sites required for Fat activity. Nonetheless, the observation that dco3 can be completely rescued by Warts overexpression, together with the epistasis of dachs to dco3, indicates that the tumor suppressor phenotype of dco3 is due to an impairment of Fat-Warts signaling, which occurs at or upstream of the action of Dachs. Altogether, these observations implicate Fat as the likely target of Dco activity in the Fat pathway (Feng, 2009).

A coordinated phosphorylation by Lats and CK1 regulates YAP stability through SCFβ-TRCP

The Yes-associated protein (YAP) transcription coactivator (a homolog of Drosophila Yorkie) is a key regulator of organ size and a candidate human oncogene. YAP is inhibited by the Hippo pathway kinase cascade, at least in part via phosphorylation of Ser 127, which results in YAP 14-3-3 binding and cytoplasmic retention. This study reports that YAP is phosphorylated by Lats on all of the five consensus HXRXXS motifs. Phosphorylation of Ser 381 in one of them primes YAP for subsequent phosphorylation by CK1delta/epsilon (Drosophila homolog: Discs overgrown) in a phosphodegron. The phosphorylated phosphodegron then recruits the SCFβ-TRCP E3 ubiquitin ligase (see Drosophila Slmb), which catalyzes YAP ubiquitination, ultimately leading to YAP degradation. The phosphodegron-mediated degradation and the Ser 127 phosphorylation-dependent translocation coordinately suppress YAP oncogenic activity. This study identified CK1delta/epsilon as new regulators of YAP and uncovered an intricate mechanism of YAP regulation by the Hippo pathway via both S127 phosphorylation-mediated spatial regulation (nuclear-cytoplasmic shuttling) and the phosphodegron-mediated temporal regulation (degradation) (Zhao, 2010).

Accumulating evidence supports the role of YAP as a key controller of organ size and as a human oncogene. Elucidating the mechanisms regulating YAP activity will have implications in the normal physiology of organ size regulation and pathogenesis of human cancer. The Hippo pathway is the only inhibitor of YAP known to date. It has been shown to play a key role in limiting organ size in Drosophila, and deregulation of several components of this pathway, such as NF2 mutation, has been implicated in human cancer. It has been shown that the Hippo pathway inhibits YAP by S127 phosphorylation-mediated 14-3-3 binding and cytoplasmic retention, therefore providing a mechanism of spatial separation of YAP from its nuclear target transcription factors, such as TEAD (Zhao, 2010).

YAP has been shown to be ubiquitinated, although the mechanism was unknown. The data presented in this study elucidated another layer of YAP regulation. By phosphorylation on S381, the Hippo pathway primes YAP for phosphorylation by CK1delta/epsilon, and subsequent ubiquitination and degradation. This provides a mechanism of temporal regulation of YAP protein levels upon activation of the Hippo pathway. Under physiological conditions like high cell density, the S381 phosphorylation-mediated degradation might be the major cause for YAP degradation. Relative S381 phosphorylation dropped dramatically when cell density increased, although relative S127 phosphorylation of YAP was increased, indicating that the S381-phosphorylated YAP could not be accumulated, possibly due to degradation. However, it is possible that there exists a S127 phosphorylation-dependent fail-safe mechanism for YAP destabilization when S381-mediated degradation is not working properly. Such a mechanism may explain why both S127 and S381 mutations are required for YAP stabilization. This study reveals that inhibition of YAP by the Hippo pathway is more complex than expected, with both spatial and temporal mechanisms. It is speculated that the spatial regulation could provide a reversible short-term inhibition of YAP, while the temporal regulation through YAP degradation may provide an irreversible long-term inhibition. Dysregulation of both mechanisms could lead to oncogenic transformation (Zhao, 2010).

It is worth noting that the S381-initiated degradation of YAP is not conserved in Drosophila Yki, because this phosphorylation site and the phosphodegron are not present in Yki, although they are conserved through vertebrates. However, this does not exclude the possibility that Yki protein stability is controlled by the Hippo pathway through other mechanisms. The phosphodegron is conserved in TAZ, a YAP paralog, and also modulates TAZ stability in a similar manner (Zhao, 2010).

Are there additional mechanisms of YAP regulation by the Hippo pathway? The possibility exists. The current studies confirmed three other Lats phosphorylation sites in YAP, but their functions are unknown. Although these sites do not seem to play an obvious role in controlling the oncogenic activity of YAP, as indicated by NIH-3T3 cell transformation assay, they may function in other contexts (Zhao, 2010).

The similarity between YAP and β-catenin is quite interesting. β-catenin is also a transcription coactivator implicated in malignant transformation. Without Wnt signaling, β-catenin is constantly degraded through SCFβ-TRCP-mediated ubiquitination. Similar to YAP, β-catenin binding with SCFβ-TRCP depends strictly on multistep phosphorylation of the phosphodegron involving CK1α and GSK-3. Perturbation of this process leads to β-catenin accumulation in colorectal cancer, HCCs, and malignant melanomas. There are similarities between YAP and β-catenin in many aspects, including their function as transcription coactivators with growth-promoting activity and as latent oncogenes. They are both subject to multistep phosphorylation and phosphodegron-dependent ubiquitination by SCFβ-TRCP, and deregulation of the degradation leads to oncogenic transformation (Zhao, 2010).

Extensive studies have been done to analyze mutations leading to β-catenin stabilization, which should shed light on future studies of YAP. In the case of β-catenin, its stabilization in cancer is frequently due to failure to recruit GSK3 as a result of inactivating mutations of adenomatous polyposis coli (APC) or axin. In some cases, stabilization of β-catenin also results from mutation in the phosphodegron and its priming phosphorylation sites. Interestingly, elevated YAP protein levels have been observed in some cancers. It will be interesting to survey possible YAP mutations in cancer samples and identify proteins regulating YAP phosphodegron phosphorylation. It will also be important to examine deregulation of YAP protein levels as a result of Hippo pathway component mutations in cancer (Zhao, 2010).

CK1 is a family of multifunctional kinases with unique substrate specificity as pS/T-X1-2-S/T. Phosphorylation by CK1 requires preceding phosphorylation of residue at the -2 or -3 position of the target residue. This requirement of a priming phosphorylation by another kinase provides a possible mechanism of signal integration in complex biological processes. For example, in the case of YAP destabilization, the requirement of CK1delta/epsilon phosphorylation following Lats phosphorylation may integrate other signals besides the Hippo pathway to regulate YAP. CK1 is often referred to as constitutively active kinase. However, it has also been reported that CK1 is regulated by subcellular localization and inhibitory autophosphorylation by stimuli such as γ irradiation and Wnt signaling. At high cell density, a clear drop of relative YAP-S381 phosphorylation and an increase of relative YAP-S127 phosphorylation are observed. The fact that both sites are phosphorylated by Lats kinase suggests that phosphorylation of S384 might induce YAP degradation. It will be interesting to investigate if cell density increases CK1 activity (Zhao, 2010).

In Drosophila, the CK1delta/epsilon homolog discs overgrown (dco) has been positioned in the Hippo pathway upstream of dachs by its regulation of the Hippo pathway downstream target genes and by genetic epistasis experiments. Recently, dco has further been shown to phosphorylate Fat, although it has not been determined if this phosphorylation directly affects Fat function and the Hippo pathway activity. However, the function of CK1delta/epsilon in regulating YAP-β-TRCP interaction is not due to inhibition of the Hippo pathway, as both YAP-4SA/S381 and YAP-S381D mutants are still inhibited by IC261. Conversely, the mechanism of CK1delta/epsilon in regulating YAP stability is unlikely to be conserved in dco, as the phosphodegron is not conserved in Yki. Nevertheless, the function of dco/CK1delta/epsilon in inhibiting Yki/YAP is conserved between Drosophila and mammals, although different mechanisms may be employed (Zhao, 2010).

YAP contains a phosphodegron, DSGXS, that is highly similar to but does not exactly match the canonical DSGXXS phosphodegron. However, the requirement of the second serine residue for β-TRCP binding is less stringent compared with the first one. In the reported phosphodegron variants, some of them require the second serine to be further away from the DSG, and, in certain cases like CDC25A, the second S is not even required. In the case of YAP, the second serine (S387) is not absolutely required, but contributes to YAP interaction with β-TRCP and YAP ubiquitination. This was shown by the residual binding between β-TRCP and the phosphorylation-deficient S387A, and the largely normal binding between β-TRCP and the phosphomimetic S387D (Zhao, 2010).

The exact YAP sequence S(-3)TDS(0)G, where S(-3) (S381) serves as a priming phosphorylation site for S(0) (S384), is conserved in some other β-TRCP substrates like CDC25A, which contains S(-6)XXS(-3)TDS(0)G. In this case, the -6 position serine phosphorylation by Chk1 is shown to be required for CDC25A binding with β-TRCP and subsequent degradation in vivo. However, in an in vitro binding assay, a peptide with phosphorylation on the S(0) but not S(-3) showed a strong binding to β-TRCP, which was not further enhanced by phosphorylation on S(-3). This in vitro binding assay using peptides sharing similar phosphodegron structure with YAP helps to exclude the function of YAP-S381 as an integral part of the phosphodegron directly involved in β-TRCP binding, but rather supports S381 as a priming phosphorylation site for S384 phosphorylation by CK1delta/epsilon. Compared with YAP, it is speculated that the main function of the S(-3) in the CDC25A phosphodegron might be a phosphorylation-relaying residue passing the signal from the -6 position to the 0 position instead of being directly involved in β-TRCP binding. Phosphodegron with a phosphorylated -3 position serine also exists in other known SCFβ-TRCP substrates, such as RE-1 silencing transcription factor (REST). Together with YAP, they may represent a class of SCFβ-TRCP substrates containing a SXDSG phosphodegron, in which the first serine serves as a priming phosphorylation site. In the case of CDC25A and REST, the kinase responsible for phosphorylating the second serine residue is unknown. The CK1 family kinases are attractive candidates for this function because of their pS/T-X1-2-S/T target consensus. It is speculated that there may be a broader role for the CK1 family in SCFβ-TRCP-mediated protein ubiquitination and degradation (Zhao, 2010).

In close proximity with the YAP phosphodegron, there is a tyrosine residue (Y391) reported to be phosphorylated by c-Abl in response to DNA damage, which results in YAP stabilization. Future studies are needed to test if the Y391 phosphorylation modulates SCFβ-TRCP-mediated YAP ubiquitination and degradation (Zhao, 2010).

Hedgehog activates fused through phosphorylation to elicit a full spectrum of pathway responses

In flies and mammals, extracellular Hedgehog (Hh) molecules alter cell fates and proliferation by regulating the levels and activities of Ci/Gli family transcription factors. How Hh-induced activation of transmembrane Smoothened (Smo) proteins reverses Ci/Gli inhibition by Suppressor of Fused (SuFu) and kinesin family protein (Cos2/Kif7) binding partners is a major unanswered question. This study shows that the Fused (Fu) protein kinase is activated by Smo and Cos2 via Fu- and CK1-dependent phosphorylation. Activated Fu can recapitulate a full Hh response, stabilizing full-length Ci via Cos2 phosphorylation and activating full-length Ci by antagonizing Su(fu) and by other mechanisms. It is proposed that Smo/Cos2 interactions stimulate Fu autoactivation by concentrating Fu at the membrane. Autoactivation primes Fu for additional CK1-dependent phosphorylation, which further enhances kinase activity. In this model, Smo acts like many transmembrane receptors associated with cytoplasmic kinases, such that pathway activation is mediated by kinase oligomerization and trans-phosphorylation (Zhou, 2011).

This study has shown that Fu is activated by phosphorylation in a Hh-initiated positive feedback loop and that Fu kinase activity alone can provoke the two key outcomes of Hh signaling in Drosophila, namely Ci-155 stabilization and Ci-155 activation. This previously unrecognized central thread of the Drosophila Hh pathway is strikingly similar to receptor tyrosine kinase (RTK) pathways or cytokine pathways, where the transmembrane receptor itself or an associated cytoplasmic tyrosine kinase initiates signal transduction via intermolecular phosphorylation. In Hh signaling, engagement of the Ptc receptor leads indirectly to changes in Smo conformation, and perhaps oligomerization that are relayed to Fu via a mutual binding partner, Cos2 (Zhou, 2011).

Three activation loop residues were identified as critical for normal Fu activity. Fu with acidic residues at T151 and T154 (Fu-EE) was not active at physiological levels in the absence of Hh but could initiate Fu activation in three different ways. First, increasing Fu- EE levels induces the full spectrum of Hh target genes and responses in wing discs and is accompanied by extensive phosphorylation, undoubtedly including S159, indicating that phosphorylation can fully activate Fu. Second, low levels of a Fu-EE derivative could synergize with an excess of wild-type Fu, provided the latter molecule had an intact activation loop and was kinase-competent, indicating that a feedback phosphorylation loop could initiate Fu activation even from a ground state containing no phosphorylated residues or their mimics. Third, Hh could activate Fu-EE or wild-type Fu, but this, unlike the above mechanisms, required Cos2 and the Cos2-binding region of Fu. Activation by Hh alters Smo conformation and increases the plasma membrane concentration of Smo-Cos2 complexes, suggesting that the role of activated Smo-Cos2 complexes may simply be to aggregate Fu molecules (Zhou, 2011).

In all of the above situations there is likely an important contribution of binding between the catalytic and regulatory regions of pairs of Fu molecules to allow cross-phosphorylation, as suggested by the impotence of the Fu-EE 1-305 kinase domain alone. The sites of inferred cross-phosphorylation, T151, S159, and S482 might most simply be direct Fu auto-phosphorylation sites but they may involve the participation of an intermediate kinase. Importantly, because Fu is the key activating stimulus and Fu is the key target for activation, there is no need to postulate additional upstream regulatory inputs into a hypothetical intermediary protein kinase. Phosphorylated residues in positions analogous to Fu S159 generally stabilize the active form of the protein kinase, whereas unphosphorylated residues at other positions, closer to the DFG motif may also, or exclusively, stabilize specific inactive conformations. By analogy, phosphorylated T151, T154, and S159 are likely to serve independent, additive functions, all of which are required to generate fully active Fu kinase. There are clearly additional phosphorylated residues on Fu, including the cluster at S482, S485, and T486. These residues are not essential for Hh or Fu-EE to generate fully active Fu when Fu is expressed at high levels. However, S485A/T486A substitutions did suppress activation of GAP-Fu in wing discs and in Kc cells, suggesting that stimulation of physiological levels of Fu, perhaps by lower levels of Hh uses S482, S485, and T486 phosphorylation to favor an active conformation of Fu or productive engagement of Fu molecules. Because the S482 region may be recognized directly as a substrate by the Fu catalytic site, this region may initially mask the catalytic site (in cis or in trans) and then reduce its affinity for the catalytic site once it is phosphorylated, permitting further phosphorylation of Fu in its activation loop (Zhou, 2011).

For a long time it was thought that Fu kinase acts only to prevent inhibition of Ci-155 by Su(fu), and Fu was postulated to accomplish this by phosphorylating Su(fu). This study mapped the sites responsible for the previously observed Hh- and Fu-stimulated phosphorylation of Su(fu) and showed that they were not important for regulating Hh pathway activity. It was found that CK1, like Fu, was required for Hh to oppose Su(fu) inhibition of Ci-155 and because each of the Fu-dependent phosphorylation sites in Fu and Su(fu) that were mapped in this study prime CK1 sites it is suspected that the critical unidentified Fu and CK1 sites for antagonizing Su(fu) will be found in the same molecule, with Ci-155 itself being a prime candidate (Zhou, 2011).

This study found that Fu does considerably more than just antagonize Su(fu). It was unexpectedly found that Fu kinase can also stabilize Ci-155 via phosphorylation of Cos2 on S572, which likely leads to reduced association of Cos2 Ci-155 activation independently of Su(fu), even when Ci-155 processing was blocked by other means (Zhou, 2011).

Some insight was gained into the key regulatory role that Fu plays in Hh signaling. The truncated partially activated Fu derivative, Fu-EE 1-473, exhibited constitutive activity when expressed at high levels but, unlike full-length Fu-EE, it was not activated by Hh. Importantly, a level of Fu-EE 1-473 expression could not be found in fumH63 mutant wing discs where Hh target genes were induced at the AP border but not ectopically. Hence, Hh regulation of Fu activity appears to be essential for normal Hh signaling. This contrasts with the normal Hh signaling observed in animals lacking Su(fu) and emphasizes that Fu is a key regulatory component that has essential actions beyond antagonizing Su(fu) (Zhou, 2011).

In mice, SUFU increases Gli protein levels and inhibits Gli activators in a manner that can be overcome by Hh, much as Su(fu) affects Ci levels and activity in flies. However, in mammalian Hh signaling there is no satisfactory mechanistic model connecting Smo activation and SUFU antagonism. This study found that mouse SUFU can substitute for all of the activities of Su(fu) in flies, including a dependence on both Fu and CK1 for Hh to antagonize silencing of Ci-155. These findings, and the observation that Drosophila Su(fu) can partially substitute for murine SUFU in mouse embryo fibroblasts, suggest that SUFU silencing of Gli proteins in mice is also likely to be sensitive to analogous changes in phosphorylation produced by at least one Hh-stimulated protein kinase. Even though the murine protein kinase most similar in sequence to Drosophila Fu is not required for Hh signaling at least three other protein kinases (MAP3K10, Cdc2l1, and ULK3) have been found to contribute positively to Hh responses in cultured mammalian cells. It will be of great interest to see if these or other protein kinases are activated by Hedgehog ligands, perhaps promoted by association with Smo-Kif7 complexes in a positive feedback loop, and whether they can antagonize mSUFU to activate Gli proteins, and perhaps even stabilize Gli proteins via Kif7 phosphorylation (Zhou, 2011).

NEMO/NLK phosphorylates PERIOD to initiate a time-delay phosphorylation circuit that sets circadian clock speed

The speed of circadian clocks in animals is tightly linked to complex phosphorylation programs that drive daily cycles in the levels of Period (Per) proteins. Using Drosophila, a time-delay circuit based on hierarchical phosphorylation was identified that controls the daily downswing in Per abundance. Phosphorylation by the Nemo/Nlk kinase at the 'per-short' phospho cluster domain on Per stimulates phosphorylation by Doubletime (Dbt/Ck1delta/epsilon) at several nearby sites. This multisite phosphorylation operates in a spatially oriented and graded manner to delay progressive phosphorylation by Dbt at other more distal sites on Per, including those required for recognition by the F box protein Slimb/β-TrCP and proteasomal degradation. Highly phosphorylated Per has a more open structure, suggesting that progressive increases in global phosphorylation contribute to the timing mechanism by slowly increasing Per susceptibility to degradation. These findings identify Nemo as a clock kinase and demonstrate that long-range interactions between functionally distinct phospho-clusters collaborate to set clock speed (Chiu, 2011).

This study shows that the per-short domain functions as a discrete hierarchical phospho-cluster that delays Dbt-mediated phosphorylation at the Slimb recognition site on Per, providing new insights into how clock protein phosphorylation contributes to circadian timing mechanisms. The cumulative effect of this delay circuit is to slow down the pace of the clock by ~8 hr. It is proposed that Dbt functions in a stepwise manner to phosphorylate clusters on Per that have distinct biochemical functions and effects on the rate of Per degradation, e.g., elements such as the per-short phospho-cluster that delays Per degradation and those such as the Slimb-binding site and global phosphorylation that enhance instability. Nmo plays a major role in the relative timing of Dbt activity at these different elements because it stimulates multisite phosphorylation at the per-short delay cluster by Dbt, which slows down the ability of Dbt to phosphorylate instability elements. Thus, a large portion of the phosphorylation events dictating when in a daily cycle Per is targeted for rapid degradation is not directly linked to binding ofSlimb per se. The current findings demonstrate that presumptive long-range interactions between distinct positively and negatively acting phospho-clusters collaborate to set clock speed and helps to explain why mutations in clock protein phosphorylation sites and/or the kinases that phosphorylate them can yield both fast and slow clocks (Chiu, 2011).

A proposed mechanism for the function of the per-short domain is supported by the congruence between in vitro biochemical studies based on purified recombinant Per protein from cultured S2 cells and in vivo changes in the pace of behavioral rhythms using transgenic models. This suggests that a primary biochemical effect of the per-short domain on clock speed in the fly is via modulating the rate of Dbt-mediated phosphorylation at the Slimb phospho-degron on Per. The physiological role of T583 phosphorylation is not clear, as mutating this site does not have detectable effects on the binding of Per to Slimb in S2 cells. In this regard, it is interesting to note that the original per-short domain was identified as encompassing aa 585-601 of Per (Baylies, 1992). Thus, it is likely that the 8 hr per-short delay circuit is governed by the dynamics underlying the phosphorylated status of three sites (i.e., S596, S589, and S585) (Chiu, 2011).

At present, it is not clear how phosphorylation in the per-short cluster slows down subsequent phosphorylation by Dbt at Ser47 and other sites. Inactivating the per-short cluster leads to increases in the rate of Dbt-mediated phosphorylation at not only the N terminus, but also the C terminus of Per, suggesting that it is a major control center for regulating the relative efficiency of Dbt phosphorylation at many sites on Per. It is suggested that the per-short phospho- cluster acts as a transient 'temporal trap' for Dbt. Once the sites in the per-short domain are phosphorylated by Dbt, this somehow allows it to continue its normal rate of phosphorylation at other phospho-clusters. Although speculative, progressive increases in phosphorylation at some of these other phospho-clusters might generate time-dependent local/overall conformational changes in Per, possibly via electrostatic repulsion, eventually leading to a more open Per structure that is more accessible to phosphorylation by Dbt at the Slimb-binding site and/or a more efficient substrate for degradation. Thus, the rapid degradation of Per during the early day is likely due to a combination of synchronous increases in the phospho-occupancy of Ser47 and overall phosphorylation of Per. Other factors such as protein phosphatases and the action of Timeless also play major roles in regulating the speed of the Per phosphorylation program (Chiu, 2011).

How might phosphorylation at S596 enhance phosphorylation at S589 and S585 by Dbt? Phosphorylation by the CK1 kinase family is generally enhanced by priming. However, phosphorylation at the per-short domain by Dbt does not follow the consensus priming-dependent recognition motif for the CK1 family of kinases (i.e., S/Tp-X-X-S/T, wherein S/Tp refers to the primed site, X is any amino acid, and the italicized residues the CK1 target site, as the S596 priming site is located C terminal to the Dbt sites. Thus, it is likely that phosphorylation of S596 by Nmo stimulates Dbt phosphorylation at the per-short region in a nonpriming-dependent manner (Chiu, 2011).

Ongoing studies are aimed at understanding the biochemical events underlying the ability of phosphorylation at S596 to enhance phosphorylation by Dbt in the per-short region. The discovery of a delay phospho-circuit also sheds light on why mutations in different phosphorylation sites on Per or Frq proteins, although affecting stability, can speed up or slow down the clock. The current findings also offer a logical explanation for why mutations that lower the kinase activity of CKI, which overall is expected to slow down the rate of PER degradation, can yield fast clocks. For example, although other mechanisms have been offered to explain the short-period phenotypes that are observed for the CKI3tau mutation in hamsters and a CKIdelta mutation associated with familial advanced sleep phase syndrome (FASPS) in humans, it is possible that phosphorylation at a per-short type delay cluster is preferentially compromised by the mutant kinase, which could appear as a substrate-specific gain-of-function mutation (Chiu, 2011).

Negatively acting phospho-clusters are likely to be a general feature of the timing mechanisms regulating the daily abundance cycles of clock proteins such as Pers in animals and Frq in Neurospora. However, other regulatory modules that operate in a phase-specific manner must participate to generate an ~24 hr oscillator. Most conspicuously, clock speed is intimately linked to the Per and Frq abundance cycles necessitating daily phases of de novo synthesis to replenish the pools of previously degraded proteins.Asrecently shown, the transcriptional negative feedback aspect of Per regulating Clk-Cyc-mediated transcription is also a component of the period-setting mechanism in Drosophila. Therefore, the ~24 hr Per abundance cycle is based on a combination of 'time constraints' that are generated using different regulatory modules. It is proposed that the per short- based timer mainly functions once Per has accumulated and begins participating in transcriptional repression, controlling Per abundance once it is disengaged from the dynamics of its cognate mRNA by setting in motion a series of sequential phosphorylation events that are calibrated to stimulate Per degradation in the nucleus at the appropriate time in a daily cycle, enabling the next round of circadian gene expression. In this context, it is interesting to note that a prior study analyzing the per-short domain suggested that it functions with a nearby 'perSD' domain to increase the transcriptional repressor function of Per. It is possible that the same phosphorylation events leading to Per degradation also function to increase its potency within the repressor complex (Chiu, 2011).

These studies also identify Nemo as a clock kinase. Nemo is the founding member of the evolutionarily conserved Nemo-like kinase (Nlk) family of proline-directed serine/threonine kinases closely related to mitogen-activated protein kinases (MAPK). It was originally characterized in Drosophila as required for planar cell polarity during eye development and is now known to function in many pathways. Nmo/Nlk is localized in the nucleus and is another factor in the circadian clock that also functions in the Wnt/Wg-signaling pathway, such as CKI3/Dbt, β-TrCP/Slimb, and GSK-3β/Sgg. It will be of interest to determine whether Nlk functions in the mammalian clock. Intriguingly, the phosphorylation sites on Per are largely clustered, and several of them have the same spatial arrangement as the per-short cluster, with a predicted pro-directed kinase site at the C-terminal end of the phospho-cluster. This suggests that Nmo and/or other pro-directed kinases serve as control points to activate spatially and perhaps functionally distinct phospho-clusters. Indeed, it has recently been shown that phosphorylation at Ser661 of Per by an as yet unidentified pro-directed kinase primes further phosphorylation by Sgg at Ser657 to regulate the timing of Per nuclear entry in key pacemaker neurons (Chiu, 2011).

In summary, a central aspect of circadian clocks is the presence of one or more clock proteins that provide a dual function by behaving as phospho-based timers and linking its timer role to gene expression by operating in a phase-specific manner to recruit repressor complexes that inhibit central clock transcription factors. These studies suggest that a major part of the timing mechanism underlying these phospho-clock proteins is based on spatially and functionally discrete phospho-clusters that interact to impose calibrated and sequentially ordered biochemical time constraints. In the case of Per, the per-short phospho-cluster functions as a central timing module by slowing down the ability of Dbt to phosphorylate instability elements regulating Per degradation and, hence, when Per repressor activity is terminated and the next round of circadian gene expression begins (Chiu, 2011).

Noncanonical FK506-binding protein BDBT binds Dbt to enhance its circadian function and forms foci at night

The kinase Doubletime is a master regulator of the Drosophila circadian clock, yet the mechanisms regulating its activity remain unclear. A proteomic analysis of Doubletime interactors led to the identification of an unstudied protein designated CG17282. RNAi-mediated knockdown of CG17282 produced behavioral arrhythmicity and long periods and high levels of hypophosphorylated nuclear Period and phosphorylated Doubletime. Overexpression of Doubletime in flies suppresses these phenotypes and overexpression of CG17282 in S2 cells enhances Doubletime-dependent Period degradation, indicating that CG17282 stimulates Doubletime's circadian function. In photoreceptors, CG17282 accumulates rhythmically in Period- and Doubletime-dependent cytosolic foci. Finally, structural analyses demonstrated CG17282 is a noncanonical FK506-binding protein with an inactive peptide prolyl-isomerase domain that binds Doubletime and tetratricopeptide repeats that may promote assembly of larger protein complexes. CG17282 was names Bride Of Doubletime and was established as a mediator of Doubletime's effects on Period, most likely in cytosolic foci that regulate Period nuclear accumulation (Fan, 2013).

While FKBPs were originally identified as mediators of the immunosuppressive effects of FK506 on calcineurin and rapamycin on the Target of Rapamycin (TOR), subsequent work has suggested their involvement in a wide range of signaling processes, including ones involved in neurodegeneration and cancer. In many cases, their function derives from their catalysis of cis-trans conversions of peptide bonds involving prolines. However, BDBT lacks the necessary catalytic residues, as do several other noncanonical FKBPs. One of these noncanonical FKBPs (FKBP38) has been proposed to interact with TOR to suppress its activity, while interactions between FKBP38 and the small GTP-binding protein RHEB relieve this repression and activate TOR. Intriguingly, TOR and RHEB have recently been show to modulate the circadian clock of Drosophila (Fan, 2013).

However, FKBP proteins have also been implicated in regulation of nuclear localization and protein stability. For instance, the noncanonical FKBP-like protein (FKBPL) has been implicated in the nuclear import of steroid hormone receptors in complexes with HSP90 proteins. An interesting possibility is that BDBT is involved in regulating the import of Per/Dbt complexes to the nucleus, and that at least some of this regulation is negative, as Per exhibits increased nuclear accumulation in BDBT knockdown flies. This hypothesis is consistent with structural work, which uncovered a resemblance between BDBT and the HSP90-binding protein FKBP51. The HSP90-binding site in FKBP51 localizes to its TPR domain and all but one of the residues that account for HSP90 binding are conserved in BDBT in spite of the low sequence homology with BDBT. Since the N-terminal, PPIase-like domain of BDBT binds to Dbt in HEK293 cells, it is possible that BDBT assembles a Dbt/Per/HSP90 complex, with Dbt bound to the PPIase-like domain, HSP90 to the TPR domain, and Per bound to Dbt (Fan, 2013).

FKBPs have also been implicated in the regulation of the stabilities of proteins with which they form a complex. A role in enhancement of Per's phosphorylation-dependent proteolysis is particularly attractive for BDBT, as it would explain the RNAi knockdown phenotype in head extracts (elevated levels of hypophosphorylated Per) and the enhancement of Dbt-dependent degradation of Per in S2 cells. The cytosolic BDBT foci in Drosophila photoreceptors accumulate at a time (ZT13-19) when Per transitions from a destabilized cytosolic form to a stabilized nuclear form, and the data supporting the involvement of BDBT in enhancement of Per proteolysis suggest that BDBT may be a negative regulator of this transition (i.e., BDBT antagonizes Per accumulation and nuclear localization). The BDBT foci are intriguing in light of the finding by Neyer of Per/Tim cytosolic foci, which form prior to accumulation of Per and Tim in S2 cell nuclei (Meyer, 2006). It was proposed that processes in these foci trigger the nuclear accumulation of both Per and Tim. Since the suggestion from this work is that BDBT foci antagonize nuclear accumulation of Per and no obvious Per foci were observed that colocalize with the BDBT foci, it is possible that BDBT antagonizes focal accumulation of Per or immediately triggers the degradation of Per in these foci. In this scenario, in contrast to the situation in S2 cells, Per might accumulate in foci in vivo only when BDBT is not present or active in the foci, and Per's presence in foci in vivo may therefore be difficult to detect because it rapidly accumulates and localizes to nuclei when BDBT is not active in the foci. It is also possible that focal accumulation of BDBT negatively regulates BDBT activity toward Dbt, since highest levels of BDBT foci are detected at ZT19, when Per is rapidly accumulating in nuclei and therefore any BDBT inhibition of Per nuclear accumulation might be inhibited (Fan, 2013).

Because the mammalian orthologs of Dbt (CKIepsilon and CKIdelta) are also essential for the molecular mechanism of the mammalian circadian clock, it is possible that the mechanism in which bdbt participates is conserved in mammals. While this manuscript was in preparation, FKBP and FKBP-like proteins were reported to form complexes with mammalian CKIepsilon and CKIdelta (Kategaya, 2012), although neither these proteins nor any other protein in the mammalian genome is an ortholog of BDBT. The lack of homology between the PPIase-like region in BDBT, which mediates binding to DBT, and the ones found in vertebrates, was initially surprising. However, the binding experiments indicate that the BDBT binding site in DBT spans its well-conserved N-terminal kinase domain and the poorly conserved C-terminal tail. Thus, it seems likely that the binding modes between BDBT and Dbt on one hand and the ones between the vertebrate homologs of BDBT and CKIepsilon and CKIdelta differ substantially. While it is not known if this interaction has any role in the mammalian circadian clock, these results offer the tantalizing prospect that this class of proteins and their roles are conserved in the mechanisms of the mammalian and Drosophila clocks (Fan, 2013).


DEVELOPMENTAL BIOLOGY

Adult

In order to test whether dbt is expressed in clock cells in the brain, in situ hybridizations to adult head sections were performed with per, tim, and dbt antisense RNAs. All three genes are expressed in photoreceptor cells composing the eyes. tim shows a discrete pattern in brain and is expressed at highest levels in the lateral neurons (LNs). per and dbt are expressed in a wider region between the optic lobe and the central brain, which includes the LNs. The pattern of PER mRNA expression is identical to the anti-ß-galactosidase staining previously seen in a per promoter ß-galactosidase transgenic line, and the pattern of TIM mRNA staining is the same as that observed with anti-Tim antibody. Although no clear differences between the patterns of per and dbt expression were detected, the technique does not resolve signals at the level of single cells; the possibility that some cells express per or dbt alone cannot be excluded. Thus, per and dbt appear to be largely expressed in the same cells in the brain, which include the tim-expressing cells. Interestingly, the RNA signals for all three genes are located predominantly at the periphery of the nucleus in the photoreceptors, although the significance of this is not yet clear (Kloss, 1998).

Because mRNA levels of the two previously identified Drosophila clock genes, per and tim, have been shown to oscillate with a 24 hr period in heads of adult wild-type flies, it was of interest to determine whether levels of DBT mRNA oscillate as well. Although analysis of wild-type pupae has already suggested that the abundance of DBT RNA remains essentially constant throughout the day and night, these measurements represent RNA levels found in whole pupae. Oscillations in the levels of the PER and TIM transcripts are considered to be such a central feature of a normally functioning clock, that it was necessary to repeat this analysis under conditions where PER and TIM oscillations could be robustly observed (i.e., in adult fly heads). Wild-type flies were entrained to a 12:12 LD cycle, and flies were collected at 4 hr intervals for 3 days. Heads were isolated from the flies collected at each time point and used to prepare total RNA. RNase protection analyses have demonstrated that while PER and TIM mRNA levels display strong (approximately 10-fold) circadian oscillations, levels of DBT mRNA remain essentially constant. Therefore, there are two unique features of dbt that distinguish it from per and tim: strong hypomorphic mutations of this gene are homozygous lethal, and levels of the DBT transcript apparently do not oscillate (Kloss, 1998).

Effects of Mutation or Deletion

Since the original description of discs overgrown (dco)/double-time (Jursnich, 1990) many new alleles of the gene have been produced by various mutagenic procedures. The phenotypes of these new alleles were examined as well as of some new deficiencies and of the few alleles described earlier, dco3 (Jursnich, 1990), in homozygotes and heteroallelic combinations. The effects of dco mutations on imaginal discs are seen most clearly in the wing disc, since this disc is fairly flat and any morphological or growth abnormalities are easily detectable in whole mounts. Most genotypes involving alleles other than dco3 or dcoP1447 show various degrees of abnormality, ranging from complete absence of discs to apparently normal discs. In intermediate cases, the folding pattern of the epithelium is very abnormal, and the discs often show a dark, granular zone in the wing pouch region. These phenotypes are produced by various EMS-induced alleles ; P-element insertions, and deletions induced by X-ray and P-element excision. All of the genotypes giving a discless or small-disc phenotype cause lethality in the embryo, larva or pupa before adult differentiation. These phenotypes result from combinations of strong loss-of-function dco alleles. Most of those genotypes that display an abnormal disc phenotype, caused by combinations of weaker dco alleles, also lead to death at some time before adult differentiation; however, some allow survival to late pupae, and in one case (dco2/dcoP915) the pupae show a phenotype including expanded tarsi that is otherwise typical of dco3 combinations. In dco3 homozygotes or heteroallelic combinations of dco3 with any of the other known dco alleles or deficiencies, the larval period is prolonged by many days. During this time, the imaginal discs grow continuously to several times the wild-type final size. The mutant discs retain their epithelial structure, hence the overgrowth should be classified as hyperplastic rather than neoplastic. The wing discs develop a central region of excess growth in which the epithelial folds are thinner and more convoluted than normal, as noted previously (Jursnich, 1990). The larval period is also prolonged and growth of discs slowed down in homozygous or heterozygous combinations of dco alleles that eventually produce normal size discs with abnormal folding. dco3 pharate adults show morphogenetic aberrations including duplicated structures Many heteroallelic combinations with dco3 allow survival to late pupal or pharate adult stage (i.e., fully differentiated adults that die without leaving the pupal case). These animals typically have swollen tarsal segments and abnormal heads in which the ptilinum appears enlarged and fails to retract. (The ptilinum is a cuticular balloon on the front of the head that is inflated by blood pressure to push open the door of the puparium in order to allow the adult to emerge, and is then retracted). Eyes are often missing, and are sometimes replaced by duplicated antennae. Excess bristles are often seen on the antennae. One genotype (dco3/Df(3R)PGX8) produces greatly enlarged tarsi giving a 'spadefoot' appearance. Most notably, some of the legs in this genotype show distally duplicated or even triplicated ventralized structures, such as duplicated sex combs and claws. The wings of these animals fail to expand and therefore any pattern abnormalities are difficult to discern and can only be analyzed in clones (Zilian, 1999).

The allele dcoP103 seems to have unique properties: dcoP103/dco2 produces normal imaginal discs and allows survival to adulthood, but the adults show expanded wings and localized outgrowths from the antennae and the distal leg segments. Combinations of dcoP103 with dco i3-193 or dcoP538 produce abnormal imaginal discs, allow survival to adulthood, and the adults have abnormally narrow wings. One other combination (dcoP103 /dcoP915 ) allows survival to adulthood with no apparent abnormalities. Another set of phenotypes is seen in surviving adults heteroallelic with the P-element insertion dcoP1447 . Thus, dcoP1447 /dco3 shows several head abnormalities including excess vibrissae, enlarged antennae and a widened prefrons often associated with reduced eyes, as well as an expanded wing with excess vein material along vein 2 and often an incomplete posterior cross-vein. The head phenotype is also seen at low frequency in dcoP1447/Df(3R)PGX8. In contrast, dcoP1447/dco18 adults have reduced wings, and they often also show incomplete posterior veins. Heteroallelic combinations of dcoP1447 with alleles other than dco3 exhibit an adult phenotype whose penetrance and expressivity depend strongly on the allelic combination and are frequently much higher in females than in males. dco- clones are growth-inhibited and fail to survive in discs Null, or alternatively, strong dco alleles show greatly reduced cell proliferation in imaginal discs and thus produce very small discs that eventually degenerate because of apoptosis (see below) and give rise to the discless phenotype. Some dco3 phenotypes show partially duplicated leg structures that are reminiscent of phenotypes produced by leg disc clones that cannot receive the Dpp signal. To test whether these effects on growth and apoptosis of strong dco alleles were clone- or disc-autonomous and caused by a defect in Dpp signaling, the activity of Dpp target genes on the adult phenotype was examined. Mitotic clones of dcole88, (a null allele), were induced in imaginal discs at various times during larval development. Would the absence of dco activity in these clones have an effect on cell growth or survival? Inspection of wing discs from wandering third-instar larvae shows that dco- clones, induced relatively late (2 days before analysis) can be observed but are less than half the size of their twin clones, which consist of up to about 16 cells. In contrast, nearly no cells of dco- clones induced one day earlier are detectable while their twin clones are of the expected normal size. When clones are induced 4 days before analysis, no dco- cells are observed and twin clones consist of hundreds of cells. Clearly, cells that lack a functional dco gene are unable to undergo continued growth and cell proliferation and die after only two or three divisions. It thus appears that these clones do not die because of a growth disadvantage with respect to competing wild-type cell. The results further demonstrate that the growth inhibition and apoptosis of dco- cells is cell-autonomous, which suggests that dco- cells are unable to respond to a signal required for growth or survival. Consistent with the above findings, no dco- clones survive to adulthood unless they are induced in imaginal discs shortly before pupariation. In most cases, the loss of dco- clones does not affect the final adult pattern, probably because surrounding cells compensate for the loss by extra proliferation. Occasionally, if many clones are induced very early in larval discs, compensation is incomplete and the bristle pattern is disturbed or, in extreme cases, the scutellum is severely affected. Both the limited growth of dco - cells observed in larval discs and the survival of histoblast clones to adulthood can be explained by the perdurance of wild-type Dco protein in the mutant cells. Additional experiments show that dco is not essential for the transduction of the Dpp signal, and effects of dco on growth are not mediated by Dpp (Zilian, 1999).

In order to test the clonal autonomy of the dco3 overgrowth phenotype, dco3 clones were generated at various larval stages as for the dcole88 null allele. No effect on Dpp target genes is detectable in such clones of discs from wandering third-instar larvae. However, in contrast to dcole88 clones, dco3 clones not only continue to grow and proliferate but fail to stop growth when the discs have reached their normal size. Such clones are larger than their twin clones, although the size of dco3 cells is not affected , and exhibit moderate overgrowth. This moderate overgrowth is not due to accelerated cell proliferation in dco3 cells because the ratios of the sizes of wild-type clones induced at various times before analysis and those of dco3 clones induced at the same times before analysis are the same. In agreement with the above results, dco3 clones survive to adulthood and show excess tissue as revealed, for example, in wings, and especially in the wing vein regions. No excess tissue appears in dco3 adult clones originating from histoblasts, presumably because of the perdurance of the wild-type Dco protein. If more clones are induced, the larval life is considerably prolonged by up to two days and the clones exhibit massive overgrowth such that the discs are clearly enlarged. Such animals die as pharate adults, as do dco3 mutants; they exhibit almost identical disc and pharate adult phenotypes. These results show that dco3 disc cells are still able to transduce growth and survival signals. Moreover, since most larval tissue does not proliferate after clone induction, it follows that dco3 disc cells are defective in responding to the signal that stops growth of the mature larval disc. This conclusion is further consistent with the recessive nature of the dco3 allele and its cell-autonomous effect on growth arrest. In addition, since pupariation is delayed in such mosaic animals, it has been concluded that dco + appears to be required in discs for the proper timing of pupariation (Zilian, 1999).

The abnormal imaginal discs produced in many dco genotypes have a localized area that appears granular and dark in transmitted light, a characteristic of degeneration as previously reported in overgrowing dco imaginal discs (Jursnich, 1990). Studies by both light and electron microscopy show clearly that, in these areas, individual cells undergo apoptosis as evident from the occurrence of fragmentation, condensation and basal extrusion. The basally extruded fragments accumulate under the basal lamina. This type of cell degeneration is typical of apoptosis and associated with mutations that cause tissue loss in imaginal discs (Zilian, 1999).

The fact that dco encodes a ser/thr protein kinase, together with many aspects of its mutant phenotypes, suggests that the protein may function in one or more signal transduction pathways. All known protein kinases are regulated by external signals and regulate downstream components by phosphorylation. The activity of CKI delta/epsilon is known to be regulated by phosphorylation in other organisms. Moreover, it has recently been shown that Cki1, the S. pombe homolog of CKI delta/epsilon, inhibits phosphatidylinositol 4-phosphate 5-kinase (PIP5K) and hence the synthesis of phosphatidylinositol 4,5- bisphosphate (PIP2) (Vancurova, 1999). PIP2 itself is a second messenger as well as an important substrate of phospholipase C, which converts PIP2 to additional second messengers, activating, for example, protein kinase C. skittles (sktl), the gene encoding a Drosophila homolog of PIP5K, is required for cell viability and germline development (Hassan, 1998). Similarly, Dco functions are required for cell survival in imaginal discs as well as for germline development since females lay no eggs if their germline is homozygous for dcole88 (assuming a chromosome free of other lethal mutations). Therefore, it is possible that Sktl is a substrate of Dco. If so, the level of PIP2 would be regulated by Sktl and Dco in opposite ways and must be critical for these processes. In addition, the observation that the activity of mammalian CKIepsilon can be regulated in vivo by autophosphorylation/dephosphorylation of its C-terminal domain (Rivers, 1998), suggests that one substrate of Dco might be Dco itself. Moreover, as phosphorylation of the C-terminal portion of CKIe inhibits the kinase activity (Rivers, 1998), this domain might well be the target of several signaling pathways regulating Dco activity (Zilian, 1999).

For doubletime (dbt) alleles no mutant adult phenotype other than the shortened or prolonged circadian rhythm has been reported (Kloss, 1998). Consistent with this mild phenotype, both dbt mutations are missense mutations at locations not expected to affect the catalytic site of the kinase domain. They probably result in altered affinities for the protein substrate Per, which mediates their effect on the circadian rhythm. Similarly, none of the dco point mutations appears to affect the catalytic activity of the Dco kinase, consistent with the production by these mutations of a wide variety of mutant phenotypes. While Per has been shown to be a substrate of Dco in vivo, the strong morphogenetic effects of Dco cannot be mediated only through Per because per null mutants are viable and show none of the dco phenotypes reported in this study. For the elucidation of all functions of Dco, it will be important to find any additional substrates that might act in signal transduction pathways controlling cell survival, proliferation, and growth arrest. Dco is required for both growth and growth arrest in discs (Zilian, 1999).

Although dco plays an essential role in growth and proliferation, this is not its sole morphogenetic function in imaginal discs. In general, mutant dco alleles inhibit growth of discs in homozygous and transheterozygous combinations. However, one allele, dco3, hardly inhibits growth, if at all, but rather fails to arrest growth of discs when they have reached their normal size. Since dco3 animals can be rescued completely to wild type by a single dco + transgene, the failure in growth arrest of dco3 discs corresponds to a loss of function. As this property of dco3 is further cell-autonomous in clones, it is concluded that the ability of dco3 cells to transduce a signal, required to stop growth at the end of larval life, is significantly reduced while the generation of the signal is not affected. Differentiation of dco3 animals to pharate adults implies that growth arrest of dco3 discs is only delayed but not abolished, which indicates that Dco 3 is hypomorphic for the growth arrest function of Dco. The foregoing interpretation of Dco function is also consistent with the observation that transheterozygous combinations of dco3 with dco alleles that are hypomorphic or amorphic for the Dco growth or survival function show disc overgrowth, if these alleles are also hypomorphic for the Dco growth-arrest function, a condition met by all null and most P alleles. This analysis of dco3 transheterozygotes suggests that the two point alleles, dco2 and dco18, are also hypomorphic for the disc overgrowth phenotype. It is further inferred from dco3 mutants that some features of the Dco CKI protein are essential only for the transduction of the growth arrest, but not of the growth or survival signal. It remains to be shown if complementary mutations of Dco can be generated that affect only its growth or survival but not its growth-arrest function. Such mutations over dco3 would complement to produce wild-type flies. Homo- or hemi-zygous dco3 discs continue to grow and reach a size considerably larger than that of normal discs. This overgrowth is accompanied by massive apoptosis and a prolonged larval life. Apparently, larval life continues as long as growth of imaginal discs has not been arrested, which suggests a coupling of pupariation to growth arrest of discs. Moreover, while larval disc cells that fail to proliferate undergo apoptosis, this fate is suppressed in non-dividing pupal cells. Apoptosis in overgrowing dco3 discs indicates that it may be induced in regions of hyperplastic growth as well. Apoptosis followed by intercalary growth in discs probably also gives rise to the duplications and triplications observed in hemizygous dco3 pharate adults as well as the outgrowths and patterning phenotypes found in dco mutants that survive to adulthood. The more dramatic dco3 phenotypes might result from excessive intercalary growth in these mutants (Zilian, 1999).

It is concluded that Dco is part of at least two signal transduction pathways of disc morphogenesis; one responding to a growth or survival signal, the other to a signal that arrests growth when a disc has reached its normal size. It is evident from the discless phenotype of strong dco mutants and from the analysis of dco null clones, that cells in which Dco function has been sufficiently reduced die after only a few cell divisions. As cell growth and inhibition of apoptosis are thought to be intimately linked, these phenotypes can be explained by loss of function in either cell growth or cell survival, and wild-type Dco might function by stimulating growth and proliferation or by inhibiting apoptosis. Coupling of cell growth and survival could occur intracellularly through branched pathways activated by the same extracellular signal, for example insulin, or extracellularly through two distinct, but coordinately expressed, signals like insulin and a survival factor. One of the substrates of mammalian delta and epsilon isoforms of CKI is the tumor suppressor p53, which regulates both proliferation and apoptosis (Knippschild, 1997). Such a mechanism could be operating in Drosophila, although a fly counterpart of p53 is not yet known (Zilian, 1999).

It is unclear which growth factors may regulate the activity of Dco. The main growth factor known to be required for growth of imaginal discs is insulin, and hypomorphic insulin receptor (inr) mutants show small disc phenotypes similar to those of hypomorphic dco larvae (Chen, 1996). But whereas some dco phenotypes resemble inr phenotypes, strong inr mutants die during embryogenesis. In contrast, dco null mutants develop to third-instar larvae and may survive for as long as three weeks at 25 degrees C. Therefore, if Dco activity is regulated by the insulin pathway, it may function in only some but not all of its branched pathways (Yenush, 1996). In addition to insulin, a family of growth factors required for growth of imaginal discs has recently been identified (Kawamura, 1999). These IDGFs (Imaginal Disc Growth Factors) are produced in the larval fat body and support survival of disc cells in culture, but are also necessary, in combination with insulin, for proliferation (Kawamura, 1999). Perhaps Dco reacts to these factors or even serves to integrate insulin and IDGF signals. It is thus conceivable that Dco activity in discs depends on both type of signals, which might explain the similarity of hypomorphic inr and dco disc phenotypes. It is also possible that recognition of some substrates by Dco requires both signals, while its recognition of other substrates depends on only one of them. That Dco is probably a component of several distinct pathways is further suggested by its additional role in the circadian rhythm. It should be emphasized that growth and survival of most larval tissues does not depend on Dco. Hence, dco null mutants are able to survive as discless third-instar larvae for a very long time. Most of them are unable to pupariate, probably because pupariation is coupled to the arrest of overall disc proliferation when discs have reached their normal size. One can speculate and consider dco as an atavistic mutation because it prevents metamorphosis of the larval worm and considerably prolongs its life span. In C. elegans mutations in the insulin pathway are known to result in the formation of a developmentally arrested larval form that can survive for a long time, the dauer larva (for a review see Wood, 1998). It is thus tempting to speculate that the considerably extended larval life span of dco null mutants might have a similar cause (Zilian, 1999).

Three alleles of double-time have been isolated: short- (dbtS) and long-period (dbtL) mutants alter both behavioral rhythmicity and molecular oscillations generated by previously identified clock genes, period and timeless; a third allele, dbtP, causes pupal lethality and eliminates circadian cycling of per and tim gene products in larvae. In dbtP mutants, Per proteins constitutively accumulate, remain hypophosphorylated, and no longer depend on Tim proteins for their accumulation. It is proposed that the normal function of Doubletime protein is to reduce the stability and thus the level of accumulation of monomeric Per proteins. This would promote a delay between per/tim transcription and Per/Tim complex function, which is essential for molecular rhythmicity (Price, 1998). Ethyl methane sulfonate (EMS) mutagenesis was used to induce new clock mutations affecting the period length of locomotor activity rhythms in homozygous or heterozygous flies. Screening of heterozygous phenotypes was performed because all known clock mutations that affect period length in Drosophila, Neurospora, Arabidopsis, mice, and hamsters are semidominant. The locomotor activity of individual flies, each bearing heterozygous or homozygous mutagenized chromosomes, was monitored under constant darkness (DD) to reveal free-running period length. From a screen of ~ 15,000 second and third chromosomes two lines were isolated that contain mutations in double-time, so named because the first mutant allele that was isolated (dbtS) dramatically shortens the behavioral period (Price, 1998).

Flies that are heterozygous for the dbtS mutation (dbtS/+) produce locomotor rhythms with an average period of 21.8 hr in DD, while homozygous flies (dbtS/dbtS) produce locomotor rhythms with an average period of 18.0 hr. Flies that are heterozygous for a second allele, dbtL (dbtL/+) produce locomotor rhythms with an average period of 24.9 hr, and homozygous flies (dbtL/dbtL ) produce locomotor rhythms with 26.8 hr period. Because dbtS/+ and dbtL/+ flies have shorter and longer periods, respectively, than wild-type controls, but not as short or long as homozygous mutant flies, dbtS and dbtL are semidominant. Homozygous dbtS and dbtL flies can be entrained by an imposed 12 hr light:12 hr dark cycle (LD 12:12) since they exhibit 24 hr periodicity under such conditions. Analysis of several hundred locomotor activity records from homozygous dbtS and dbtL flies has indicated complete penetrance of the mutant phenotypes (Price, 1998).

dbtS and dbtL were also tested for aberrant circadian rhythms of eclosion (emergence of the adult fly from the pupal case) to determine whether dbt mutations affect this phenotype as previously observed for per and tim. Obviously, eclosion occurs only once in the lifetime of an individual fly, but when viewed in terms of a population of flies of diverse ages, it occurs repeatedly and rhythmically. In constant daylight (DD), the period of the dbtS eclosion rhythm is shorter than the rhythm of the wild-type population. Peaks of eclosion occurred progressively earlier in dbtS as compared to wild type over the 5-day interval tested. For dbtL, a longer period rhythm is obtained as compared to wild type. The similar effects of the dbtS and dbtL mutations on two behavioral outputs of the Drosophila circadian clock are consistent with an effect on the central pacemaker mechanism rather than on a specific output pathway. In this regard, the effects of dbt mutations on rhythmicity are comparable to those of period and timeless mutations (Price, 1998).

To investigate whether dbt period-altering alleles change the molecular oscillation of known clock components, Per and Tim protein time courses were examined on Western blots. One day of a twelve hour light dark cycle (LD) and two days of constant darkness (DD) were examined for wild-type, dbtS, and dbtL genotypes. Overall, the levels of expression of both Per and Tim are not grossly altered. However, in LD, both proteins oscillate with a slight phase advance in dbtS, and phase delay in dbtL. In DD, the proteins oscillate with a period length corresponding to the locomotor activity rhythms of the mutants. For dbtS in DD, sharp transitions from hyperphosphorylated to hypophosphorylated Per occur between circadian time (CT) CT6 and CT10, next between CT22 and CT2, and on the last cycle between CT18 and CT22, giving an average periodicity of 18 hr. Wild-type controls show hyper-to-hypophosphorylated Per transitions between CT6 and CT10 in both cycles, giving a 24 hr period. Although shifts in mobility are less dramatic in dbtL, transitions appear to occur between CT10 and CT14, and subsequently between CT14 and CT18, giving a 28 hr period. For Tim, mobility differences for all genotypes are much smaller, but the strong phase differences among the genotypes, and period differences in RNA expression patterns, indicate that period is likely altered as for Per (Price, 1998).

A closer inspection of dbtS reveals that both Per and Tim disappear prematurely in an LD cycle. Forms of Per with lowest electrophoretic mobility, which have been associated with highest levels of Per phosphorylation, appear earlier than in wild type. In contrast, in dbtL, both Per and Tim are detectable for an extended period of time in DD, and the appearance of low-mobility forms of Per is delayed. In contrast to wild-type flies, persistence of Per is detected in the absence of Tim after lights-on in an LD cycle in dbtL. This effect suggests unusual persistence of monomeric Per proteins after Tim is eliminated by light. The higher level of Per from ZT2-6 (ZT, zeitgeber time, indicates time in LD cycles) is not simply due to increased Per levels in dbtL, since a side-by-side comparison of Per proteins in wild type and dbtL at ZT0 shows roughly equal amounts of Per. Thus, there seems to be an increase in Per stability in dbtL. Conversely, dbtS may cause premature degradation of both Per and Tim (Price, 1998).

PER and TIM mRNA cycling can also be altered by mutation of dbt. Patterns of RNA cycling were followed in dbtL mutants and in wild-type flies. The first day of sampling occurred in LD, with subsequent days followed in DD. Although the initial LD cycles of PER and TIM mRNA expression occurred with essentially the same phase in dbtL and wild type, dbtL gave three complete molecular cycles in ~3.5 days of DD, while wild-type flies produced three cycles in ~2.5-3 days of DD. Peaks of PER and TIM mRNA accumulation occur with an ~27 hr periodicity in dbtL, and an ~23 hr periodicity in wild type. Because peaks of RNA expression are not always coincident for per and tim in dbtL, and the oscillations are of reduced amplitude, it is possible that the molecular rhythms are less stable in the mutant. However, the simplest interpretation of the data is that per and tim cycle together in the mutant with an ~27 hr period (Price, 1999).

In order to isolate a P-element insertion mutant of dbt, Drosophila strains containing P-element insertions on the right arm of the third chromosome were screened for failure to complement the original dbtS mutation. One strain, referred to as dbtP, behaves like a deficiency that eliminates dbt. dbtS/dbtP flies produce locomotor activity rhythms with a period of 19 hr, while dbtP/+ flies have wild-type periods (23.8 hr). Similarly, dbtL/dbtP flies have locomotor activity rhythms of 26.6 hr. The P element is therefore likely to result in a large reduction, or even absence, of dbt gene products. The finding that dbtP fails to complement both dbtS and dbtL indicates that the latter mutations affect the same gene. Recessive lethality is associated with the dbtP strain, as no adults of the genotype dbtP/dbtP are found. Most third instar homozygous dbtP larvae pupate, but they die later in pupal development (Price, 1998).

Short- and long-period mutations (dbtS and dbtL), which alter period length of Drosophila circadian rhythms, produce single amino acid changes in conserved regions of the predicted kinase. A mutant causing pupal lethality (dbtP) eliminates rhythms of per and tim expression and constitutively overproduces hypophosphorylated Per proteins, abolishing most dbt expression. DBT mRNA appears to be expressed in the same cell types as are per and tim and shows no evident oscillation in wild-type heads. Dbt is capable of binding to Per in vitro and in Drosophila cells, suggesting that a physical association of Per and Dbt regulates Per phosphorylation and accumulation in vivo (Kloss, 1998).

Phosphorylation is an important feature of pacemaker organization in Drosophila. Genetic and biochemical evidence suggests involvement of the casein kinase I homolog doubletime (dbt) in the Drosophila circadian pacemaker. Two novel dbt mutants have been characterized. Both cause a lengthening of behavioral period and profoundly alter period (per) and timeless (tim) transcript and protein profiles. The Per profile shows a major difference from the wild-type program only during the morning hours, consistent with a prominent role for Dbt during the Per monomer degradation phase. The transcript profiles are delayed, but there is little effect on the protein accumulation profiles, resulting in the elimination of the characteristic lag between the mRNA and protein profiles. These results and others indicate that light and post-transcriptional regulation play major roles in defining the temporal properties of the protein curves and suggest that this lag is unnecessary for the feedback regulation of per and tim protein on per and tim transcription (Suri, 2000).

Both mutations, when presented in the context of the highly similar yeast casein kinase I HRR25, severely reduce kinase activity on peptide substrates. The long-period phenotypes are likely caused by insufficient Dbt activity, so it takes longer to reach some required level of Per phosphorylation. It is also assumed that both mutants are expressed at a level similar to that of wild-type Dbt (Suri, 2000).

Both dbth and Dbtg/+ have ~29 hr periods and are similar in all other respects, suggesting that the phenotypes are not idiosyncratic features of the mutations but reflect the role of Dbt in the pacemaker. Although the mutant flies entrain to imposed 24 hr photoperiods, the LD locomotor activity patterns indicate that there is no anticipation of the morning or evening light/dark transitions, and the evening activity peak is delayed by several hours into the night. The altered LD patterns are probably a consequence of the longer periods. Indeed, flies that carry pers as well as dbth have a period of ~22.5 hr and manifest robust anticipation of both morning and evening transitions as well as an advanced evening activity peak. Both dbt mutant LD profiles resemble that of the 29 hr period perl mutant strain, consistent with this altered period notion (Suri, 2000).

The molecular features of the perl circadian program are difficult to compare with those of wild-type flies, because the mutant rhythms are weak and of low amplitude as well as long period even under 12 hr LD entraining conditions. In contrast, Per and Tim cycling in the long-period dbt mutants is robust. Protein levels are comparable with those in wild-type flies during the night, and levels in the two mutant strains appear even higher than wild-type levels during the daytime. Previous work suggests a role for Dbt-catalyzed phosphorylation in targeting Per for degradation: this probably reflects slower protein turnover during the morning in the dbt mutants. The Tim phosphorylation pattern in the mutants did not show any noticeable difference from the wild-type pattern. These observations suggest that the modest mutant effects on the Tim profiles are indirect, perhaps through a primary effect of the dbt mutants on Per (Suri, 2000).

Per phosphorylation is still readily observable in both mutant lines. In fact, there is a hint that Per is even hyperphosporylated in these strains. Although this might reflect phosphorylation events that never take place in a wild-type background, less active Dbt mutants might be expected to depress the magnitude as well as the kinetics of the temporal phosphorylation program. This suggests that Per might not be a direct Dbt substrate in vivo but is only influenced indirectly, through intermediates that are direct Dbt targets. For example, Dbt may phosphorylate and activate a direct Per kinase or a specific protease. In this context, Per has not yet been shown to be a direct Dbt substrate. It is also possible that Dbt is a functionally relevant but minor Per kinase. In this case, the bulk of the Per mobility shift on SDS-PAGE is a consequence of other kinases. Because Per persists for several hours longer in the mutants than in wild-type flies, the other kinases would continue to function and give rise to even more highly phosphorylated species than are usually observed. These would be an indirect consequence of weak dbt activity and delayed degradation. A final possibility is that the enhanced and delayed Per phosphorylation simply reflects some misregulation of Dbt activity (Suri, 2000).

Careful analysis of the Per and Tim protein profiles in the long-period dbt mutants suggests that Dbt acts in the late night and morning phase of the molecular cycle: the mutants leave the early evening protein profile almost unaltered. This indicates that dbt probably targets nuclear, monomeric Per. It has also been suggested that Dbt acts in the early night to destabilize cytoplasmic Per, thus delaying nuclear entry and repression. The dbt mutants reported here do not significantly change this early night, presumptive cytoplasmic phase of accumulation. It is possible that Dbt prefers free Per over Per complexed to Tim. If free Per is a better substrate, then Dbt mutants should show a greater effect in the late night and early morning, after a large fraction of Tim has disappeared. Alternatively, Dbt might influence only marginally the Per accumulation phase for some other reason. But dbt mutant larvae accumulate high levels of hypophosphorylated Per, which suggests that Dbt is the major Per kinase and strongly influences Per accumulation as well as degradation. There is evidence, however, that much of this Per accumulation occurs in cells and tissues where Per is not normally detectable, making the connection with the normal Per-Tim cycle uncertain (Suri, 2000).

To assess the effect of the dbt mutants on transcription, per and tim mRNA cycling was assayed in wild-type and dbt mutant flies. Both mutant profiles are delayed by 4-5 hr. This is presumably because of the delayed disappearance of Per as well as Tim, which has been suggested to repress per and tim transcription. This relationship is very similar to that previously reported for the perS mutant strain; in this case, the clock proteins disappear more quickly, leading to an advance in the RNA profiles. The perS effect is more pronounced on Per than on Tim, consistent with the notion that monomeric Per might be the major transcriptional repressor. In any case, comparable results in the three mutants indicate a solid relationship between the timing of the decline in protein levels and the timing of the subsequent increase in per and tim transcription (Suri, 2000).

Based on these observations, a possible model for Dbt function in the Drosophila pacemaker is presented. In the cytoplasm, normal destabilization of Per delays substantial buildup of Per-Tim complexes and the consequent nuclear transport of the dimeric Per-Tim complex. In the nucleus, Per destabilization relieves repression. In Dbt mutants, Per degradation is much slower. This prolongs repression and delays the per and tim mRNA upswing in the next cycle (Suri, 2000).

There is an impressive relationship between the per and tim RNA profiles in comparison to the evening locomotor activity peak. In all cases, these RNA and locomotor activity begin to increase at approximately the same time, i.e., around ZT7 in the middle of the daytime. Mutants or physiological manipulations that affect the timing of the RNA profiles affect the timing of the evening activity peak in parallel. This fits with the emerging view, from mammalian as well as Drosophila work, that cycling transcription plays an important role in circadian output as well as within the central pacemaker oscillator. A further implication of these relationships is that the protein oscillations from one day affect behavior as well as the RNA profiles on the next one: the morning decline and eventual disappearance of Per and Tim terminate a protein cycle from the previous day, which then causes the subsequent increases in both RNA levels and locomotor activity (Suri, 2000).

In contrast, the delayed Per and Tim disappearance in the mutants has little if any effect on the subsequent protein accumulation phase (ZT13-ZT20) under these standard LD conditions; it is hardly affected, and both proteins peak at approximately the same time as they do in the wild-type flies (ZT19-ZT21). Because of the delayed RNA rise in the mutants, the per and tim RNA accumulation profiles almost coincide with those of the proteins, between ZT15 and ZT21. This indicates that the timing of the RNA rise is insufficient to time the protein rise. The increase in protein levels may reflect protein half-life regulation, which is uncoupled from the underlying mRNA levels, at least under some circumstances (Suri, 2000).

The coincidence of the protein and RNA curves also raises doubts about the importance of the 4-6 hr lag between these two accumulation profiles. The data presented in this study indicate that the lag is dispensable for robust behavioral and molecular oscillations. This is especially relevant for the RNA fluctuations. Despite evidence that at least per mRNA fluctuations may not be necessary for core oscillator function, they normally correlate with other molecular and behavioral circadian fluctuations. Moreover, there are substantial data indicating that Per and Tim feedback regulate these transcriptional oscillations. There is also considerable experimental evidence as well as theoretical models, to suggest that the normal 4-6 hr lag between the RNA and protein curves is essential for generating these robust, high-amplitude transcriptional oscillations. The general view is that the protein accumulation delay gives enough time for transcription to increase substantially, before protein levels have increased sufficiently to inhibit transcription. The presence of robust transcriptional oscillations without the delayed protein accumulation makes this scheme less likely. It redirects focus toward some post-transcriptional delay (e.g., the timing of nuclear entry of the Per-Tim dimer), which is predicted to be functional and important for transcriptional feedback regulation. It is important to note that these conclusions are based on biochemical experiments with whole-head extracts. It is still possible that the mRNA-protein lag may be important in the specific pacemaker neurons of Drosophila (Suri, 2000).

All of these experiments were performed under LD conditions. When the light comes on at ZT24, it causes a rapid decline in Tim levels. In DD conditions, therefore, Tim levels are much higher in the early subjective day, as expected. But a major, unanticipated difference was that the Per and Tim profiles in the dbt mutant flies are profoundly delayed in DD, as evidenced by the late appearance of faster-migrating species. This occurs without a comparable change in the RNA profiles, giving rise to a quasi-normal lag between RNA and protein. The light-mediated advance of the protein curves and the absence of a comparable light reset of the RNA profile reinforce the independent regulation of the accumulation phase of the clock RNAs and proteins: only the RNA profiles are influenced by the declining phase of the protein cycle of the previous day, whereas only the protein profiles appear to be reset by the light entrainment stimulus. The data are therefore consistent with a post-translational route of light entrainment, perhaps mediated by some aspect of the normal light effect on Tim. This presumably contributes to the daily advance of the dbt mutant clock under LD conditions, which counteracts the 5 hr period-lengthening effect that would take place under DD conditions (Suri, 2000).

Further understanding of the role of Dbt in the clock will require experiments that directly address Dbt function and regulation. For example, it is possible that temporal regulation of Dbt activity makes a major contribution to the temporal phosphorylation profile and more generally to the normal timing of the circadian program. Additionally, the extent to which Dbt modifies other pacemaker proteins is not clear. It is possible that these other putative Dbt substrates may also be intimately connected to the pacemaker mechanism. Addressing these issues would provide a much deeper understanding of the role of phosphorylation in the pacemaker (Suri, 2000).

Circadian (24 hour) Period (Per) protein oscillation is dependent on the double-time (dbt) gene, a casein kinase Iepsilon homolog. Without dbt activity, hypophosphorylated Per proteins over-accumulate, indicating that dbt is required for Per phosphorylation and turnover. There is evidence of a similar role for casein kinase Iepsilon in the mammalian circadian clock. A new dbt allele, dbtar, has been isolated that causes arrhythmic locomotor activity in homozygous viable adults, as well as molecular arrhythmicity, with constitutively high levels of Per proteins, and low levels of Timeless (Tim) proteins. Short-period mutations of per, but not of tim, restore rhythmicity to dbtar flies. This suppression is accompanied by a restoration of Per protein oscillations. These results suggest that short-period per mutations, and mutations of dbt, affect the same molecular step that controls nuclear Per turnover. It is concluded that, in wild-type flies, the previously defined Per 'short domain' may regulate the activity of DBT on Per (Rothenfluh, 2000).

In a screen for mutations affecting Drosophila locomotor activity rhythms, a new dbt allele, dbtar, was recovered that results in arrhythmic flies when homozygous. Neither the strongly hypomorphic dbtP allele, nor a deficiency, Df(3R)tll-g, which removes the dbt gene, are able to complement the new mutation. At the molecular level, dbtar stops the oscillation of Per and Tim proteins, and causes under-accumulation of Tim, and over-accumulation of Per. These phenotypes are similar to those of dbtP mutants, suggesting that dbtar is a reduced-function allele. However, the degree of phosphorylation of constitutively produced Per in dbtar ranges from hypo- to hyperphosphorylated, whereas over-accumulating Per is hypophosphorylated in dbtP. dbtar flies therefore retain a higher level of DBT-mediated kinase activity than dbtP. The fact that ~70% of dbtar homozygotes are viable, compared with the complete pupal lethality of dbtP and dbtdco alleles also supports higher kinase activity of DBTAR. The single missense mutation associated with dbtar results in a His126 to Tyr mutation. His126 is highly conserved in casein kinase Iepsilon family members, but Tyr126 is found in casein kinase Igamma. The specificity of DbtAR may, therefore, be altered, resulting in some, but not sufficient, Per phosphorylation for turnover (Rothenfluh, 2000).

dbtar shows some residual, weak, long-period rhythms, when heterozygous with the dbtP allele or Df(3R)tll-g. To see if the frequency of rhythmicity could be increased, dbtar was crossed into various short-period mutant backgrounds. The two short-period per alleles, perT (16 hours), and perS (19 hours), substantially reduce the frequency of arrhythmia: dbtar/dbtP flies are rescued to almost complete rhythmicity by perT, perS, and even perT/+. Rescue of dbtar/Df(3R)tll-g is less pronounced, but the ~90% arrhythmicity observed in this genotype is suppressed by perT to over 50% rhythmicity. The suppression of arrhythmia is also accompanied by an increased strength of the rhythms. This is the first report of a rescue of genetically determined arrhythmia by a second-site mutation (Rothenfluh, 2000).

Unlike the 20.5 hour heterozygous perT background, the 20.5 hour timS1 background is not able to rescue rhythms in dbtar/dbtP and dbtar/Df(3R)tll-g heterozygotes. There is, therefore, a specific genetic interaction between short-period per alleles and dbtar, and dbtar is not generally rescued toward rhythmicity by any period-shortening allele. Specificity of this rescue is also indicated by the finding that a 28-hour period per allele, perSLIH, does not suppress dbtar arrhythmia. Furthermore, rescue of arrhythmicity is also specific to dbtar, because perT heterozygotes do not suppress the arrhythmia from the tim01 mutation (Rothenfluh, 2000).

How can this allele-specific suppression of dbtar arrhythmia be explained? In perS flies, PerS disappears prematurely from nuclei , and Per from perS and perT flies falls to trough levels prematurely at the end of the night. Evidently per-short mutations increase the turnover of nuclear Per proteins. Because dbt affects the stability of cytoplasmic and nuclear Per, dbtar and per-short alleles may both affect nuclear Per stability, although in opposite directions. PerS and PerT might be turned over in the nucleus despite reduced Dbt activity in dbtar flies, thus completing the molecular cycle by terminating Per auto-repression, and resulting in molecular and behavioral rhythms. Conversely, in per+;dbtar flies, the program of nuclear Per protein degradation may be obstructed, resulting in continued repression of per and tim. Altered regulation of this sort should be reflected in the Tim under-accumulation observed in dbtar homozygotes (Rothenfluh, 2000).

To test whether the behavioral rescue is associated with restoration of molecular oscillations, Per and Tim protein time courses were examined on Western blots from perT;dbtar/dbtP flies. Per and Tim levels oscillate, and Per reaches trough levels at ZT 10 (in LD), and CT 18 (first day in DD). In this time span progressive phosphorylation of Per is also observed, even though the transition from hypo- to hyper-phosphorylation takes ~20 hours, compared to 8-12 hours in wild-type flies. This reduced rate of phosphorylation is probably a result of the dbtar mutation, and might explain the 31-hour behavioral period of perT;dbtar/dbtP flies (Rothenfluh, 2000).

Normally, lights-on in the morning results in Tim degradation, followed by progressive phosphorylation and degradation of hyperphosphorylated Per in 5-7hours. In the presence of light, hyperphosphorylated forms of Per are found that are not eliminated from dbtar/dbtP flies. In perT;dbtar/dbtP flies, however, Per levels are reduced by exposure to light and hyperphosphorylated Per proteins are lost, indicating that PerT proteins are more easily degraded than wild-type Per proteins when DBT activity is compromised. Since progressive phosphorylation of PerT is observed in perT;dbtar/dbtP flies, and PerT proteins turned over following exposure to light in an LD cycle are hyperphosphorylated, Per proteins should still require phosphorylation for degradation in this background. A hypophosphorylated form of PerT was found in the presence of light in these flies that might be freshly synthesized cytoplasmic Per. The dbtP mutation results in stable, hypophosphorylated cytoplasmic Per, and similarly, dbtar may also increase Per's cytoplasmic stability (Rothenfluh, 2000).

To test whether the increased degradation of PerT proteins occurs in nuclei of perT;dbtar/dbtP flies, head sections were stained for Per protein. There is a pronounced diminution of nuclear Per from ZT 2-10 in perT;dbtar/dbtP photoreceptors, whereas similar levels of Per are observed at these two time points in dbtar and in dbtar/dbtP photoreceptors. It is concluded that increased nuclear turnover of PerT allows completion of the molecular cycle in the presence of the Per-stabilizing mutation dbtar (Rothenfluh, 2000).

The specific molecular mechanism affected by the interaction of short period mutations of per and dbtar is unknown. However, earlier work has established that the perS mutation maps to a ~30 amino acid domain of Per in which most amino acid substitutions produce similar short-period phenotypes. A simple deletion of 17 amino acids {SERDSVMLGEISPHDDY} from this Per 'short domain' also reduces period-length as in perS, and the perT mutation has been mapped to this interval of Per. Together the results indicate that in wild-type flies, Per's short domain somehow inhibits the action of DBT. Because DBT and Per physically bind to each other in vitro, in cultured Drosophila cells, and in vivo, the short domain might influence a pattern of physical association between Per and DBT that retards Per phosphorylation in wild-type flies. Alternatively, because Per is repeatedly phosphorylated in vivo, and hyperphosphorylation appears to be required for Per degradation, the short domain may influence a temporal sequence of Per phosphorylation. Additional evidence that per-short mutations and dbt affect the same step in the cycle comes from the finding that per-short and dbt-short double mutant combinations show an unusual non-additive phenotype (Rothenfluh, 2000).

The Drosophila double-time (dbt) gene, which encodes a protein similar to vertebrate epsilon and delta isoforms of casein kinase I, is essential for circadian rhythmicity because it regulates the phosphorylation and stability of period (per) protein. In this study, the circadian phenotype of a short-period dbt mutant allele (dbtS) was examined. The present study shows that dbt affects posttranscriptional regulation of Per at the level of nuclear accumulation, in addition to the previously demonstrated effects on cytoplasmic stability. dbt therefore affects multiple aspects of the Per temporal program, and it is possible that further analysis will reveal additional aspects of clock biochemistry that are regulated by dbt. The circadian period of the dbtS locomotor activity rhythm varies little when tested at constant temperatures ranging from 20° to 29°C. However, perL;dbtS flies exhibit a lack of temperature compensation like that of the long-period mutant (perL) flies. Light-pulse phase-response curves were obtained for wild-type, the short-period (perS), and dbtS genotypes. For the perS and dbtS genotypes, phase changes are larger than those for wild-type flies, the transition period from delays to advances is shorter, and the light-insensitive period is shorter. Immunohistochemical analysis of per protein levels has demonstrated that per protein accumulates in photoreceptor nuclei later in dbtS than in wild-type and perS flies, and that it declines to lower levels in nuclei of dbtS flies than in nuclei of wild-type flies. Immunoblot analysis of per protein levels has demonstrated that total per protein accumulation in dbtS heads is neither delayed nor reduced, whereas RNase protection analysis has demonstrated that per mRNA accumulates later and declines sooner in dbtS heads than in wild-type heads. These results suggest that dbt can regulate the feedback of per protein on its mRNA by delaying the time at which it is translocated to nuclei and altering the level of nuclear Per during the declining phase of the cycle (Bao, 2001).

CKIε/discs overgrown promotes both Wnt-Fz/β-catenin and Fz/PCP signaling in Drosophila

The related Wnt-Frizzled(Fz)/β-catenin and Fz/planar cell polarity (PCP) pathways are essential for the regulation of numerous developmental processes and are deregulated in many human diseases. Both pathways require members of the Dishevelled (Dsh or Dvl) family of cytoplasmic factors for signal transduction downstream of the Fz receptors. Dsh family members have been studied extensively, but their activation and regulation remains largely unknown. In particular, very little is known about how Dsh differentially signals to the two pathways. Recent work in cell culture has suggested that phosphorylation of Dsh by Casein Kinase I ε may act as a molecular 'switch', promoting Wnt/β-catenin while inhibiting Fz/PCP signaling (Cong, 2004). This study demonstrates in vivo in Drosophila through a series of loss-of-function and coexpression assays that CKIε acts positively for signaling in both pathways, rather than as a switch. The data suggest that the kinase activity of CKIε is required for peak levels of Wnt/β-catenin signaling. In contrast, CKIε is a mandatory signaling factor in the Fz/PCP pathway, possibly through a kinase-independent mechanism. Furthermore, the primary kinase target residue of CKIε on Dsh has been identified. Thus, the data suggest that CKIε modulates Wnt/β-catenin and Fz/PCP signaling pathways via kinase-dependent and -independent mechanisms (Klein, 2006).

Cell-culture assays have suggested that CKIε positively regulates Wnt-Fz/β-catenin signaling and that it antagonizes Fz/PCP signaling. To confirm that CKIε is required for Wnt/β-catenin signaling in vivo, loss of function (LOF) alleles of discs overgrown (dco/doubletime, the Drosophila CKIε gene) were examined for phenotypes indicative of Wingless signaling defects. Consistent with previous data demonstrating a requirement for dco in disc growth, strong dco/CKIε alleles (dcodbt-P) gave clones too small to analyze for disruption of Wg target-gene expression. Thus dco/CKIε mutant clones were generated with the Minute technique. In these, expression of the Wg target gene senseless (sens) was lost in mutant cells. Accordingly, dco/CKIε adult wing clones show loss of margin bristles and/or parts of the wing margin, consistent with a positive requirement for dco in Wg signaling. However, Wg targets that require lower levels of Wg signaling (e.g., Dll) were not affected, indicating that dco/CKIε is only required for peak Wg signaling levels. Consistent with this finding, genome-wide RNAi screens for Wg signaling components identified dco/CKIε as a factor required for peak levels of β-catenin reporter expression (Klein, 2006).

The Fz/PCP pathway can easily be studied in Drosophila. The precise ommatidial arrangement in the eye and the orientation of hairs on the wing depend on correct Fz/PCP input. The two best-studied PCP signaling factors are Fz and Dsh, which also act in canonical Wnt/β-catenin signaling. To study the role of dco/CKIε in PCP, various heteroallelic dco combinations were analyzed. In several of these (e.g., dcodbt-P/dcodbt-AR), typical PCP defects were seen. Clones of a strong dco/CKIε allele show classical PCP phenotypes in the wing, with reoriented wing hairs, and in the eye, with ommatidal chirality and orientation defects (dco/CKIε clones also displayed ommatidia with photoreceptor loss, likely as a result of the cell viability requirement of Dco/CKIε (Klein, 2006).

In vivo LOF analyses allow led to the conclusion that dco/CKIε is required for peak levels of Wg signaling, but does not appear to be a mandatory Wnt/β-catenin signaling component. In addition, the data identify dco/CKIε as a new factor required in Fz/PCP signaling (Klein, 2006).

To dissect the function of dco/CKIε in Wg and PCP signaling, the effects of overexpressing CKIε were examined. In the eye, dco/CKIε was overexpressed with sevenless(sev)-Gal4 (in R3/R4 cells that are critical for PCP establishment, which causes PCP phenotypes. In the wing, decapentaplegic(dpp)-Gal4-driven expression in a proximal-distal stripe along the A-P compartment boundary can be used to identify positive and negative effects on both Wg and PCP signaling. dpp>CKIε displayed a mild but consistent PCP defect, a characteristic hair swirl near the intersection of the dpp stripe and wing margin. It also caused a small number of extra margin bristles, typical of increased Wg signaling (Klein, 2006).

Given that these phenotypes were mild, attempts were made to enhance them. Because CKIε requires an activating dephosphorylation event, which can be induced by Fz signaling, the effect was tested of coexpressing CKIε with either Fz or Fz2 in the dpp stripe. Overexpression of Fz (dpp>Fz) causes a characteristic reorientation of wing hairs that point away from the expression domain, but does not induce ectopic margin bristles. Strikingly, coexpression of Fz and CKIε leads to a dramatic synergy, with enhanced PCP defects and a large number of extra margin bristles in the expression domain, indicative of a positive CKIε role in both Wg and PCP signaling. Consistently, expression of Fz or CKIε alone is not sufficient to induce visible changes in Wg target-gene expression, but coexpression of CKIε and Fz cell-autonomously induces ectopic Senseless-positive cells (Klein, 2006).

dpp>Fz2 induces many ectopic margin bristles near the intersection of dpp expression and the wing margin (at 25°C;). Because dpp-driven coexpression of Fz2 and CKIε at 25°C is lethal, the dpp>Fz2, CKIε coexpression was examined in flies raised at 18°C (allowing for weaker Gal4-driven expression). dpp>Fz2 at 18°C induces only few margin bristles, a phenotype that is enhanced upon coexpression with CKIε; dpp>Fz2, CKIε coexpression also induces PCP-like hair swirls, although this effect could be indirect given that this combination induces wing-margin-like vein tissue, which could repolarize parts of the wing blade). Taken together with the LOF analyses, these data demonstrate a positive, synergistic role for Dco/CKIε not only in Wg signaling, but also in Fz/PCP signaling (Klein, 2006).

To confirm that dco/CKIε acts positively for both pathways in vivo, genetic interactions of dco with known Wg and PCP signaling factors were examined in the eye. Overexpression of Fz (sevFz) causes strong PCP defects, a phenotype that is significantly suppressed by the removal of a single copy of dco. Overexpression of Strabismus (Stbm; also known as Van Gogh or Vang), an antagonist of Fz/PCP signaling, with sevGal4 (sevStbm) causes mild PCP defects, which are enhanced by removal of a copy of dco, again supporting a positive role for dco in Fz/PCP signaling (Klein, 2006).

The effect of removing a copy of dco in the context of Dsh overexpression (sev>Dsh). sev>Dsh causes both PCP defects and loss of photoreceptors, with the latter resembling the effect of sev>Wg. Removing a copy of dco strongly suppresses the loss-of-photoreceptor phenotype, supporting a positive role for dco for peak Wg signaling. Assessing the effect of the removal of a copy of dco on the PCP in sev>Dsh is not possible, because the loss-of-photoreceptor phenotype masks PCP defects in many ommatidia (Klein, 2006).

In addition, genetic analyses was performed with overexpressed CKIε. sev>CKIε eyes exhibit mild PCP defects and a small percentage of ommatidia with a change in photoreceptor number. Removal of a single copy of dsh suppressed the PCP and photoreceptor number defects, consistent with Dco/CKIε acting positively together with Dsh to elicit both phenotypes. Consistent with the coexpression results in the wing, these interactions support a positive role for dco/CKIε in both Wg and Fz/PCP signaling (Klein, 2006).

On the basis of the genetic interaction data with Dsh and cell-culture and in vitro kinase assays that have shown that CKIε can bind and phosphorylate Dsh, Dsh appears to be a likely phosphorylation target of CKIε. The actual site of phosphorylation on Dsh, however, has not been mapped. To narrow down the region of phosphorylation, a series of GST-Dsh constructs were generated covering all domains. In vitro kinase assays using CKIε showed specific phosphorylation of all Dsh isoforms containing the basic region and PDZ domain (GST-Dsh, GST-bPDZ, GST-ΔC), but not of those containing just the PDZ (GST-PDZ) or other parts of Dsh (Klein, 2006).

To determine the exact site of phosphorylation, the region N terminal to the PDZ domain was analzyed for conserved CKIε consensus sites, and a likely motif was identified. In this motif, S236 is predicted to be the first serine residue phosphorylated by CKIε. When this serine is mutated to alanine (within GST-bPDZ), CKIε is no longer able to phosphorylate Dsh. Interestingly, this residue is in a short region of Dsh that is important for Dsh phosphorylation, activity, and signal specificity (Klein, 2006).

Because previous studies have suggested that CKIε kinase activity is required for its ability to transduce Wnt/β-catenin signals, a kinase-dead isoform of CKIε, Dco/CKIεD132N (which affects the ATP binding site) was tested in vivo. This mutant was unable to transduce Wg signals, but, surprisingly, it induced strong GOF PCP phenotypes in both the eye and wing. sev>CKIεD132N eyes display clean PCP phenotypes. Wings from dpp>CKIεD132N flies also display PCP defects, with wing hairs that point away from the expression domain, demonstrating a GOF Fz/PCP phenotype (Klein, 2006).

To investigate the potential for a kinase-independent role of Dco/CKIε, Fz and CKIεD132N (with dppGAL4) were coexpressed. In contrast to coexpression of Fz and wild-type CKIε, CKIεD132N did not display extra margin bristles, indicating that kinase activity is important for Wg signaling. Similarly, in contrast to coexpression of Fz2 and wild-type CKIε, dpp>Fz2, CKIεD132N (at 18°C) did not cause an increase in margin bristles as compared to dpp>Fz2 alone. At 25°C, dpp>Fz2, CKIεD132N was not lethal, as compared to dpp>Fz2 together wild-type CKIε (Klein, 2006).

In summary, these data demonstrate a requirement for the Dco/CKIε kinase activity in Wg signaling, with CKIεD132N often acting as a dominant negative, but suggest that kinase activity is not required for Dco/CKIε activity during Fz/PCP signaling (Klein, 2006).

The CKIε requirement for Dsh phosphorylation was examined. In an unbiased Drosophila S2-cell-based screen for kinases that are required for the PCP-signaling-associated Dsh phosphorylation, dco/CKIε was identified as a kinase required in this context. Strikingly, the kinase-dead CKIε isoform, CKIεD132N, promotes PCP-signaling-associated Dsh phosphorylation as much as wild-type CKIε. These data suggest that although the presence of CKIε protein is important for this phosphorylation event, its kinase activity is not required, consistent with the in vivo expression data with Dco/CKIεD132N (Klein, 2006).

A possible caveat to these data is that, rather than acting on downstream targets in a kinase-independent manner, CKIεD132N could act to titrate away factors that inhibit the endogenous CKIε. To test this, the effect of CKIεD132N on the phosphorylation of Dsh was examined in S2 cells in the absence of endogenous CKIε. These data show that endogenous CKIε is not mediating the CKIεD132N effect, and thus CKIε is likely to act in a kinase-independent manner in the PCP context (Klein, 2006).

The in vivo data support a positive requirement of dco/CKIε for peak levels of Wnt/β-catenin signaling and a strict requirement in the Fz/PCP pathway. Whereas the kinase activity of CKIε is required for Wnt/β-catenin signaling, the analysis suggests that it is not required for Fz/PCP signaling. These findings differ from the proposed inhibitory effect of CKIε on Fz/PCP signaling in cell culture. It is possible that the PCP readout in cell culture, namely activation of JNK, reflects only a subset of PCP activities of Dsh, not representing an accurate measure of overall PCP activity. Alternatively, CKIε could act as a constitutively active kinase when expressed in cell culture, whereas its activity is regulated in vivo. Thus, for Fz/PCP signaling in vivo, the primary role for CKIε may not be as an active kinase, but rather as a stabilizer of a complex that allows for PCP-specific Dsh phosphorylation. This is supported by the data that the kinase activity of CKIε is not required for Fz/PCP signaling and that kinase-dead CKIε still stimulates phosphorylation of Dsh (Klein, 2006).

CKIε phosphorylates a specific residue in Dsh, S236, in a short region known to be phosphorylated by multiple kinases and suggested to be important in the regulation of Dsh signal specificity. This supports the proposed possibility of in vivo competitive phosphorylation as a mechanism for Dsh regulation. The region upstream of the PDZ domain appears to act as a docking site for Dsh binding proteins. Differential phosphorylation of this region could alter the binding properties of Dsh. In support of this possibility, protein-protein interaction studies have identified a large number of proteins that bind to the Dsh PDZ domain. It is unlikely that all these interactions occur at the same time, and phosphorylation is a potential mechanism to regulate this. Further experiments are needed to finely map the many potential phosphorylation target residues and the corresponding kinases and demonstrate their in vivo significance (Klein, 2006).

Delineation of a Fat tumor suppressor pathway

Recent studies in Drosophila of the protocadherins Dachsous and Fat suggest that they act as ligand and receptor, respectively, for an intercellular signaling pathway that influences tissue polarity, growth and gene expression, but the basis for signaling downstream of Fat has remained unclear. This study characterizes functional relationships among Drosophila tumor suppressors and identifies the kinases Discs overgrown and Warts as components of a Fat signaling pathway. fat, discs overgrown and warts regulate a common set of downstream genes in multiple tissues. Genetic experiments position the action of discs overgrown (doubletime) upstream of the Fat pathway component dachs, whereas warts acts downstream of dachs. Warts protein coprecipitates with Dachs, and Warts protein levels are influenced by fat, dachs and discs overgrown in vivo, consistent with its placement as a downstream component of the pathway. The tumor suppressors Merlin, expanded (ex), hippo, salvador (sav) and mob as tumor suppressor (mats) also share multiple Fat pathway phenotypes but regulate Warts activity independently. These results functionally link what had been four disparate groups of Drosophila tumor suppressors, establish a basic framework for Fat signaling from receptor to transcription factor and implicate Warts as an integrator of multiple growth control signals (Cho, 2006).

Since Dachs is required for loss of Wts protein in fat mutants, and Dachs encodes a large Myosin protein, a model was considered in which Dachs acts as a scaffold to link Wts to proteins that promote Wts proteolysis, analogous to the roles of Costal2 in Hedgehog signaling, or APC in Wnt signaling. This model predicts that Dachs should be able to bind to Wts. To evaluate this possibility, tagged forms of Dachs and Wts were coexpressed in cultured cells and assayed for coimmunoprecipitation. These experiments identified a specific and reproducible interaction between Dachs and Wts (Cho, 2006).

Recent studies have identified the transcriptional coactivator Yorkie (Yki) as a downstream component of the Hippo pathway and a substrate of Wts kinase activity. Phosphorylation of Yki by Wts inactivates Yki, and overexpression of Yki phenocopies wts mutation. The determination that the Fat tumor suppressor pathway acts through modulation of Wts thus predicts that Yki should also be involved in Fat signaling. When the influence of Yki overexpression was examined on Fat target genes, expression of Wg in the proximal wing, Ser in the proximal leg and fj in the wing and eye were each upregulated by Yki overexpression, consistent with the inference that Fat tumor suppressor pathway signaling acts through Yki (Cho, 2006).

In order to identify additional components of the Fat tumor suppressor pathway, advantage was taken of the observation that loss of fat in clones of cells is associated with an induction of Wingless (Wg) expression in cells just proximal to the normal ring of Wg expression in the proximal wing, reflective of its role in distal-to-proximal wing signaling. It was reasoned that this influence on Wg expression could be used to screen other Drosophila tumor suppressors for their potential to contribute to Fat signaling. Analysis of mutant clones in the proximal wing identified dco, ex, mats, sav, hpo and wts as candidate components of the Fat tumor suppressor pathway. As for fat, mutation of each of these genes is associated with induction of Wg expression specifically in the proximal wing, whereas Wg expression is not affected in more distal or more proximal wing cells. Although Wg expression often seems slightly elevated within its normal domain, the effect of these mutations is most obvious in the broadening of the Wg expression ring. The induction of Wg expression does not seem to be a nonspecific consequence of the altered growth or cell affinity associated with these mutations, since Wg expression is unaffected by expression of the growth-promoting microRNA gene bantam or by expression of genes that alter cell affinity in the proximal wing (Cho, 2006).

dco encodes D. melanogaster casein kinase I delta/epsilon. The overgrowth phenotype that gave the gene its name is observed in allelic combinations that include a hypomorphic allele, dco3, and it is this allele that is associated with induction of Wg. Null mutations of dco actually result in an 'opposite' phenotype: discs fail to grow, and clones of cells mutant for null alleles fail to proliferate. This is likely to reflect requirements for dco in multiple, distinct processes, as casein kinase I proteins phosphorylate many different substrates, and dco has been implicated in circadian rhythms, Wnt signaling and Hedgehog signaling (Cho, 2006).

Mer and ex encode two structurally related FERM domain-containing proteins. ex was first identified as a Drosophila tumor suppressor, whereas Drosophila Mer was first identified based on its structural similarity to human Merlin. Mutation of Mer alone causes only mild effects on imaginal disc growth, but Mer and ex are partially redundant, and double mutants show more severe overgrowth phenotypes than either single mutant. Consistent with this, elevation of Wg expression was observed in ex mutant clones (7/10 proximal wing clones induced Wg) and not in Mer mutant clones (0/8 clones), whereas Mer ex double mutant clones showed even more severe effects on Wg than ex single mutant clones. Because of the partial redundancy between Mer and ex, when possible, focus was placed for subsequent analysis on Mer ex double mutant clones (Cho, 2006).

Wts, Mats, Sav and Hpo interact biochemically, show similar overgrowth phenotypes and regulate common target genes. Mats, Sav and Hpo are all thought to act by regulating the phosphorylation state and thereby the activity of Wts. Mutation of any one of these genes is associated with upregulation of Wg in the proximal wing. The effects of sav (47/84 clones in the proximal wing induced Wg) and hpo (23/31 clones) were weaker than those of mats (19/19 clones) and wts (92/97 clones), but this might result from differences in perdurance or allele strength. Because sav, hpo and mats all act through Wts, focus for most of the subsequent analysis was placed on wts (Cho, 2006).

The observation that mutation of dco, Mer, ex, mats, sav, hpo or wts all share the distinctive upregulation of Wg expression in the proximal wing observed in fat mutants suggests that the functions of these genes are closely linked. To further investigate this, the effects of these tumor suppressors were characterized on other transcriptional targets of Fat signaling. Expression of the Notch ligand Ser is upregulated unevenly within fat mutant cells in the proximal region of the leg disc. A very similar upregulation occurred in dco3, Mer ex, and wts mutant clones. fj is a target of Fat signaling in both wing and eye imaginal discs, and fj expression was also upregulated in dco3, Mer ex, or wts mutant clones. The observation that these genes share multiple transcriptional targets in different Drosophila tissues implies that they act together in a common process (Cho, 2006).

The hypothesis that Fat pathway genes and Hippo pathway genes are linked predicts that not only should Fat target genes be regulated by Hippo pathway genes, but Hippo pathway target genes should also be regulated by Fat pathway genes. The cell cycle regulator CycE and the inhibitor of apoptosis Diap1 (encoded by thread) have been widely used as diagnostic downstream targets to assign genes to the Hippo pathway. Notably, then, clones of cells mutant for fat showed upregulation of both Diap1 and CycE protein expression. Genes whose expression is upregulated within fat mutant cells (such as wg, Ser and fj) have been shown previously to be induced along the borders of cells expressing either fj or dachsous (ds), and Diap1 is also upregulated around the borders of ds- or fj-expressing clones. That thread is affected by fat at a transcriptional level was confirmed by examining a thread-lacZ enhancer trap line. The regulation of Diap1 by the Hippo pathway is thought to be responsible for a characteristic eye phenotype in which an excess of interommatidial cells results from their failure to undergo apoptosis; an increase was also observed in interommatidial cells in fat mutant clones. Upregulation of both Diap1 and CycE is also observed in Mer ex double mutant clones. In dco3 mutant clones, consistent upregulation was detected only for Diap1, and CycE was upregulated only weakly and inconsistently. dco3 also has weaker effects on Wg and fj expression; the weaker effects of dco3 could result from its hypomorphic nature. ex has recently been characterized as another Hippo pathway target, and an ex-lacZ enhancer trap that is upregulated in wts or Mer ex mutant clones is also upregulated in fat or dco3 mutant clones. Analysis of ex transcription by in situ hybridization also indicated that ex is regulated by fat. Altogether, this analysis of Hippo pathway targets further supports the conclusion that the functions of the Fat pathway, the Hippo pathway and the tumor suppressors Mer, ex and dco are linked (Cho, 2006).

Genetic epistasis experiments provide a critical framework for evaluating the functional relationships among genes that act in a common pathway. The relationships was evaluated between each of the tumor suppressors linked to the Fat pathway and dachs, using both wing disc growth and proximal Wg expression as phenotypic assays. dachs is the only previously identified downstream component of the Fat tumor suppressor pathway. It acts oppositely to fat and is epistatic to fat in terms of both growth and gene expression phenotypes (Cho, 2006).

dachs is also epistatic to dco3 for overall wing disc growth and for proximal Wg expression. The epistasis of dachs to dco3 implies that the overgrowth phenotype of dco3 is specifically related to its influence on Fat signaling, as opposed to participation of dco in other pathways. By contrast to the epistasis of dachs to dco3, both wts and ex are epistatic to dachs for disc overgrowth phenotypes, and wts and Mer ex are epistatic to dachs in their influence on proximal Wg expression. Together, these epistasis experiments suggest that dco acts upstream of dachs, whereas Mer ex and wts act downstream of dachs (Cho, 2006).

Because wts and Mer ex have similar phenotypes, their epistatic relationship cannot be determined using loss-of-function alleles. However, overexpression of ex inhibits growth and promotes apoptosis, which suggests that ex overexpression affects ex gain-of-function. Clones of cells overexpressing ex are normally composed of only a few cells, and over time most are lost, but coexpression with the baculovirus apoptosis inhibitor p35 enabled recovery of ex-expressing clones. These ex- and p35-expressing clones were associated with repression of proximal Wg expression during early- to mid-third instar, as has been described for dachs2, consistent with ex overexpression acting as a gain-of-function allele in terms of its influence on Fat signaling. In epistasis experiments using overexpressed ex and mutation of wts, wts was epistatic; Wg was induced in the proximal wing. Additionally, when wts is mutant, coexpression with p35 was no longer needed to ensure the viability and growth of ex-expressing clones, indicating that wts is also epistatic to ex for growth and survival. Consistent with this conclusion, others have recently described phenotypic similarities between Mer ex and hpo pathway mutants and have reported that hpo is epistatic to Mer ex (Cho, 2006).

When Fat was overexpressed, a slight reduction was detected in Wg expression during early- to mid-third instar, suggesting that overexpression can result in a weak gain-of-function phenotype. Clones of cells overexpressing Fat but mutant for dco3 still showed reduced Wg levels, whereas clones of cells overexpressing Fat but mutant for warts showed increased Wg levels. Although experiments in which the epistatic mutation is not a null allele cannot be regarded as definitive, these results are consistent with the conclusion that wts acts downstream of fat and suggest that dco might act upstream of fat (Cho, 2006).

The epistasis results described above suggest an order of action for Fat tumor suppressor pathway genes in which dco acts upstream of fat, fat acts upstream of dachs, dachs acts upstream of Mer and ex, and Mer and ex act upstream of wts. However, the determination that one gene is epistatic to another does not prove that the epistatic gene is biochemically downstream, as it is also possible that they act in parallel but converge upon a common target. Thus, to better define the functional and hierarchical relationships among these genes, experiments were initiated to investigate the possibility that genetically upstream components influence the phosphorylation, stability or localization of genetically downstream (that is, epistatic) components. Focus in this study was placed on the most downstream of these components, Wts. As available antibodies did not specifically recognize Wts in imaginal discs, advantage was taken of the existence of functional, Myc-tagged Wts-expressing transgenes (Myc:Wts) to investigate potential influences of upstream Fat pathway genes on Wts protein. In wing imaginal discs, Myc:Wts staining outlines cells, suggesting that it is preferentially localized near the plasma membrane, and it was confirmed that expression of Myc:Wts under tub-Gal4 control can rescue wts mutation. Notably, mutation of fat results in a reduction of Myc:Wts staining. As Myc:Wts is expressed under the control of a heterologous promoter in these experiments, this must reflect a post-transcriptional influence on Wts protein. fat does not exert a general influence on the levels of Hippo pathway components; fat mutant clones had no detectable influence on the expression of hemagglutinin epitope-tagged Sav (HA:Sav) (Cho, 2006).

The decrease in Wts protein associated with mutation of fat contrasts with studies of the regulation of Wts activity by the Hippo pathway, which have identified changes in Wts activity due to changes in its phosphorylation state. To directly compare regulation of Wts by Fat with regulation of Wts by other upstream genes, Myc:Wts staining was examined in ex, sav and mats mutant clones. In each of these experiments, the levels and localization of Myc:Wts in mutant cells was indistinguishable from that in neighboring wild-type cells (Cho, 2006).

Since Myc:Wts appears preferentially localized near the plasma membrane, it was conceivable that the apparent decrease in staining reflected delocalization of Wts, rather than destabilization. To investigate this possibility, Wts levels were examined by protein blotting. Antisera against endogenous Wts recognized a band of the expected mobility in lysates of wing imaginal discs or cultured cells, and this band was enhanced when Wts was overexpressed. The intensity of this band was reproducibly diminished in fat or dco3 homozygous mutant animals but was not diminished in fat or dco3 heterozygotes or in ex mutants. Conversely, levels of Hpo, Sav, Mer or Mats were not noticeably affected by fat mutation (Cho, 2006).

The determination that Wts is affected by Fat, together with the genetic studies described above, place Wts within the Fat signaling pathway, as opposed to a parallel pathway that converges on common transcriptional targets. Indeed, given that even hypomorphic alleles of wts result in disc overgrowth, the evident reduction in Wts levels might suffice to explain the overgrowth of fat mutants. As a further test of this possibility, Wts levels were examined in fat dachs double mutants. As the influence of Fat on gene expression and growth is absolutely dependent upon Dachs, if Fat influences growth through modulation of Wts, its influence on Wts levels should be reversed by mutation of dachs. Examination of Myc:Wts staining in fat dachs clones and of Wts protein levels in fat dachs mutant discs confirmed this prediction (Cho, 2006).

Prior observations, including the influences of fat and ds on gene expression, and the ability of the Fat intracellular domain to rescue fat phenotypes, suggested that Fat functions as a signal-transducing receptor. By identifying kinases that act both upstream (Dco) and downstream (Wts) of the Fat effector Dachs and by linking Fat to the transcriptional coactivator Yki, these results have provided additional support for the conclusion that Fat functions as a component of a signaling pathway and have delineated core elements of this pathway from receptor to transcription factor. Fat activity is regulated, in ways yet to be defined, by Ds and Fj. The influences of Fat on gene expression, growth, and cell affinity, as well as on Wts stability, are completely dependent on Dachs, indicating that Dachs is a critical effector of Fat signaling. Since Dachs can associate with Wts or a Wts-containing complex, it is suggested that Dachs might act as a scaffold to assemble a Wts degradation complex. The observations that Fat, Ds and Fj modulate the subcellular localization of Dachs, that Wts is preferentially localized near the membrane and that Dachs accumulates at the membrane in the absence of Fat, suggest a simple model whereby Fat signaling regulates Wts stability by modulating the accumulation of Dachs at the membrane and thereby its access to Wts. The working model is that dco3 is defective in the phosphorylation of a substrate in the Fat pathway, but the recessive nature of dco3, the genetic epistasis experiments, and biochemical experiments argue that this substrate is not Wts, and further work is required to define the biochemical role of Dco in Fat signaling (Cho, 2006).

In addition to identifying core components of the Fat pathway, the results establish close functional links between the Fat pathway, the Hippo pathway and the FERM-domain tumor suppressors Mer and Ex. The common phenotypes observed among these tumor suppressors can be explained by their common ability to influence Wts. However, they seem to do this in distinct ways, acting in parallel pathways that converge on Wts rather than a single signal transduction pathway. The Fat pathway modulates levels of Wts, apparently by influencing Wts stability. By contrast, the Hippo pathway seems to regulate the activity of Wts by modulating its phosphorylation state. Thus, Wts seems to act as an integrator of distinct growth signals, which can be transmitted by both the Fat pathway and the Hippo pathway. It has been suggested that Mer and Ex also act through the Hippo pathway, although present experiments cannot exclude the possibility that Mer and Ex act in parallel to Hpo. Moreover, it should be noted that Mats might regulate Wts independently of Hpo and Sav and hence function within a distinct, parallel pathway. Although it is simplest to think of parallel pathways, there is also evidence for cross-talk. fj and ex are both components and targets of these pathways. Thus, they can be regarded as feedback targets within their respective pathways, but their regulation also constitutes a point of cross-talk between pathways. Another possible point of cross-talk is suggested by the observation that levels of Fat are elevated within Mer ex mutant clones. Although the potential for cross-talk complicates assessments of the relationships between tumor suppressors, the observations that fat, dco3 and dachs affect Warts protein levels in vivo, whereas ex, hippo, sav and mats do not, argues that there are at least two distinct pathways that converge on Warts. This conclusion is also consistent with the observations that ex, hippo, sav and mats can influence Wts phosphorylation in cultured cell assays, but Fat, Dachs and Dco do not (Cho, 2006).

Although the Fat and Hippo pathways converge on Wts, Hippo pathway mutants seem more severe. Thus, hpo, wts or mats mutant clones show a distinctive disorganization and outgrowth of epithelial tissues that is not observed in fat mutant clones, and they show a greater increase in interommatidial cells. This difference presumably accounts for the previous failure to recognize the tight functional link between Fat and Hippo signaling, and it can be explained by the finding that Wts levels are reduced but not completely absent in fat mutant cells. Thus, fat would be expected to resemble a hypomorphic allele of wts rather than a null allele, and consistent with this, a hypomorphic allele, wtsP2, results in strong overgrowth phenotypes. The effects of Yki overexpression on growth and target gene expression can be even stronger than those of fat or wts mutations, which suggests that Yki levels become limiting when upstream tumor suppressors are mutant (Cho, 2006).

fat encodes a protocadherin, which in the past has led to speculation that its influences on growth and cell affinity might result from Fat acting as a cell adhesion molecule. However, all of the effects of fat on growth and affinity require dachs, which is also required for the effects of fat on transcription. Additionally, targets of Fat signaling include genes that can influence growth and affinity; recent studies identified an influence of fat on E-cadherin expression, and as describe in this study, Fat influences CycE and Diap1 expression. Thus, one can account for the influence of fat on growth and affinity by its ability to regulate gene expression. fat interacts genetically with other signaling pathways, including EGFR and Wnt, and in some cells Fat signaling also influences the expression of ligands (such as Wg and Ser) for other signaling pathways. Regulation of these ligands contributes to fat overgrowth phenotypes, but since clonal analysis indicates that fat is autonomously required for growth control in most imaginal cells, the principal mechanism by which fat influences growth presumably involves the regulation of general targets (Cho, 2006).

Normal tissue growth and patterning depend on a relatively small number of highly conserved intercellular signaling pathways. The Fat pathway is essential for the normal regulation of growth and PCP in most or all of the external tissues of the fly and also participates in local cell fate decisions. In this regard, its importance to fly development can be considered comparable to that of other major signaling pathways. Although the biological roles and even the existence of a Fat pathway in mammals remain to be demonstrated, there is clear evidence that the mammalian Warts homologs Lats1 and Lats2 act as tumor suppressors and that a mammalian Yorkie homolog, YAP, can act as an oncogene. Moreover, other genes in the Drosophila Fat pathway have apparent structural homologs in mammals. Thus, it is likely that mammals also have a Fat tumor suppressor pathway that functions in growth control (Cho, 2006).

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. In addition, it was found 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 and a set of these CKI sites are primed by GSK3β phosphorylation of the PPPSP motif. 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 gishe01759 attenuates but not completely blocks Wg responsive gene expression. The residual Wg signaling activity in gishe01759 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).

Planar polarity is positively regulated by casein kinase Iepsilon in Drosophila

Members of the casein kinase I (CKI) family have been implicated in regulating canonical Wnt/Wingless (Wg) signaling by phosphorylating multiple pathway components. Overexpression of CKI in vertebrate embryos activates Wg signaling, and one target is thought to be the cytoplasmic effector Dishevelled (Dsh), which is an in vitro target of CKI phosphorylation. Phosphorylation of Dsh by CKI has also been suggested to switch its activity from noncanonical to canonical Wingless signaling. However, in vivo loss-of-function experiments have failed to identify a clear role for CKI in positive regulation of Wg signaling. By examining hypomorphic mutations of the Drosophila CKIepsilon homolog discs overgrown (dco)/double-time, this study shows that it is an essential component of the noncanonical/planar cell polarity pathway. Genetic interactions indicate that dco acts positively in planar polarity signaling, demonstrating that it does not act as a switch between canonical and noncanonical pathways. Mutations in dco result in a reduced level of Dishevelled phosphorylation in vivo. Furthermore, in these mutants, Dishevelled fails to adopt its characteristic asymmetric subcellular localisation at the distal end of pupal wing cells, and the site of hair outgrowth is disrupted. Finally, dco function in polarity was found to be partially redundant with CKIalpha (Strutt, 2006).

Identification of domains responsible for ubiquitin-dependent degradation of dMyc by glycogen synthase kinase 3beta and casein kinase 1 kinases

Ubiquitin-mediated degradation of dMyc, the Drosophila homologue of the human c-myc proto-oncogene, is regulated in vitro and in vivo by members of the casein kinase 1 (CK1) family and by glycogen synthase kinase 3beta (GSK3beta). Using Drosophila S2 cells, it was demonstrated that CK1alpha promotes dMyc ubiquitination and degradation with a mechanism similar to the one mediated by GSK3beta in vertebrates. Mutation of ck1alpha or ck1epsilon (discs overgrown) or sgg/gsk3beta in Drosophila wing imaginal discs results in the accumulation of dMyc protein, suggesting a physiological role for these kinases in vivo. Analysis of the dMyc amino acid sequence reveals the presence of conserved domains containing potential phosphorylation sites for mitogen kinases, GSK3beta, and members of the CK1 family. Mutations of specific residues within these phosphorylation domains regulate dMyc protein stability and confer resistance to degradation by CK1alpha and GSK3beta kinases. Expression of the dMyc mutants in the compound eye of the adult fly results in a visible defect that is attributed to the effect of dMyc on growth, cell death, and inhibition of ommatidial differentiation (Galletti, 2009).

In vivo downregulation of GSK3β and CK1α or CK1ɛ kinases in wing imaginal discs results in the accumulation of dMyc protein, an effect particularly visible in the hinge and notum regions but not in cells adjacent to the zone of nonproliferative cells (ZNC). Reduction of GSK3β and CK1α activates Wingless (Wg) signaling, which in turns negatively regulates dmyc RNA in the ZNC. This functional relationship might explain the lack of expression of dMyc protein in clones falling in the wing pouch area and in the ZNC. This positional effect also suggests that dMyc activity is regulated by patterning signals active during the development of the wing imaginal discs (Galletti, 2009).

This analysis of the dMyc amino acid sequence uncovered novel conserved domains, which serve as potential phosphorylation substrates for CK1s or GSK3β kinases. Biochemical characterization of these domains indicated that a combination of amino acid substitutions (S201A, S205A, and S207A) in the dMyc-PI sequence produces a protein with a shorter half-life than dMyc-WT. In vivo expression of the dMyc-MPI mutant did not confer the typical ommatidial roughness that is induced by the expression of dMyc-WT. Moreover, expression of dMyc-PI failed to induce apoptosis in the eye imaginal discs, an effect normally associated with dMyc-WT overexpression. In conclusion, the data suggest that dMyc-PI produces a protein that is less stable than dMyc-WT. In vertebrates, phosphorylation of c-Myc on Ser-62 by MAPK/ERK, JNK N-terminal kinase, or CDK4 increases its stability. The dMyc-PI sequence does not contain a bona fide ERK phosphorylation site (PXSP). However, Ser-201 lies in a favorable context for phosphorylation by the ribosomal S6 kinase-p90 RSK. RSK-p90 belongs to a class of Ser/Thr kinases, activated by ERK and insulin signaling, that phosphorylates the S6 protein component of the 40S ribosomal subunit in response to mitogenic stimulation, resulting in enhanced translation. Interestingly, it has been reported that RSK-p90 activation by ERK is capable of switching on mTOR signaling via inactivation of the TSC1/2 complex, suggesting a role for this kinase in protein synthesis and mass accumulation. No evidence for this regulatory mechanism has been described thus far in Drosophila. It is hypothesized that growth factors may stabilize Myc protein, possibly through phosphorylation by the RSK-p90 kinase, and promote ribosomal biogenesis, in accordance with the prominent role played by dMyc in the production of mass and growth regulation (Galletti, 2009).

Biochemical analysis of the protein stability of dMyc-PII, dMyc-PV, and dMyc-AB showed an increased half-life of these mutants compared to dMyc-WT. The sequence within the dMyc-PII domain (S324A-T328A-S330A) contains potential targets for phosphorylation by GSK3β at Ser-324 [324-S/T-XXX-S/T-(PO4)+4], which requires a priming event of phosphorylation at the +4 position (Thr-330). This phosphorylation event also acts as priming for other kinases (i.e., CK1s) and creates an optimum consensus site for phosphorylation by CK1s at Thr-330 [S/T-(PO4)-XX-330-S/T]. This study found that alanine substitutions of amino acids 324, 328, and 330 conferred resistance to dMyc protein degradation upon phosphorylation by the CK1α and GSK3β kinases. These experiments show that mutation of the residues S324, T328, and S330 confers to the dMyc-PII mutant a resistance to degradation mediated by the ubiquitin ligase Ago. Moreover, it was found that dMyc-MPV, which is degraded by CK1α and GSK3β kinases, is somewhat resistant to degradation by Ago, suggesting that CK1α- and GSK3β-mediated phosphorylation of dMyc is not sufficient to induce its degradation by Ago but perhaps by another unknown ubiquitin ligase (Galletti, 2009).

These data also demonstrate that the dMyc-AB plays an important role in the regulation of dMyc protein stability. Mutation of acidic amino acids imparted to dMyc resistance to degradation primed by CK1α and GSK3β kinases. It has been proposed that acidic domains act as docking sites for the CK1 and CK2, enabling proper positioning of the kinases to recognize their substrates. It is speculated that the conserved acidic amino acid stretch in Myc protein helps the binding of CK1 and CK2 kinases and favors Myc phosphorylation. In support of this hypothesis, the dMyc-PV amino acid sequence (residues 405, 407, and 409), located within the AB (amino acids 404 to 414), was found to be highly homologous to the PEST domain of c-Myc (amino acids 226 to 270). This domain was previously demonstrated to be relevant for c-Myc stability and to act as a potential substrate for CK2 phosphorylation. These biochemical data show that mutations of the dMyc-PV and the AB domains confer increased stability to dMyc protein and suggest that the acidic sequence functions similarly to the PEST domain to control dMyc stability. Notably, Ser-407 constitutes an optimum consensus site for phosphorylation by CK2 (S/T-407-XX-D/E). This observation agrees with the hypothesis that in mammals CK2 is involved in the regulation of c-Myc degradation by targeting the PEST domain (Galletti, 2009).

In vivo expression of the stable mutants dMyc-PII, dMyc-PV, and dMyc-AB resulted in a visible eye defect, accompanied by a reduction of the head capsule and a diminution of the number of the ommatidia. This was particularly visible for dMyc-PV and -AB. Cellular analysis of third-instar larvae eye imaginal discs revealed that expression of these mutants induced apoptosis during disc development. Apoptosis was detected not only within the compartment of dMyc expression (cell autonomous) but also in the neighboring cells (non-cell autonomous). This is a well-documented phenomenon and illustrates the role of dMyc in cell competition, where cells expressing high dMyc kill slower-proliferating neighboring cells nonautonomously through an unidentified mechanism (Galletti, 2009).

In conclusion, multiple phosphorylation events may work hierarchically to prime Myc phosphoamino acids for binding by multiple kinases. It is proposed that different kinases respond to a 'phosphorylation code' that is required to properly control Myc protein stability. This code will depend on an upstream program that in turn activates these kinases. The identification of other phosphorylation residues in dMyc will help in drawing a complete map of phosphorylation activities and will elucidate the events necessary for robust regulation of Myc protein stability. For example, it is speculated that components of growth signaling pathways, such as ras or insulin, may influence the activities of different combinations of kinases, thus affecting phosphorylation at different amino acids to control dMyc protein stability. In support of this hypothesis, preliminary data was produced showing that activation of the DILP (for Drosophila insulinlike peptides) pathway increases dMyc protein stability in vivo through the inactivation of GSK3β kinase, suggesting that the metabolic and nutrient pathways affect growth by partially controlling dMyc protein expression (Galletti, 2009).

Differential requirement of Salvador-Warts-Hippo pathway members for organ size control in Drosophila melanogaster

The Salvador-Warts-Hippo (SWH) pathway contains multiple growth-inhibitory proteins that control organ size during development by limiting activity of the Yorkie oncoprotein. Increasing evidence indicates that these growth inhibitors act in a complex network upstream of Yorkie. This complexity is emphasised by the distinct phenotypes of tissue lacking different SWH pathway genes. For example, eye tissue lacking the core SWH pathway components salvador, warts or hippo is highly overgrown and resistant to developmental apoptosis, whereas tissue lacking fat or expanded is not. This study explores the relative contribution of SWH pathway proteins to organ size control by determining their temporal activity profile throughout Drosophila eye development. Eye tissue lacking fat, expanded or discs overgrown displays elevated Yorkie activity during the larval growth phase of development, but not in the pupal eye when apoptosis ensues. Fat and Expanded do possess Yorkie-repressive activity in the pupal eye, but loss of fat or expanded at this stage of development can be compensated for by Merlin. Fat appears to repress Yorkie independently of Dachs in the pupal eye, which would contrast with the mode of action of Fat during larval development. Fat is more likely to restrict Yorkie activity in the pupal eye together with Expanded, given that pupal eye tissue lacking both these genes resembles that of tissue lacking either gene. This study highlights the complexity employed by different SWH pathway proteins to control organ size at different stages of development (Milton, 2010).

The SWH pathway controls Drosophila eye size by limiting growth during the larval stage of development and by restricting proliferation and promoting apoptosis during pupal development. Eyes lacking core SWH pathway components (e.g. sav, wts or hpo) are significantly larger than eyes lacking the non-core components ft, ex, dco or Mer. Owing to this disparity, it has been hypothesized that ft and ex only partially affect SWH pathway activity, whereas sav, wts and hpo have stronger effects, or, alternatively, that non-core components affect pathway activity in a temporally restricted fashion. Analysis of tissue recessive for ft, ex or dco3 revealed that Yki activity was elevated during larval eye development when tissues are actively growing and proliferating, but not during pupal development when apoptosis ensues, supporting the idea that Ft, Ex and Dco influence SWH pathway activity in a temporally restricted fashion. However, when tissue lacking both Mer and ft, or Mer and ex, was analysed, Yki activity was found to be elevated during both larval and pupal development, similar to the Yki activity profile observed in tissue lacking core SWH pathway proteins. This is consistent with previous reports showing that Mer acts in parallel to both Ft and Ex, and that these proteins can compensate for each other to control SWH pathway activity. Therefore, Ft and Ex do contribute to SWH pathway regulation in the pupal eye to ensure appropriate exit from the cell cycle and developmental apoptosis, but these functions can be executed by Mer in their absence, suggesting a degree of plasticity in the regulation of Yki activity by non-core SWH pathway proteins. The ability of Mer to compensate for Ft or Ex cannot simply be explained by compensatory increases in Mer protein in pupal eye tissues lacking ft or ex, since Mer expression levels were found to be unaltered in these tissues (Milton, 2010).

Previous analyses of tissue lacking both ft and ex showed that these proteins function, at least in part, in parallel to control growth of larval imaginal discs. The current analysis of ft,ex double-mutant tissue suggests that these proteins are likely to function together to control Yki activity in the pupal eye. Yki activity was not elevated in tissue lacking ft, ex or both genes, showing that these genes cannot compensate for each other in the pupal eye. This is consistent with the notion that Ft influences the activity of downstream SWH pathway proteins by multiple mechanisms, an idea that is supported by THE analysis of the requirement of the atypical myosin, Dachs, for Ft signalling in the pupal eye. During larval imaginal disc development, Ft can influence Yki activity by repressing Dachs activity, which in turn can repress the core SWH pathway protein Wts. Analysis of pupal eye tissue that lacks both Mer and ft, or Mer, ft and dachs, showed that Yki activity was elevated in each scenario. This shows that in the pupal eye, the ability of Ft to compensate for Mer is not reliant on Dachs, and implies that Ft can employ different modes of signal transduction throughout eye development. However, because Ft and Mer can compensate for each other it is not possible to formally conclude that normal signal transduction by Ft in the pupal eye occurs independently of Dachs (Milton, 2010).

Expression of Ex is tightly controlled in response to alterations in SWH pathway activity at both the transcriptional and post-transcriptional levels. Interestingly, it was also found that Ex expression is controlled in a temporal fashion throughout eye development; Ex is expressed at relatively high levels in the larval eye, but at very low levels in the pupal eye. Despite the fact that Ex expression is very low in the pupal eye, it clearly retains function at this stage of development because it can compensate for loss of Mer to restrict Yki activity. The dynamic expression profile of Ex suggests that factors that influence its expression play an important role in defining overall eye size in Drosophila. At present, only two transcriptional regulatory proteins have been shown to influence the expression of ex: Yki and Sd. There are conflicting reports on whether Yki and Sd control basal expression of ex in larval imaginal discs. It is clear, however, that Yki and Sd collaborate to drive ex expression when the activity of the SWH pathway is suppressed, presumably as part of a negative-feedback loop. Despite the fact that basal ex expression is low in the pupal eye, the ex promoter is still responsive to Yki, as Ex expression is substantially elevated in pupal eye clones lacking hpo or Mer and ex. Future investigation of the ex promoter will help to clarify understanding of the complex fashion by which expression of the ex gene is controlled, and should aid understanding of eye size specification in Drosophila (Milton, 2010).

This study emphasises the complexity of the means by which the activity of core SWH pathway proteins is regulated by non-core proteins such as Ft, Ex, Mer and Dco. The signalling mechanisms employed by non-core proteins appear to differ at discrete stages of development in order to achieve appropriate organ size during the larval growth period of eye development, and to subsequently sculpt the eye by regulating apoptosis during pupal development (Milton, 2010).


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discs overgrown: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 10 February 2014

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