cubitus interruptus

Protein interactions (part 2/2)

Hedgehog stimulates maturation of Cubitus interruptus into a labile transcriptional activator

In Drosophila, signaling by the protein Hedgehog (Hh) alters the activity of the transcription factor Cubitus interruptus (Ci) by inhibiting the proteolysis of full-length Ci (Ci-155) to its shortened Ci-75 form. Ci-75 is found largely in the nucleus and is thought to be a transcriptional repressor, whereas there is evidence to indicate that Ci-155 may be a transcriptional activator. However, Ci-155 is detected only in the cytoplasm, where it is associated with the protein kinase Fused (Fu), with Suppressor of fused [Su(fu)], and with the microtubule-binding protein Costal-2. It is not clear how Ci-155 might become a nuclear activator. Mutations in Su(fu) cause an increase in the expression of Hh-target genes in a dose-dependent manner while simultaneously reducing Ci-155 concentration by some mechanism other than proteolysis to Ci-75. Conversely, eliminating Fu kinase activity reduces Hh-target gene expression while increasing Ci-155 concentration. It is proposed that Fu kinase activity is required for Hh to stimulate the maturation of Ci-155 into a short-lived nuclear transcriptional activator and that Su(fu) opposes this maturation step through a stoichiometric interaction with Ci-155 (Ohlmeyer, 1998).

Hh signaling thus elicits several changes that are required to convert Ci into an effective transcriptional activator. Hh spares Ci-155 from Protein kinase A- and Slimb-dependent protolysis to Ci-75 (see supernumerary limbs), perhaps by modifying the phosphorylation status of Ci, and promotes dissociation of the Ci-155 complex from microtubules. It is proposed that in wing discs some of this 'primed' Ci-155 is not associated with Su(fu) and can activate dpp and ptc transcription but not anterior en expression. Most of the primed Ci-155 in wing discs and perhaps all of the primed Ci-155 in embryos is inactive while it is in complex with Su(fu) and signaling by Hh and Fused kinase are necessary for Ci-155 to become a transcriptional activator. This active form of Ci appears to be unstable and so is not detectable in the nuclei of cells responding to Hh. The lower levels of Ci-155 that are found in wing discs close to the source of Hh, as compared with levels in more distant regions of the Hh-signaling domain, may be explained by this model if more Hh is required to stimulate the Fu-kinase-dependent step in Ci activation than to protect Ci-155 from proteolytic degradation to Ci-75. This dosage dependence may account for the restricted range of engrailed induction relative to dpp and ptc in wing discs and the single-cell range of Hh signaling in embryonic ectoderm (Ohlmeyer, 1998).

Transgenic inhibitors identify two roles for Protein Kinase A in Drosophila development: Regulation of Cubitus interruptus

Analysis of protein kinase A (PKA) has been initiated in Drosophila using transgenic techniques to modulate PKA activity in specific tissues during development. GAL4/UAS-regulated transgenes were constructed in active and mutant forms that encode PKAc, the catalytic subunit of PKA, and PKI(1-31), a competitive inhibitor of PKAc. Evidence that the wild-type transgenes are active and the phenotypes produced by a number of GAL4 enhancer-detector strains are summarized. The effects of transgenes encoding PKI(1-31) are compared with those encoding PKAr*, a mutant regulatory subunit that constitutively inhibits PKAc because of its inability to bind cyclic AMP. Both inhibitors block larval growth, but only PKAr* alters pattern formation by activating the Hedgehog signaling pathway. Therefore, transgenic PKI(1-31) should provide a tool to investigate the role of PKAc in larval growth regulation without concomitant changes in pattern formation. The different effects of PKI(1-31) and PKAr* suggest two distinct roles, cytoplasmic and nuclear, for PKAc in Hedgehog signal transduction. Alternatively, PKAr* may target proteins other than PKAc, suggesting a role for free PKAr in signal transduction, a role inhibited by PKAc in reversal of the classical relationship of these subunits (Kiger, 1999).

Phenotypes produced by PKI(1-31) and PKAr* are surprisingly different. The phenotypic effects of PKI(1-31) appear to represent a subset of those of PKAr*. Both retard or otherwise block larval growth. PKAr* alone affects patterning in embryos and imaginal discs by activating Hedgehog signaling, and it alone causes abnormal differentiation in imaginal discs (which may reflect minor aberrations in patterning). The origin of this difference might reside in some fundamental difference in the biological properties of PKI(1-31) and PKAr* or perhaps in their relative stabilities in different cell types. However, PKI(1-31) is demonstrably active in wing imaginal discs and in other tissues since it is capable of inhibiting ectopic PKAcF (epitope tagged PKA catalytic subunit). Regardless of the origin of the difference, it would appear that PKI(1-31) specifically targets larval growth (Kiger, 1999).

With regard to Hedgehog signaling, a possible target of PKAr* and PKI(1-31) in the cytoplasm would be the complex responsible for the proteolysis of the transcription factor Ci, where PKAr* and PKI(1-31) would inhibit phosphorylation of the PKA sites necessary for proteolysis of Ci155 to the repressor form Ci75. The ability of PKAr* to interact with anchoring or other proteins might give it greater access to this complex than PKI(1-31), accounting for the failure of the latter to activate Hedgehog target genes (Kiger, 1999).

Another possible explanation for the different actions of PKAr* and PKIF(1-31) is that free PKAr* (and by implication free PKAr) has a target other than PKAc through which it activates Hedgehog signaling. Precedent for such a role exists. In Dictyostelium, free PKAr binds and activates a cAMP-specific phosphodiesterase that is postulated to have functional homology to the cAMP-specific phosphodiesterase encoded by dunce. The Dictyostelium phosphodiesterase is also activated by bovine PKAr1a, and a synthetic monomeric form of this regulatory subunit is a more potent activator than the dimeric form. (The Dictyostelium PKAr protein lacks a dimerization domain, and its PKA exists as a heterodimer). In this scenario, in the absence of a cAMP signal, PKAc would bind to PKAr, inhibiting this novel activity. Reduction in the level of PKAc, e.g., in a mitotic clone of cells homozygous for a lethal allele of DC0, would lead to free PKAr that would activate Hedgehog signaling (Kiger, 1999 and references).

In an alternative scenario, the effect of PKAr* on Hedgehog target genes could be caused by its ability to deplete nuclear PKAc, a role that cannot be fulfilled by PKI(1-31). Since the normal role of PKI(1-77) is not only to inhibit, but to export, nuclear PKAc, it is possible that PKAc plays another critical role in the nucleus in addition to its catalytic role in phosphorylation. For example, PKAc might function as a corepressor with Ci75 to block transcription of Hedgehog target genes. Consistent with this hypothesis PKI(1-60) has been shown to activate Ci-mediated chloramphenicol acetyltransferase transcription from a model Gli enhancer in Drosophila Kc cells, a finding can be attribute solely to inhibition of proteolysis of cytoplasmic Ci155. It may be that PKAc can function as a corepressor even if its catalytic site is occupied by PKI(1-31). Corepression by PKI(1-31):PKAc and Ci75 might block transcription of target genes, even in the presence of Ci155 produced by concommitant inhibition of Ci155 proteolysis in the cytoplasm. Small changes in the ratio of Ci155 and Ci75 are believed to be critical for activation of Hedgehog target genes. In addition, PKI(1-77) may differ from PKI(1-31) because only the former reduces basal transcription from cAMP-stimulated promoters. If PKAc has such an additional role, then the R224 mutant must have lost this function, as well as its ability to bind PKAr* and PKIF, since PKAcR224F produces no abnormal phenotypes and has no effect on viability. On the other hand, the hypothesized nuclear role of PKAc might be catalytic if nuclear PKAc is in some way inaccessible to nuclear PKI(1-31) (Kiger, 1999 and references).

These considerations suggest that normal Hedgehog signal transduction may require both inhibition of cytoplasmic PKAc activity and export of nuclear PKAc. The Drosophila homolog of PKI(1-77) would be a good candidate for carrying out these functions. The fact that PKI(1-77) seems to play some role in regulating the cell cycle may help to explain why PKI(1-31) has different effects on endoreplicating cells and mitotic cells. Resolving the nature of the roles played by PKAc in the cytoplasm and in the nucleus may lead to simultaneous understanding of the effects seen here on pattern formation and on cell growth (Kiger, 1999 and references).

Direct comparisons of the effects of PKI(1-31) and of PKI(1-77) are needed to provide more insight into how different PKAc inhibitors are functioning. PKAc transgenes with specific catalytic site mutations should provide evidence for or against a noncatalytic nuclear role for PKAc. PKAr* transgenes with domain-specific mutations should provide insight into the role of PKAR* in Hedgehog signaling. Identification of a Drosophila homolog of PKI(1-77) and study of its regulation will be important to achieve a clear understanding of the roles of PKAc. From a practical standpoint, PKI(1-31) transgenes should provide a useful tool for investigating the role of PKA in larval growth regulation, independent of PKA's effects on pattern formation. Mutations that permit larvae to survive the effect of PKI(1-31) and develop to adults should help to identify elements controlling larval growth. Conversely, mutations that sensitize adults or embryos to PKI(1-31) may reveal elements important for pattern formation (Kiger, 1999).

Genetic evidence for a protein kinase A/Cubitus interruptus complex that facilitates processing of Cubitus interruptus in Drosophila

Hedgehog (Hh) activates a signal transduction pathway regulating Cubitus interruptus (Ci). In the absence of Hh, full-length Ci (Ci-155) is bound in a complex that includes Costal2 (Cos2) and Fused (Fu). Ci-155 is phosphorylated by protein kinase A (PKA), inducing proteolysis to Ci-75, a transcriptional repressor. Hh signaling blocks proteolysis and produces an activated Ci-155 transcriptional activator. The relationship between PKA and the Ci/Cos2/Fu complex is unclear. PKAc is generally thought to be part of a PKA holoenzyme where its activity is inhibited by PKAr and regulated by cAMP. Since Hh signaling leads to inhibition of Ci-155 phosphorylation and proteolysis, one might expect that this would coincide with a drop in ambient cAMP level and inhibition of PKAc activity by PKAr. No change in cAMP levels, however, has been detected in a mouse cell line sensitive to Sonic hedgehog signaling when exposed to Sonic hedgehog. Experiments addressing this question in Drosophila have employed ectopic expression of PKAc*, a constitutively active mutant mouse catalytic subunit impaired in interaction with PKAr. Low levels of PKAc* expression appear to not affect normal Hh signaling, while high levels override Hh signaling and promote Ci-155 proteolysis (Kiger, 2001).

Ectopic expression of the PKAc inhibitors PKAr* and PKI(1-31) produces quite different responses from Hh target genes: PKAr* mimics the effect of mitotic clones deficient in PKAc (homozygous for loss-of-function DC0 mutants), while PKI(1-31) has no effect, despite its demonstrated ability to inhibit endogenous PKAc. The basis for the different effects of PKAr* and PKI(1-31) has been addressed through studies of mutant forms of PKAr* and PKAc. The possibility that free PKAr* (and free wild-type PKAr) has a target other than PKAc through which it induces Hh signaling is tested. Precedence for such a role can be found in Dictyostelium, where free PKAr binds to, and activates, a cAMP-specific phosphodiesterase. In this view, a mitotic clone deficient in PKAc would induce Hh signaling because of the free PKAr created therein. It has been shown that ectopic expression of a catalytic site mutant of PKAc, PKAcA75, produces an unexpected bipartite phenotype compared to that of ectopic PKAc. To wit, PKAcA75 is null with respect to PKAc phenotypes and is dominant negative with respect to activation of Hh target genes. These observations cast new light on the role of PKAc in Hh signaling and indicate that PKAc is part of a larger complex that includes Ci-155. This insight makes it possible to understand those attributes of PKAr*, lacking in PKI(1-31), that allow PKAr* to block PKAc involved in phosphorylation of Ci-155 (Kiger, 2001).

Thus, an examination was performed of Hh target gene expression caused by mutant forms of PKA regulatory (PKAr) and catalytic (PKAc) subunits and by the PKAc inhibitor PKI(1-31). The mutant PKAr*, defective in binding cAMP, has been shown to activate Hh target genes solely through its ability to bind and inhibit endogenous PKAc. Surprisingly, PKAcA75, a catalytically impaired mutant, also activates Hh target genes. To account for this observation, it is proposed that PKAc phosphorylation targeting Ci-155 for proteolysis is regulated within a complex that includes PKAc and Ci-155 and excludes PKI(1-31). This complex may permit processive phosphorylation of Ci-155 molecules, facilitating their processing to Ci-75. Phosphorylation of a number of sites on Ci-155 is required for its proteolysis. If so, Ci-155 must be able to rotate to access the active site of the bound PKAc and other components of the complex must be required to hold it in place. Such steric constraints could prevent PKI(1-31) from finding its PKAc binding site, explaining the inability of this inhibitor to induce Hh target gene expression (Kiger, 2001).

Determining what other components make up the PKAc/Ci complex will be an important step in understanding Hh signal transduction. Since most cytoplasmic Ci-155 is bound in a Ci/Cos2/Fu complex, PKAc must be part of this complex or part of another complex that is closely tied to it. The association of PKAr type I and PKAc in vitro is exquisitely balanced by the binding of MgATP to the holoenzyme at the PKAc catalytic site. A decrease in MgATP concentration leads to rapid dissociation of the holoenzyme. Since PKAcA75 is mutant at a residue required for binding of MgATP, it seems probable that PKAcA75 is unable to form a holoenzyme with PKAr type I, just as it is unable to block phosphorylation of most PKAc substrates. Indeed, the fact that PKAr*, a type I subunit, is less effective than PKAcA75 in inducing Hh target genes could be due to PKAr type I not being part of the PKA/Ci complex. On the other hand, binding of PKAr type II to PKAc does not require MgATP, suggesting that PKAcA75 may enter the complex bound to PKAr type II. If PKAr type II is part of the complex, then the presence of an A-kinase anchor protein (AKAP) might also be expected. At least five AKAPs have been described in Drosophila that tether PKA type II to plasma membranes or the cytoskeleton. It is also possible that an inhibitor protein might escort PKAc to the complex but not be part of the complex (Kiger, 2001).

Knowing the components of the complex will permit choices to be made between possible models. In one model, in the absence of a Hh signal, ambient cAMP level in the complex would be high enough to dissociate PKAr and PKAc and permit processive phosphorylation of Ci-155. It is envisioned that dissociated PKAr and PKAc would remain bound within the complex. A Hh signal then might act to reduce cAMP level and allow bound PKAc and bound PKAr to reassociate, preserving Ci-155 from phosphorylation. In another model, active PKAc may be bound within the complex, and a Hh signal might activate a phosphatase to convert phosphorylated Ci-155 into a transcriptional activator. Whether or not cAMP is involved in Hh signaling remains an open question (Kiger, 2001).

Nuclear trafficking of Cubitus interruptus in the transcriptional regulation of Hedgehog target gene expression

Transcriptional activation of Hedgehog (Hh) target genes requires Cubitus interruptus, a 155 kDa cytoplasmic zinc finger protein (Ci155), that, in the absence of Hh signaling, is processed to form a nuclear repressor (Ci75). Although many proteins are regulated by modulation of their nuclear localization, this possibility has been difficult to evaluate for Ci155, since its presence in the nucleus has not been demonstrated. Past studies of regulated nuclear localization have focused primarily on nuclear import, but specific nuclear export recently has emerged as another important regulatory mechanism. Hh signaling reduces phosphorylation of Ci155, and this reduction in turn appears to decrease processing into Ci75. Blocking processing with proteasome inhibitors or altered Ci proteins, however, is insufficient for activation of Hh targets. Hh signaling increases the rate of Ci155 nuclear import, resulting in significant nuclear accumulation. Even in the absence of signaling, nuclear accumulation of Ci155 suffices for significant induction of Hh targets, and active nuclear export of Ci155 is an essential mechanism for maintenance of the unstimulated state (C.-H. Chen, 1999).

Hh signaling was examined in the cl-8 cell line, which has been shown to be a homogeneous source for biochemical characterization of Hh signaling processes. The proteasome inhibitors lactacystin and MG132 cause a reduction in Ci75 levels and an increase in Ci155. Proteasome inhibitors thus are active in cl-8 cells, and Ci processing is proteasome dependent. Proteasome inhibition not only blocks Ci155 processing but also causes a marked shift toward slower-migrating species; this shift is even more pronounced in the presence of additional phosphatase inhibitors. Previous genetic studies have implicated a Cdc4-related ubiquitin targeting protein, Slimb, in the Hh pathway and in regulation of Ci processing, suggesting the possibility of direct ubiquitinylation of Ci. However, although Ci-ubiquitin conjugates were detected using cotransfection and overexpression of differently tagged Ci and ubiquitin proteins in cl-8 cells, the formation of such conjugates is not under Hh regulation and could not be detected when Ci protein was expressed at physiological levels. In analyzing the dramatically shifted migration of Ci155 species, it was found that phosphatase treatment of immunoprecipitates reduces the heterogeneity in migration to produce a single faster-migrating Ci155 species, suggesting that the anomalous migration of Ci155 species can be accounted for entirely by phosphorylation, even under conditions of proteasome inhibition that cause dramatic accumulation of slower-migrating forms. Therefore, proteasome action in Ci155 processing, if direct, would appear to operate through a ubiquitin-independent pathway. Alternatively, the proteasome might act upstream, ultimately resulting in the activation of a protease capable of processing Ci155 directly (C.-H. Chen, 1999 and references).

To identify possible differences between Hh-stimulated and proteasome inhibitor lactacystin-treated cells, the localization of Ci155 and Ci75 was examined by subcellular fractionation. In untreated cells, Ci75 is concentrated in nuclei and Ci155 in the cytoplasm. The apparent absence of Ci155 from nuclear fractions of unstimulated cells is in contrast to previously reported results (Aza-Blanc, 1997) but was reproducible; the difference in results likely is due to differences in the subcellular fractionation procedures used. In striking contrast to its apparent absence in the nuclei of unstimulated cells, Ci155 is readily detected in the nuclear fractions of Hh-stimulated cells. Hh stimulation thus induces a significant increase in nuclear Ci155 relative to unstimulated cells. To corroborate these results and rule out possible artifacts in the subcellular fractionation experiments, the localization of Ci155 was examined by indirect immunofluorescence. A significantly higher level of immunofluorescence signal is detected in the nuclei of Hh-stimulated cells. This Hh-induced increase in nuclear Ci155 is observed consistently and is quantified by measurements of average pixel intensities within the nucleus. The low levels of nuclear Ci155 detected by immunofluorescence, together with cytoplasmic retention of the bulk of Ci155, are consistent with and may account for previous difficulties in detection of nuclear Ci155 by immunofluorescence in embryos or imaginal discs. The subcellular fractionations also indicate low levels of nuclear Ci155. Subcellular fractionation further reveals that the high levels of Ci155 induced by lactacystin do not accumulate in the nucleus unless also stimulated by Hh. The lack of significant levels of nuclear Ci155 in lactacystin-treated cells correlates with the lack of increased reporter expression, thus suggesting that nuclear Ci155 is the form of Ci active in induction of ptc transcription. It is noted that Ci75 is decreased but not completely abolished by lactacystin alone or by lactacystin plus Hh, suggesting that the proteasome may also contribute to degradation of Ci75 (C.-H. Chen, 1999).

To investigate the regulation of Ci155 localization, cl-8 cells were treated with leptomycin B (LMB), a fungal metabolite that inhibits nuclear protein export by specifically binding to the CRM1 receptor for nuclear export signals. LMB treatment of cl-8 cells for 32 hr causes nuclear accumulation of Ci155 to a level similar to that induced by Hh signaling; transfected cells expressing high levels of Ci show a predominant nuclear accumulation within 2 hr of LMB treatment, and this nuclear accumulation exceeds that produced by Hh stimulation. Subcellular fractionation of cells treated for 6 hr with LMB in the absence of Hh stimulation also confirms the accumulation of endogenous Ci155 in the nucleus. These results suggest that Ci155 normally enters the nucleus in the absence of Hh stimulation and that endogenous Ci155 and particularly the high levels of Ci155 produced by transfection owe their cytoplasmic localization to nuclear export mediated by CRM1. A region just carboxy terminal to the site of Ci155 cleavage (residues 675-860) has been identified as a cytoplasmic localization signal (Aza-Blanc, 1997); the current studies confirm that fusion of GFP to this region alters the predominantly nuclear localization of GFP to a cytoplasmic localization. This region previously was proposed to tether Ci155 in the cytoplasm through association with other components of a cytoplasmic complex (Aza-Blanc, 1997). However, treatment with LMB renders GFPCyt nuclear in its localization, thus suggesting that residues 675-860 contain a nuclear export signal instead of a cytoplasmic tether. As observed for transfected Ci, Hh stimulation does not appreciably alter the localization of GFPCyt (C.-H. Chen, 1999).

Both Hh signaling and LMB treatment induce Ci155 accumulation in the nucleus, but they do so with markedly different kinetics. Whereas most of the Hh-dependent nuclear accumulation is achieved in less than 4-6 hr, LMB treatment is much slower in producing significant nuclear accumulation, which eventually reaches levels similar to those induced by Hh at 32 hr. The rapid increase produced by Hh signaling is observed even in the presence of LMB, suggesting that LMB does not interfere with the normal mechanisms that increase nuclear Ci155 and that the rate of Ci155 nuclear import is increased by Hh signaling. The relatively slow kinetics of LMB-induced nuclear accumulation of Ci155 suggests that, in the absence of Hh stimulation, Ci155 is subject to a cytoplasmic activity that restricts its rate of nuclear import. However, this mechanism fails to prevent the almost complete nuclear accumulation of Ci155 within 2 hr when it is expressed at high levels. The cytoplasmic association of Ci155 with several other proteins in a microtubule-bound complex prompted testing for whether higher levels of these other proteins might restore this restriction of Ci155 nuclear import. Indeed, coexpression of high levels of Ci with Cos2 results in a largely cytoplasmic localization for Ci155, even after 32 hr of LMB treatment. This effect is not observed upon coexpression of Su(fu), although high-level Su(fu) do appear to stabilize and increase the levels of cytoplasmic Ci155 (C.-H. Chen, 1999).

ptc reporter expression is induced by LMB treatment in the absence of Hh stimulation. This LMB-induced reporter activity at all stages is lower than Hh-induced activity, but the ratio of LMB-induced to Hh-induced reporter activities increases with time and in parallel with the increasing nuclear accumulation of Ci155. By 48 hr of incubation, LMB treatment alone produces a 28-fold induction, more than 25% of the induction associated with Hh stimulation. The ability of LMB to significantly induce ptc reporter activity in the absence of Hh stimulation suggests that increasing Ci155 nuclear concentration is sufficient to activate Hh target gene expression and that increasing the rate of Ci155 nuclear import may be a major mechanism for Hh induction of target gene expression. The lag in reporter activity induced by LMB relative to Hh stimulation is not due to a detrimental effect of LMB on the health of cl-8 cells, since LMB does not appreciably interfere with the Hh induction of ptc reporter when cells are treated with both LMB and Hh. This lag might be accounted for in part by the slower kinetics of Ci155 nuclear accumulation, by the persistence of Ci75 repressor in the nuclei of LMB-treated cells, or possibly by other mechanisms (C.-H. Chen, 1998).

Several mechanisms by which Ci activity is regulated can now be enumerated. The first of these is cleavage of Ci155 to generate the Ci75 nuclear repressor, a constitutive process that is blocked by Hh stimulation (Aza-Blanc, 1997). This observation led to the hypothesis that a critical consequence of Hh signaling is to lift Ci75 repression (Aza-Blanc, 1997). The current studies are consistent with this hypothesis but also demonstrate that lifting of Ci75 repression with the use of proteasome inhibitors in the absence of Hh stimulation is not sufficient to induce expression of the ptc reporter. Furthermore, altered Ci proteins that cannot be processed are nevertheless capable of a significant response to Hh stimulation. The major role of Ci155 processing thus appears to be maintenance of the inactive state of Hh targets in the absence of Hh signaling, and other mechanisms are required for target gene activation (C.-H. Chen, 1998).

A second constitutive mechanism for maintenance of the inactive state of the pathway is active nuclear export of Ci155, which prevents its accumulation in the nucleus. This export of Ci155, which emerged from studies with LMB, also may help ensure a dynamic response to changes in Hh stimulation so that transcription of Hh targets can be rapidly extinguished upon loss of Hh signaling. It should be noted that in contrast to Ci155 processing, which produces a repressor for Hh targets, nuclear export simply prevents nuclear accumulation of the Ci155 activator. As outlined above, this export process operates at least in part through nuclear export signal(s) located within residues 675-860 of the Ci protein (C.-H. Chen, 1998).

A third mechanism identified in these studies is the rapid nuclear import of Ci155 that is triggered by Hh stimulation. It is suggested that this Hh-induced import plays a significant role in activation of Hh target gene expression, since nuclear accumulation of Ci155 induced by LMB treatment in the absence of Hh stimulation also results in significant activation of the ptc reporter. This increase in import appears not to be caused simply by the increased overall levels of Ci155 that are associated with Hh stimulation, since the high levels of Ci155 produced by lactacystin treatment remain dependent upon Hh stimulation for a significant increase in nuclear levels. These results do not support the idea that nuclear increase of endogenous Ci155 in Hh-stimulated cells is due to a titration of other cytoplasmic components, and they suggest instead that a Hh-triggered event other than increase in total Ci155 is required for the increase in nuclear import. Much remains to be learned about this Hh-stimulated increase in nuclear import, but these studies suggest that Cos2 may be involved since Cos2 limits nuclear accumulation of Ci155 when both are expressed at high levels in the presence of LMB (C.-H. Chen, 1999). Previous work further suggests that Hh stimulation may antagonize the Cos2 block of Ci155 nuclear import by releasing Cos2 and other associated components from microtubules (Robbins, 1997).

A fourth mechanism is the recent proposal that Hh stimulation transforms Ci155 from a stable, inactive form to a labile, transcriptionally active form; this transition is thought to be opposed by the action of Su(fu) and facilitated by a negative effect of Fu on Su(fu). Consistent with such a regulatory mechanism, is the observed suppression of ptc reporter activity by cotransfection with a construct for expression of Su(fu), either with or without additional cotransfection of a Ci construct. In addition, the inability of LMB alone to match Hh stimulation in induction of reporter activity, even after lengthy incubations that produce equivalent or higher levels of nuclear Ci155, would be consistent with a partial failure of Ci155 molecules to complete the transition to this active form. A similar explanation might also account for the observed failure of LMB to induce higher levels of reporter expression when combined with Hh, even though the nuclear levels of Ci155 resulting from such a combined treatment are somewhat higher. Two additional observations suggest that Su(fu) suppression of reporter expression acts by a mechanism distinct from that of Cos2. (1) Unlike Cos2 but in keeping with previous observations (Ohlmeyer, 1998), a high level of Su(fu) from a construct transfected into cl-8 cells paradoxically increases cytoplasmic levels of Ci155. (2) Unlike Cos2, Su(fu) does not retard LMB-induced nuclear accumulation of Ci155 from a transfected construct. The Fu/Su(fu)-governed transition thus may represent a fourth mechanism for regulation of Ci activity, although further work will be required to provide a molecular definition of the 'activated state' of Ci and of the cellular roles of Su(fu) and Fu in governing this transition (C.-H. Chen, 1999).

All of these mechanisms are likely to contribute to control of Hh pathway targets during development. Evidence presented here and elsewhere indicates that at least some of these mechanisms are coordinately regulated by Hh signaling. The cytoplasmic, microtubule-associated complex seems likely to play a central role in this coordination, since each mechanism can be linked to one or more components of the complex. For example, in cos2 mutant clones, higher levels of Ci155 are observed, suggesting a role for Cos2 in processing, and the current studies further indicate a potential role for Cos2 in regulating nuclear import. Fu function likewise has been linked both to processing and to a role in governing the Ci155 transition to an activated state. A more detailed understanding of Hh pathway activation via Ci will require further investigation of the interactions between components of this complex and of the dynamics induced by Hh signaling (C.-H. Chen, 1999 and references).

The subcellular localization and activity of Drosophila Cubitus interruptus are regulated at multiple levels

Cubitus interruptus (Ci), a Drosophila transcription factor, mediates Hedgehog (Hh) signaling during the patterning of embryonic epidermis and larval imaginal discs. In the absence of Hh signal, Ci is cleaved to generate a truncated nuclear form capable of transcriptional repression. Hh signaling stabilizes and activates the full-length Ci protein leading to strong activation of downstream target genes, including patched and decapentaplegic. A number of molecules have been implicated in the regulation of Ci. Mutations in these molecules affect changes in Ci protein level, and also influence the extent of Ci proteolysis and the expression of Ci target genes. This paper examines the regulation of Ci subcellular localization and activity. A bipartite nuclear localization signal (NLS) within Ci has been characterized. It is proposed that the subcellular distribution of Ci is affected by two opposing forces; the action of the NLS and that of at least two regions targeting Ci to the cytoplasm. The data also show that loss of PKA or Costal-2 activity does not fully mimic Hh signaling, demonstrating that Ci proteolysis and Ci activation are two distinct events that are regulated through different paths. It is proposed that there are three levels of apparent Ci activity, corresponding to three zones along the AP axis with different sets of gene expression and different levels of Hh signaling (Q. T. Wang, 1999).

Comparison of the sequences of Ci, Gli and Tra-1 draws attention to a stretch of amino acids starting at the end of the zinc finger domain. This amino acid stretch contains two basic clusters separated by 10 amino acids, typical features of a bipartite NLS. The first basic cluster (R596-K600) lies within the last zinc finger, while the second (K611-K614) lies completely outside the zinc finger domain and is separated from the zinc finger domain by 6 non-conserved amino acids. To test whether this NLS-like sequence is functional, amino acids K581-L616 were fused to beta-galactosidase (beta-gal) and the fusion protein was expressed in S2 cells. While b-gal is restricted entirely to the cytoplasm, beta-gal-NLS shows strong nuclear staining. Therefore, the identified sequence functions as an NLS on a heterologous protein, and is sufficient to direct active nuclear transport. Next a test was performed to see whether the identified sequence is functional in vivo. Because full-length Ci is cytoplasmic, no change in its subcellular localization would be expected if the NLS on full-length Ci were disrupted. Previous studies have shown that when the sequences C-terminal to the zinc finger domain are removed, whether as a consequence of proteolysis or a result of artificial truncations, the rest of the protein exhibits nuclear staining. Therefore CiNZn and CiNZnDeltaNLS, which differ by only 7 amino acids, were constructed: the former contains (while the latter lacks) the second basic cluster. Only the second basic cluster was removed, because mutation of the first basic cluster results in disruption of the last zinc finger. It has been postulated that for proteins containing bipartite NLSs, the functional NLS is reconstituted from the two basic clusters by protein folding. Removal of either one of the clusters, therefore, should disrupt the function of the whole NLS (Q. T. Wang, 1999).

When expressed in vivo, CiNZnDeltaNLS exhibits substantially reduced nuclear staining, when compared to CiNZn, which is predominantly nuclear. This suggests that the second basic cluster is functional in vivo and that its presence is necessary for full activity of the NLS. Some residual nuclear staining persists, suggesting that the first basic cluster on its own or some cryptic sequence on the peptide can still direct nuclear import, though the efficiency is greatly compromised. Amino acids 703-835 are sufficient to counteract the NLS but do not account for all cytoplasmic targeting The characterized NLS is able to direct efficient nuclear transport of CiNZn and beta-gal-NLS, yet full-length Ci is cytoplasmic despite containing this NLS. These results suggest that sequences C-terminal to the zinc finger domain target Ci to the cytoplasm, thereby overcoming the function of the NLS. A C-terminal domain encompassing N703-M850 is known to be capable of restricting GFP, which normally diffuses freely, to the cytoplasm. The cytoplasmic targeting effect of this region was further tested by fusing P617-R836 (C1) to the beta-gal-NLS construct and analyzing the subcellular distribution of beta-gal in transfected S2 cells. This region is indeed able to overcome the action of the NLS and to keep the fusion protein cytoplasmic. Such a cytoplasmic targeting force must have come from sequences C-terminal to L685, because ci-NZnHA, which is truncated after L685, is nuclear. L685-R836 (C1-A) was deleted from full-length Ci (Ci DC1-A ) and its subcellular distribution was examined in vivo. Interestingly, Ci DC1-A is cytoplasmic. These results indicate that N703-R836 are sufficient for cytoplasmic targeting, and that other C terminal sequences can also target Ci to the cytoplasm (Q. T. Wang, 1999).

By SDS-PAGE, Ci exhibits an apparent molecular mass of greater than 155 kDa and is not readily resolved into isoforms. However, when an extract of cultured cl-8 cells is resolved by isoelectric focusing (IEF), Ci is detected as a group of charge isoforms with pI values around 7. At least 6 major isoforms are easily and consistently detected. Samples prepared from wild-type (OreR) wing discs give a similar western pattern. In fact, when OreR and cl-8 samples are resolved in the same lane, all bands superimposed and no additional species are visible. Phosphorylation is one of the common events that could result in charge isoforms. A test was performed to see whether the isoforms observed for Ci are due to phosphorylation. When a cl-8 extract is treated with l protein phosphatase (l-PPase), the proportion of the Ci isoform with the highest pI (the unphosphorylated) increased at the expense of those with lower pI values (the more phosphorylated forms). Therefore, the bands observed in cl-8 extract are phosphorylation isoforms, and a single band can be identified as the unphosphorylated protein (Q. T. Wang, 1999).

Previous studies have demonstrated that protein kinase A plays an important role in Ci proteolysis. In PKA loss-of-function clones, Ci protein level is greatly elevated. An extract from discs carrying large numbers of such clones exhibits reduces proteolysis. Furthermore, Ci mutated at putative PKA sites is resistant to cleavage. The resolution of Ci into phosphorylation isoforms enables a direct test of the action of PKA upon Ci. When cl-8 cells are treated for an hour with H-89, a potent PKA inhibitor, a slight increase in the amount of full-length Ci is observed, accompanied by a slight decrease of Ci-75. This suggests that the basal activity of PKA is low in cl-8 cells. When cl-8 cells were treated with the PKA stimulator forskolin, within an hour a decrease of Ci-155 is observed, but there is no marked change in the IEF pattern of Ci. However, there is a dramatic shift in the western pattern when cells are simultaneously treated with forskolin and MG132. The amount of the unphosphorylated isoform remained constant, while the highly phosphorylated isoforms accumulate (Q. T. Wang, 1999).

Ci proteolysis is inhibited in fu mutants and cos2 mutants. In wild-type wing imaginal discs, full-length Ci is found at high levels in a stripe along the AP compartment boundary and at low levels throughout the rest of the anterior compartment. High level Ptc is expressed in a thin stripe along the boundary. In discs mutant for fu, the high level Ci stripe is expanded, and the Ptc stripe is more diffuse with a modest protein level. In loss-of-function cos2 clones, Ci protein level is elevated and these clones cell-autonomously express high level Ptc. To examine whether Ci protein level in fu and cos2 mutants is up-regulated through inhibition of proteolysis, extracts from fu and cos2 hypomorphic discs were analyzed by SDS-PAGE and western blot. Proteolysis of Ci is not detectable in cos2 hypomorphic wing extracts and is significantly inhibited in extracts from both class I and class II fu mutant discs. Despite the inhibition of Ci proteolysis in fu mutants, such animals display evidence of compromised Ci activity, both in reduced ptc expression and fusion between LV3 and 4 in adult wings (Q. T. Wang, 1999).

Cells in the anterior compartment of wing pouch express different sets of ci target genes depending on their distance from the AP boundary. Based on the expression profile of Ci and its target genes, cells along the AP axis can be divided into three zones. The Anterior Zone consists of cells more than 8-9 cell diameters away from the boundary. Cells in this zone express low level Ci, low level Ptc and no Dpp. The Intermediate Zone, marked by expression of high level Ci, low level Ptc and high level Dpp, corresponds to cells between 8-9 and 2-3 cell diameters away from the boundary. Cells immediately adjacent to the boundary (within 2-3 cell diameters) fall into the Boundary Zone, marked by medium level Ci, high level Ptc, and medium level Dpp. The Ci regulated enhancer element/reporter 4bslacZ is also expressed in this zone. Expression of ci target genes is a consequence of the overall activity of many individual Ci peptides, which is determined by both the potency of each peptide and the number of peptides present. In the following discussion, the overall Ci activity observed for a cell, judged by the expression of target genes, is termed the 'apparent activity' of Ci. The specific activity, or potency, of each peptide is defined as its 'activation state'. In a wild-type wing disc, there are three levels of apparent Ci activity corresponding to the three zones. In the Boundary Zone, Ci has the highest apparent activity, activating both ptc and 4bslacZ to high levels. dpp expression in this region is likely subjected to a partial repression by anterior En. In the Intermediate Zone, Ci exhibits an intermediate level of apparent activity, inducing strong expression of dpp but not high level ptc nor 4bslacZ. In the Anterior Zone, Ci has the lowest apparent activity (Q. T. Wang, 1999).

Although there are three levels of apparent Ci activity, evidence has been found for only two activation states. 4bslacZ, whose sensitivity allows the monitoring Hh-induced Ci activation, is expressed only in the Boundary Zone, suggesting that Ci is activated only in this zone. Ci in the activated state, therefore, is given the name Ciboundary. The high level of Ci in the Intermediate Zone indicates that cells in this zone must receive some level of Hh signaling, which inhibits Ci proteolysis. Despite the high protein level, Ci in the Intermediate Zone is not sufficiently activated to induce 4bslacZ expression, and is given the name Cidefault. The lack of proteolysis in the Intermediate Zone allows Cidefault to accumulate, resulting in high level expression of dpp. In the Anterior Zone, the majority of Ci is proteolytically cleaved into Ci-75. Although the Anterior Zone and the Intermediate Zone differ in Ci protein levels and the expression patterns of target genes, at present there is no evidence that they differ in the state of Ci activation. In fact, when Ci stabilization is mimicked in the Anterior Zone by making PKA loss-of-function clones, the expression of high level dpp is also mimicked. This observation is consistent with the idea that the two zones share the same activation state of Ci (Cidefault) but differ in the levels of full-length Ci. In summary, the three levels of apparent Ci activity correspond to Ci boundary, high level Cidefault, and low level Cidefault, respectively (Q. T. Wang, 1999).

The boundary between the Anterior Zone and the Intermediate Zone corresponds to a division between cells with low Ci levels and those with high Ci levels, and is likely to coincide with the anterior border of Hh signaling. Between this line and the AP boundary, cells in both the Intermediate Zone and the Boundary Zone receive some level of Hh signaling and show stabilization of Ci. (The relatively lower Ci level in the Boundary Zone probably reflects partial transcriptional repression by anterior En. However, the activation from Cidefault to Cibounday happens only in the Boundary Zone, suggesting that it takes place when the level of Hh signaling is above a certain threshold. The level of Hh signaling changes across the anterior compartment. In the Boundary Zone, cells express high level Ptc, which both transduces and sequesters Hh signaling. The presence of high level Ptc creates a steep decline of the Hh signal. Consequently, cells receiving Hh are divided into those receiving high level Hh signal (the Boundary Zone) and those receiving lower level Hh signal (the Intermediate Zone). Its role in regulating Hh distribution makes Ptc essential for proper regulation of the apparent activity of Ci (Q. T. Wang, 1999).

Study of Hh-induced Ci activation has been complicated by the fact that in almost all the assays, a high level of Ci protein seems to suffice for the activation of Ci target genes. This is well illustrated in the case of loss-of-function PKA clones, in which elevated Ci protein levels are associated with strong activation of Hh target genes such as ptc and dpp. It has been difficult to tell whether the activation of downstream genes is due to elevated Ci alone, or if Ci is activated in addition to being stabilized in these clones. Progress was made in a recently published study through a combination of manipulating the expression of an uncleavable deletion construct of Ci and examining smo loss-of-function clones in the posterior compartment (Methot, 1999). Importantly, it demonstrates that inhibition of Ci proteolysis is not sufficient to activate Ci. However, the assays as described do not address the question of whether in vivo stabilization and activation of Ci are regulated simultaneously as two consequences of the same process, or regulated separately through different mechanisms. The sensitivity of 4bslacZ allows this question to be addressed. While a modest level of Ci in cells receiving high level Hh signal can activate 4bslacZ and clones lacking ptc activate 4bslacZ, high level Ci in PKA or cos2 loss-of-function clones cannot. It is concluded that Ci is stabilized but not activated in PKA or cos2 mutant clones. In other words, these mutations mimic one aspect of Hh signaling but not the other, and Ci in the clones exists as Ci default instead of Ci boundary (Q. T. Wang, 1999).

Nuclear import of Cubitus interruptus is regulated by Hedgehog via a mechanism distinct from Ci stabilization and Ci activation

The Hedgehog (Hh) signal is transduced via Cubitus interruptus (Ci) to specify cell fates in the Drosophila wing. In the absence of Hh, the 155 kDa full-length form of Ci is cleaved into a 75 kDa repressor. Hh inhibits the proteolysis of full-length Ci and facilitates its conversion into an activator. Recently, it has been suggested that Hh promotes Ci nuclear import in tissue culture cells. The mechanism of Ci nuclear import in vivo and the relationship between nuclear import, stabilization and activation have been studied. Ci rapidly translocates to the nucleus in cells close to the anteroposterior (AP) boundary and this rapid nuclear import requires Hh signaling. The nuclear import of Ci is regulated by Hh even under conditions in which Ci is fully stabilized. Furthermore, cells that exhibit Ci stabilization and rapid nuclear import do not necessarily exhibit maximal Ci activity. It has been previously shown that stabilization does not suffice for activation. Consistent with this finding, the results suggest that the mechanisms regulating nuclear import, stabilization and activation are distinct from one another. cos2 and pka, two molecules that have been characterized primarily as negative regulators of Ci activity, also have positive roles in the activation of Ci in response to Hh (Q. Wang, 2000).

In order to analyze the nuclear import of Ci in vivo, imaginal discs were subjected to whole-mount organ culture experiments, which allowed studies to be performed in different genetic backgrounds and under conditions that preserve the spatial context of Hh signaling. Because imaginal discs are sacs with only two layers of epidermal cells, drugs that inhibit cellular processes work as well in this context as on tissue culture cells. When protein export is blocked with LMB, a potent inhibitor of protein nuclear export, the amount of Ci peptides that enter the nucleus can be monitored, and a remarkable difference is observed in cells close to and those away from the AP boundary. Ci accumulates in the nucleus only in cells close to the boundary, and this has been further shown to be Hh dependent, since mutations in smo prevent nuclear accumulation of Ci. The rate of Ci nuclear import in the presence of Hh is very rapid in vivo, and the subcellular distribution of Ci shifts from predominantly cytoplasmic to predominantly nuclear within as short a time as half an hour of treatment. The rate difference of Ci nuclear import in cells receiving and not receiving Hh is also significant. The cytoplasmic localization of Ci is unchanged in cells away from the boundary after three hours of treatment. From these results, it can be concluded that the rate of Ci nuclear import is dramatically increased by Hh signaling in vivo (Q. Wang, 2000).

pka mutation has a negative effect on the expression of hh/ci target genes. Mutations in pka are associated with disc overgrowth and duplication, signs of up-regulated Ci activity. The level of full-length Ci is elevated in pka loss-of-function clones and these clones ectopically express ptc and dpp. PKA activity is required for the cleavage of Ci. Nevertheless, PKA is not involved in nuclear import of Ci (Q. Wang, 2000).

Another molecule that negatively regulates Ci stability and hh target gene expression is Cos2. To directly test the role of cos2 in Ci nuclear import, discs carrying cos2 clones were treated with LMB. All clones exhibit cell-autonomous nuclear accumulation of Ci regardless of the clone’s distance from the AP boundary. cos2 encodes a kinesin-like molecule that is part of a multi-protein complex including Ci. Over-expression of cos2 blocks nuclear entry of Ci in tissue culture cells, and it has been proposed that Cos2 regulates the microtubule-association of the complex and consequently the ability of Ci to translocate to the nucleus (Q. Wang, 2000).

Discs mutant for fu show signs of compromised Ci activity, including fusion between wing veins 3 and 4, lack of late en expression in the anterior compartment, and diffuse ptc expression at lower levels. Examination of these discs after LMB treatment reveals a lack of Ci nuclear accumulation, suggesting that fu function is required for rapid Ci nuclear import. It has been shown previously that overexpression of ci can rescue the wing vein phenotype in fu mutants. Consistent with this finding, the Ci nuclear import phenotype is also partially rescued in fu discs in which a UAS-ci transgene is over-expressed along the AP boundary via ptcGAL4 (Q. Wang, 2000).

The role of cos2 in Hh signal transduction has been studied by examining the expression of ptc in cos2 clones. High level ptc expression is dependent on Hh signaling and is normally found only in the Boundary Zone, namely the 2-3 rows of cells immediately anterior to the compartment boundary. Although cos2 clones away from the boundary can ectopically induce ptc expression, a small number of such clones do not express ptc. A striking variability in the expression of ptc is observed for cos2 clones abutting the boundary: approximately 50% of cos2 clones overlapping the Boundary Zone disrupt the wild-type high-level Ptc stripe. It is not known why cos2 activity is more critical along the boundary than away from the boundary. Nonetheless, this result suggests that cos2 is required for Ci to become fully active in the Boundary Zone in response to a high level of Hh signaling. A requirement for cos2 is more evident when an in vivo reporter of Ci activity, 4bslacZ, whose enhancer element contains only four Ci binding sites and four Scalloped binding sites, was examined and whose response to Ci exhibits a more stringent requirement for Hh signaling than that of ptc. Not only do cos2 clones away from the boundary fail to ectopically induce 4bslacZ, but clones abutting the boundary invariably disrupt the wild-type 4bslacZ stripe. Given the cell-autonomous stabilization and nuclear import of Ci in cos2 clones, it is inferred that the disruption of ptc and 4bslacZ expression in such clones is due to a lack of Ci activation (Q. Wang, 2000).

The role of pka in Ci activation was assayed in the posterior compartment of discs, in which all cells are exposed to a high level of Hh ligand. An actin promoter was used to drive a low level of ci expression throughout the discs. In discs with ubiquitous ci, high-level ptc expression is observed in the posterior compartment cells (P cells) but not in the anterior compartment cells (A cells). pka mutant P cells in this background do not express ptc, indicating that the loss of pka function disrupts Hh signal transduction and compromises maximal Ci activity. Consistent with this result, loss of pka function also disrupts 4bslacZ expression in the Boundary Zone (Q. Wang, 2000).

While the loss of ptc and 4bslacZ expression in pka clones could reflect a disruption of Ci activity, it could also be due to negative effects on other proteins that bind these enhancers. To distinguish between these two possibilities, wild-type Ci and a PKA site-mutant form of Ci, Ci(m1-3), were compared for their abilities to induce ectopic 4bslacZ in the posterior compartment. Wild-type Ci exhibits a sensitive response to Hh and, at a modest protein level, induces robust 4bslacZ expression in the posterior compartment but not in the anterior. In contrast, Ci(m1-3) shows no response to Hh and does not induce 4bslacZ in either compartment. Thus, the integrity of specific PKA sites within Ci is essential for it to respond to high levels of Hh and become fully active. Since pka is dispensable for the acceleration of Ci nuclear import in Hh-receiving cells, the requirement of PKA phosphorylation for maximal Ci activity presumably reflects a requirement for Ci activation (Q. Wang, 2000).

The ubiquitin ligase Hyperplastic discs negatively regulates hedgehog and decapentaplegic expression by independent mechanisms

Photoreceptor differentiation in the Drosophila eye disc progresses from posterior to anterior in a wave driven by the Hedgehog and Decapentaplegic signals. Cells mutant for the hyperplastic discs gene misexpress both of these signaling molecules in anterior regions of the disc, leading to premature photoreceptor differentiation and overgrowth of surrounding tissue. hyperplastic discs encodes a HECT domain E3 ubiquitin ligase that is likely to act by targeting Cubitus interruptus and an unknown activator of hedgehog expression for proteolysis (Lee, 2002).

If hyd regulates dpp expression by altering Ci activity, loss of hyd should lead to upregulation of full-length, active Ci. Increased levels of full-length Ci are indeed observed in hyd mutant clones in the anterior of the eye disc. However, this could be due to misexpression of hh in the same clones. To determine whether hyd has a direct effect on Ci, hyd;hh double mutant clones anterior to the morphogenetic furrow were examined. High levels of full-length Ci accumulated in these clones, confirming that Hyd normally reduces Ci levels independently of Hh activity (Lee, 2002).

The F-box protein Slmb has been shown to promote processing of Ci to Ci75 as a component of an SCF ubiquitin ligase complex. Therefore the effects were compared of slmb and hyd mutations on Ci levels in the wing disc. Ci155 is much more dramatically increased in slmb clones than in hyd clones. An interesting difference was also observed between hyd and slmb in the regulation of dpp. dpp expression is increased in hyd mutant clones close to the AP border, but is very rarely activated outside this domain. In contrast, slmb mutant clones activated dpp expression only when they lay outside the wing pouch, perhaps because of activation of Wg signaling, which represses dpp expression, within the wing pouch. Consistent with these third instar phenotypes, anterior duplications like those resulting from loss of slmb are not observed in adult wings containing hyd mutant clones, although outgrowths did arise from internal regions of the wing. Such duplications would require dpp to be misexpressed at a distance from its normal domain of expression. Ptc expression, which requires activation of the full-length form of Ci, was not altered in either hyd or slmb mutant clones. Slmb and Hyd thus appear to have distinct effects on Ci protein accumulation and activity, suggesting that they have either different substrates or different effects on the same substrate (Lee, 2002).

Cubitus interruptus acts to specify naked cuticle in the trunk of Drosophila embryos

One function of the Wingless signaling pathway is to determine the naked, cuticle cell fate choice in the trunk epidermis of Drosophila larvae. The zinc finger transcripton factor Teashirt (Tsh) binds to the transactivator domain of Armadillo to modulate Wingless signaling output in the embryonic trunk and contributes to the naked cell fate choice. The Hedgehog pathway is also necessary for the correct specification of larval epidermal cell fate, which signals via the zinc finger protein, Cubitus interruptus (Ci). Ci also has a Wingless-independent function, which is required for the specification of the naked cell fate; previously, it had been assumed that Ci induces naked cuticle exclusively by regulation of wg. Wg and Hh signaling pathways may be acting combinatorially in the same, or individually in different, cells for this process, by regulating common sets of target genes. (1) The loss of the naked cuticular phenotype in embryos lacking ci activity is very similar to that induced by a late loss of Wg function. (2) Overexpression of Ci causes the suppression of denticles (as Wg does) in absence of Wg activity in the anterior trunk. Using epistasis experiments, it has been concluded that different combinations of the three proteins Tsh, Ci, and Arm are employed for the specification of naked cuticle at distinct positions both along the antero-posterior axis and within individual trunk segments. Finally, biochemical approaches suggest the existence of protein complexes consisting of Tsh, Ci, and Arm (Angelats, 2001).

The cuticles of ci null embryos resemble those that lack wg function specifically during the cell fate specification phase. In both genotypes, the bands of naked cells are reduced though they are not totally lost and the number of denticles is increased, suggesting that both Wg and Ci are required for the patterning of the naked regions. Closer comparison of the denticle identities from these embryos reveals that the expansion of denticles belts correspond, in both cases, to an increase in the number of denticles of types 2, 3, and 4. Since the EGF pathway, and particularly rhomboid (rho), is required to specify these denticle identities, rho expression was examined in wgts embryos. When wgts embryos are shifted to the restrictive temperature at stage 10-11, rho expression is expanded posteriorly in a similar way as that observed in ci94 embryos. These observations support the idea that Ci has a function related to the late activity of Wg signaling (Angelats, 2001).

Thus examination of rhomboid expression and cuticle patterns shows the close similarity of phenotype between a late loss of wg function and the loss of function of ci. Following ectopic expression, Ci is able to promote the specification of naked cuticle in the absence of Wg signaling, showing that Ci is acting downstream or in parallel to Wg during the specification phase. This capacity of Ci to induce the naked cell fate was previously explained by ectopic expression of wg, but the experiments described here show that Ci can also act directly for the specification of the naked cell fate choice especially in the anterior trunk segments. It is believed that UASCi produces high levels of full-length Ci, resulting in the saturation of its normal negative regulation, producing naked cuticle (Angelats, 2001).

Ci, Arm, and Pangolin act in a combinatorial fashion to regulate the expression of dpp in the wing disc. Thus, in addition to the well-known regulatory effects of Hh on wg, it is proposed that downstream components of these signaling pathways may interact directly for gene regulation. Similar arguments may apply in vertebrates where Wnt signaling has been shown to be critical for the regulation of the Ci orthologs Gli2 and Gli3. These considerations and the current results support the hypothesis that Wg and Hh signaling components (Arm and Ci, respectively) have overlapping and thus common functions for patterning, at least in some cells (Angelats, 2001).

The capacity of Ci to mimic Wg activity seems to be position-specific since Ci never suppresses the denticles in the most posterior part of the abdomen (from A5 to the tail) in the absence of Wg activity. In this region of the body, the presence of another unidentified factor may modify the activity of Ci (Angelats, 2001).

Morphological examination of the wild-type trunk segments shows that a typical thoracic segment has fewer and smaller denticle belts compared to those in any abdominal segment. Consequently, thoracic segments generally possess more naked cuticle than abdominal ones. Ci and Arm exhibit differences in their ability to induce naked cuticle in different parts of the trunk. The activity of Arm is crucial for the transduction of the Wg signaling pathway and plays a pivotal role in the trunk for naked cuticular identity. Despite this, ectopic production of stabilized Arm or Wg does not replace denticles of the prothoracic beard with naked cuticle. Loss of Ci activity affects this process of patterning, suggesting that Ci activity acts with Arm signaling for the patterning of the beard. In accord with this hypothesis, ectopic Ci, with or without Wg/Arm signaling, suppresses denticles in the beard. In this context, it is interesting to note that loss of the Wg signaling component sgg induces naked cuticle in the trunk, as expected for constitutive Wg signaling, but in the prothorax no beard develops, contrary to the effects of ectopic Wg signaling (Angelats, 2001).

Tsh activity is also critical for the identity of the prothoracic segment, raising the possibility that Tsh cooperates with the Hh and Wg signaling pathways for patterning of the beard. In conclusion, it is thought that different combinations of dTcf/Arm, Ci, and Tsh proteins are acting to specify the naked cuticular choice, both in different A/P positions along the body and at distinct positions within segments, presumably by acting on common and overlapping sets of downstream target genes (Angelats, 2001).

The differential effects of ectopic Ci or ArmS10c along the A/P axis for the induction of naked cuticle may depend on the Hox proteins, which are known to act in distinct parts of the trunk for segmental identity in combination with Tsh. For example, tsh cooperates with the Sex combs reduced Hox gene for patterning of the prothorax. These results are consistent with the idea that combinations of signaling effectors, Tsh and Hox proteins determine epidermal patterning, since their binding sites are often clustered on the enhancers of target genes (Angelats, 2001).

Consistent with the idea that signaling effectors and Tsh act together during epidermal patterning, Ci, Arm, and Tsh form protein complexes in vivo. Tsh is a phosphoprotein whose phosphorylation is induced in part by Wg signaling. Additionally, hyper-phosphorylated forms of Tsh are found in the nucleus whereas hypophosphorylated forms are predominantly in the cytoplasm. By coimmunoprecipitation, only a hyperphosphorylated form of Tsh coimmunoprecipitates with Ci, suggesting that the interaction between the two proteins takes place in the nucleus. However, only hypo-phosphorylated Tsh interacts with Arm (Angelats, 2001).

These results favour the existence of bipartite complexes (Arm-Tsh and Tsh-Ci) rather than tripartite complexes in vivo. However, the existence of a complex containing these three molecules cannot be excluded (Angelats, 2001).

Genetic dissection of the Drosophila Cubitus interruptus signaling complex

Much of the understanding of the Hedgehog signaling pathway comes from Drosophila, where a gradient of Hh signaling regulates the function of the transcription factor Cubitus interruptus at three levels: protein stabilization, nuclear import, and activation. Regulation of Ci occurs in a cytoplasmic complex containing Ci, the kinesin-like protein Costal-2 (Cos2), the serine-threonine kinase Fused (Fu), and the Suppressor of Fused [Su(fu)] protein. The mechanisms by which this complex responds to different levels of Hh signaling and establishes distinct domains of gene expression are not fully understood. By sequentially mutating components from the Ci signaling complex, their roles in each aspect of Ci regulation can be analyzed. The Cos2-Ci core complex is able to mediate Hh-regulated activation of Ci but is insufficient to regulate nuclear import and cleavage. Addition of Su(fu) to the core complex blocks nuclear import while the addition of Fu restores Hh regulation of Ci nuclear import and proteolytic cleavage. Fu participates in two partially redundant pathways to regulate Ci nuclear import: the kinase function plays a positive role by inhibiting Su(fu), and the regulatory domain plays a negative role in conjunction with Cos2 (Lefers, 2001).

In fu94;Su(fu)LP mutants, it is unlikely that either Fu or Su(fu) is present in the complex: Fu protein from class II mutants fails to immunoprecipitate Cos2, and Su(fu) protein cannot be detected in Su(fu)LP mutants. In this mutant combination, the processing of Ci is not Hh regulated, and this results in uniform levels of Ci protein across the entire anterior compartment. Hh regulation of Ci nuclear import is also lost, and the Ci protein shuttles into and out of the nucleus throughout the anterior compartment. As a consequence, dpp is expressed at modest levels in all anterior compartment cells. Previous studies have shown that Cos2 is required for Ci sequestration in the cytoplasm and its proteolytic processing, but clearly Cos2 is not sufficient for all aspects of Ci regulation. In the absence of the Fu regulatory domain and Su(fu) from the complex, all anterior compartment cells behave as if they are receiving at least modest levels of Hh signaling (Lefers, 2001).

While dpp is expressed throughout the anterior compartment, there is still Hh regulation of transcriptional activation. The elevated expression of ptc and the activation of 4bs-lacZ in cells immediately adjacent to the compartment boundary are similar to the wild-type situation, and even dpp expression is higher in cells receiving Hh signaling. Thus, the process of Ci activation appears intact in the absence of Hh regulation of Ci cleavage or nuclear import. This observation contrasts with the disruption of ptc and 4bs-lacZ expression in cos21 clones, in which Ci also accumulates to high levels and enters the nucleus. The requirement of cos2 for ptc expression has been adequately confirmed. Taken together, Cos2 is necessary and sufficient for Ci activation in response to high-level Hh signaling. It is likely that Cos2 provides a platform where Ci and other components required for Ci activation, such as PKA, can assemble (Lefers, 2001).

Addition of Su(fu) to the Ci-Cos2 complex dramatically reduces the rate of Ci release from the complex as Ci does not accumulate in the nucleus in fu94 mutant discs that have been treated with leptomycin B (LMB), which blocks Ci nuclear export. No regulation of Ci nuclear import is observed, and processing of Ci into Ci75 is still inhibited. The block in Ci nuclear import by Su(fu) appears to be dependent on the presence of Cos2 as clones double mutant for fumH63;cos21 release Ci independent of Hh signaling (Lefers, 2001).

Addition of Fu to the Ci-Cos2 complex essentially restores Hh regulation of Ci nuclear import and the processing of Ci into Ci75. Therefore, in the absence of Hh signaling, Fu is required for both the cleavage of Ci into Ci75 and its retention in the cytoplasm. The major consequence of removing Su(fu) from the complex is a significant decrease in the overall levels of both Ci and Ci75. This decrease does not appear to significantly compromise Hh regulation (Lefers, 2001).

Although Cos2 provides an important tethering force, it apparently cannot hold Ci in the cytoplasm on its own. Addition of either the Fu regulatory domain or Su(fu) is sufficient to restore effective tethering. The requirement for Fu in Ci tethering is a new finding, since it has been shown that Fu plays a positive role in Ci nuclear entry by inhibiting Su(fu) via its kinase domain. Further examination of different classes of fu alleles demonstrates that Fu participates in Ci tethering through its regulatory domain. When a Fu class I mutant protein (kinase domain mutations) is added to the Ci-Cos2 core complex [fu1,Su(fu)LP], regulation of Ci nuclear entry is almost wild type. In contrast, when a Fu class II mutant protein (regulatory domain mutations) is present [fu94;Su(fu)LP or fuRX15;Su(fu)LP], the complex fails to tether Ci in the absence of Hh signaling. It has been shown that Fu interacts with Cos2 through its regulatory domain, and the proteins made by fu class II alleles fail to immunoprecipitate with Cos2. These results suggest that the interaction between Cos2 and the Fu regulatory domain is important for Cos2 to tether Ci in the absence of Su(fu) activity. This Cos2-Fu interaction may also be important for targeting Fu kinase regulation of Su(fu). Both fuRX15 and fu94, which delete different extents of the regulatory domain, might be expected to retain kinase function, yet Hh regulation of Su(fu) appears to have been lost and Ci is not released from the cytoplasm in either of these mutants. The simplest explanation is that by preventing Fu interaction with Cos2, Fu cannot perform its structural role in the complex nor can it regulate Su(fu). Thus, Fu plays two opposite roles in the regulation of Ci nuclear entry. Without Hh signaling, the regulatory domain in conjunction with Cos2 tethers Ci in the cytoplasm; upon Hh signaling, the kinase domain inhibits Su(fu) which, along with a change in the Cos2/Fu regulatory domain interaction, leads to the release of Ci (Lefers, 2001).

In addition to its role in regulating Ci release, Fu also has a role in regulating Ci proteolysis. This is not dependent on the Fu kinase domain, since in the fu class I mutation, fumH63, Ci is readily cleaved in the absence of Hh signaling. The C-terminal regulatory domain is implicated in this process; a fu class II mutation, fuA, blocks repression of hh expression in the absence of Hh signaling. With the fu94 mutation, all anterior compartment cells fail to efficiently process Ci. Using fu94;Su(fu)LP, it has been shown that this proteolytic processing defect is separable from the Ci release defect also observed in fu mutants. As with nuclear import, the structural role of Fu in Ci processing most likely involves interaction with Cos2 (Lefers, 2001).

Taking the nuclear import and proteolytic processing results together, it appears that the Fu protein is required for the complex to behave properly in the absence of Hh signaling. Elimination of the Fu regulatory domain leads to a block in Ci processing, and in combination with elimination of Su(fu), release of Ci. These are events which normally require modest levels of Hh signaling (Lefers, 2001).

While it has been possible to clearly establish a role for Su(fu) in Ci nuclear import, its role in Ci activation and cleavage is less clear. In cells double mutant for cos2;Su(fu), Ci appears to be at least partially activated since double mutant clones away from the compartment boundary ectopically express en. A reasonable interpretation of these data is that Ci activation is inhibited by Su(fu) and signaling through Cos2 relieves such inhibition (Lefers, 2001).

But this cannot be the whole story. In Su(fu)LP discs, the expression of ptc or en is still tightly regulated and does not expand into all the cells with efficient Ci nuclear import. This regulation of Ci activity is evidently not rendered by the Fu regulatory domain, since it persists in the fu94;Su(fu)LP double mutants. It seems likely that Su(fu) is partially redundant with other factors that regulate Ci activation and that these yet to be identified factors function with Cos2 in the fu;Su(fu) double mutants. Su(fu) may also play some role in Ci cleavage. In the fu94;Su(fu)LP double mutants, the level of Ci seems significantly reduced relative to fu94 single mutants. In addition, Ci protein levels are not elevated across the entire anterior compartment in fuRX15 single mutants but are in fuRX15;Su(fu)LP double mutants. The implication of Su(fu) in these other aspects of Hh regulation suggests that while it is possible to dissect the complex and assign primary roles to the various components, the complex does normally function as a whole (Lefers, 2001).

Smoothened regulates alternative configurations of a regulatory complex that includes Fused, Costal, Suppressor of Fused and Cubitus interruptus

In the Drosophila wing, Hedgehog is made by cells of the posterior compartment and acts as a morphogen to pattern cells of the anterior compartment. High Hedgehog levels instruct L3/4 intervein fate, whereas lower levels instruct L3 vein fate. Transcriptional responses to Hedgehog are mediated by the balance between repressor and activator forms of Cubitus interruptus, CiR and CiA. Hedgehog regulates this balance through its receptor, Patched, which acts through Smoothened and thence a regulatory complex that includes Fused, Costal, Suppressor of Fused and Cubitus interruptus. It is not known how the Hedgehog signal is relayed from Smoothened to the regulatory complex nor how responses to different levels of Hedgehog are implemented. Chimeric and deleted forms of Smoothened were used to explore the signaling functions of Smoothened. A Frizzled/Smoothened chimera containing the Smo cytoplasmic tail (FFS) can induce the full spectrum of Hedgehog responses but is regulated by Wingless rather than Hedgehog. Smoothened whose cytoplasmic tail is replaced with that of Frizzled (SSF) mimics fused mutants, interfering with high Hedgehog responses but with no effect on low Hedgehog responses. The cytoplasmic tail of Smoothened with no transmembrane or extracellular domains (SmoC) interferes with high Hedgehog responses and allows endogenous Smoothened to constitutively initiate low responses. SmoC mimics costal mutants. Genetic interactions suggest that SSF interferes with high signaling by titrating out Smoothened, whereas SmoC drives constitutive low signaling by titrating out Costal. These data suggest that low and high signaling (1) are qualitatively different, (2) are mediated by distinct configurations of the regulatory complex and (3) are initiated by distinct activities of Smoothened. A model is presented where low signaling is initiated when a Costal inhibitory site on the Smoothened cytoplasmic tail shifts the regulatory complex to its low state. High signaling is initiated when cooperating Smoothened cytoplasmic tails activate Costal and Fused, driving the regulatory complex to its high state. Thus, two activities of Smoothened translate different levels of Hedgehog into distinct intracellular responses (Hooper, 2003).

Analyses of the activities of truncated and chimeric forms of Smo in a variety of genetic backgrounds yielded four principal observations. (1)The FFS chimera activates the full spectrum of Hh responses, but is regulated by Wg rather than Hh. From this, it is concluded that the Smo cytoplasmic tail initiates all intracellular responses to Hh, while the remainder of Smo regulates activity of the tail. (2) The SSF chimera interferes with high signaling but has no effect on low signaling. SSF mimics Class II fu mutants and is suppressed by increasing smo+ but not fu+ or cos+. From this, it is concluded that high Hh instructs Smo to activate Fu by a mechanism that is likely to involve dimeric/oligomeric Smo. (3) The cytoplasmic tail of Smo (SmoC) derepresses endogenous Smo activity in the absence of Hh and represses endogenous Smo activity in the presence of high Hh. That is, SmoC drives cells to the low response regardless of Hh levels. This mimics cos mutants and is suppressed by 50% increase in cos+. From this, it is concluded that low Hh instructs Smo to inactivate Cos, by a mechanism that may involve stoichiometric interaction between Cos and the Smo cytoplasmic tail. (4) Chimeras where the extracellular CRD and TM domains are mismatched fail to exhibit any activity. From this, it is concluded that these two domains act as an integrated functional unit. This leads to a model for signaling where Fz or Smo can adopt three distinct states, regulating two distinct activities and translating different levels of ligand into distinct responses. Many physical models are consistent with these genetic analyses (Hooper, 2003).

Two mutant forms of Smo have been identified that regulate downstream signaling through different activities. These mutant forms of Smo mimic phenotypes of mutants in other components of the Hh pathway, as well as normal responses to different levels of Hh. These data suggest a model where Smo can adopt three distinct states that instruct three distinct states of the Ci regulatory complex. The model further suggests that Smo regulates Ci through direct interactions between Fu, Cos and the cytoplasmic tail of Smo. This is consistent with the failure of numerous genetic screens to identify additional signaling intermediates, and with the exquisite sensitivity of low signaling to Cos dosage (Hooper, 2003).

The model proposes that Smo can adopt three states, a decision normally dictated by Hh, via Ptc. The Ci regulatory complex, which includes full-length Ci, Cos and Fu, likewise can adopt three states. (1) In the absence of Hh Smo is OFF. Its cytoplasmic aspect is unavailable for signaling. The Cos/Fu/Ci regulatory complex is anchored to microtubules and promotes efficient processing of Ci155 to CiR. (2) Low levels of Hh expose Cos inhibitory sites in the cytoplasmic tail of Smo. Cos interaction with these sites drives the Ci regulatory complex into the low state, which recruits Su(fu) and makes little CiR or CiA. (3) High levels of Hh drive a major change in Smo, possibly dimerization. This allows the cytoplasmic tails of Smo to cooperatively activate Fu and Cos. Fu* and Cos* (* indicates the activated state) then cooperate to inactivate Su(fu), to block CiR production, and to produce CiA at the expense of Ci155 (Hooper, 2003).

The OFF state is normally found deep in the anterior compartment where cells express no Hh target genes (except basal levels of Ptc). In this state, the Ci regulatory complex consists of Fu/Cos/Ci155. Cos and Fu contribute to efficient processing of Ci155 to the repressor form, CiR, presumably because the complex promotes access of PKA and the processing machinery to Ci155, correlating with microtubule binding of the complex. This state is universal in hh or smo mutants, indicating that intracellular responses to Hh cannot be activated without Smo. Therefore Smo can adopt an OFF state where it exerts no influence on downstream signaling components and the OFF state of the Ci regulatory complex is its default state (Hooper, 2003).

The low state is normally found approximately five to seven cells from the compartment border, where cells are exposed to lower levels of Hh. These cells express Iro, moderate levels of dpp, no Collier and basal levels of Ptc. They accumulate Ci155, indicating that little CiA or CiR is made. Ci155 can enter nuclei but is insufficient to activate high responses. The physical state of the Ci regulatory complex in the low state has not been investigated. Cells take on the low state regardless of Hh levels when Ci is absent or when SmoC is expressed, and are strongly biased towards that state in fu(classII); Su(fu) double mutants. This state normally requires input from Smo, which becomes constitutive in the presence of SmoC. Because SmoC drives only low responses and cannot activate high responses, this identifies a low state of Smo that is distinct from both OFF and high. It is proposed that the low state is normally achieved when Smo inactivates Cos, perhaps by direct binding of Cos to Smo and dissociation of Cos from Ci155. Neither CiR nor CiA is made efficiently, and target gene expression is similar to that of ci null mutants (Hooper, 2003).

The high state is normally found in the two or three cells immediately adjacent to the compartment border where there are high levels of Hh. These cells express En, Collier, high levels of Ptc and moderate levels of Dpp. They make CiA rather than CiR, and Ci155 can enter nuclei. In this state a cytoplasmic Ci regulatory complex consists of phosphorylated Cos, phosphorylated Fu, Ci155 and Su(fu). Dissociation of Ci from the complex may not precede nuclear entry, since Cos, Fu, and Sufu are all detected in nuclei along with Ci155. Sufu favors the low state, whereas Cos and Fu cooperate to allow the high state by repressing Sufu, and also by a process independent of Sufu. This high state is the universal state in ptc mutants and requires input from Smo. As this state is specifically lost in fu mutants, Fu may be a primary target through which Smo activates the high state. SSF specifically interferes with the high state by a mechanism that is most sensitive to dosage of Smo. This suggests SSF interferes with the high activity of Smo itself. It is suggested that dimeric/oligomeric Smo is necessary for the high state, and that Smo:SSF dimers are non-productive. Cooperation between Smo cytoplasmic tails activates Fu and thence Cos. The activities of the resulting Fu* and Cos* are entirely different from their activities in the OFF state, and mediate downstream effects on Sufu and Ci (Hooper, 2003).

The cytoplasmic tail of Smo is sufficient to activate all Hh responses, and its activity is regulated through the extracellular and TM domains. This is exemplified by the FFS chimera, which retains the full range of Smo activities, but is regulated by Wg rather than Hh. The extracellular and transmembrane domains act as an integrated unit to activate the cytoplasmic tail, since all chimeras interrupting this unit fail to activate any Hh responses, despite expression levels and subcellular localization similar to those of active SSF or FFS. As is true of other serpentine receptors, a global rearrangement of the TM helices is likely to expose 'active' (Cos regulatory?) sites on the cytoplasmic face of Smo. The extracellular domain of Smo must stabilize this conformation and Ptc must destabilize it. But how? Ptc may regulate Smo through export of a small molecule, which inhibits Smo when presented at its extracellular face. Hh binding to Ptc stimulates its endocytosis and degradation, leaving Smo behind at the cell surface. Thus, Hh would separate the source of the inhibitor (Ptc) from Smo, allowing Smo to adopt the low state. Transition from low to high might require Smo hyperphosphorylation. The high state, which is likely to involve Smo oligomers, might be favored by cell surface accumulation if aggregation begins at some threshold concentration of low Smo. Alternatively, these biochemical changes may all be unnecessary for either the low or high states of Smo (Hooper, 2003).

Cubitus interruptus is targeted by Shaggy

The Drosophila protein Shaggy (Sgg, also known as Zeste-white3, Zw3) and its vertebrate ortholog glycogen synthase kinase 3 (GSK3) are inhibitory components of the Wingless (Wg) and Wnt pathways. Sgg is also a negative regulator in the Hedgehog (Hh) pathway. In Drosophila, Hh acts both by blocking the proteolytic processing of full-length Cubitus interruptus, Ci (Ci155), to generate a truncated repressor form(Ci75), and by stimulating the activity of accumulated Ci155. Loss of sgg gene function results in a cell-autonomous accumulation of high levels of Ci155 and the ectopic expression of Hh-responsive genes, including decapentaplegic and wg. Simultaneous removal of sgg and Suppressor of fused, Su(fu), results in wing duplications similar to those caused by ectopic Hh signaling. Ci is phosphorylated by GSK3 after a primed phosphorylation by protein kinase A (PKA), and mutating GSK3 phosphorylation sites in Ci blocks its processing and prevents the production of the repressor form. It is proposed that Sgg/GSK3 acts in conjunction with PKA to cause hyperphosphorylation of Ci, which targets Ci for proteolytic processing, and that Hh opposes Ci proteolysis by promoting its dephosphorylation (Jia, 2002).

During Drosophila limb development, posterior (P)-compartment cells express and secrete Hh that induces adjacent anterior (A)-compartment cells to express target genes including dpp, wg (leg only) and patched (ptc) by regulating the transcription factor Ci. In A-compartment cells distant from the AP compartment boundary, Ci is processed to generate a truncated repressor form (Ci75) that represses a subset of Hh-responsive genes including dpp. In A-compartment cells adjacent to the AP compartment border, Hh signaling blocks Ci processing to generate Ci75, and causes the accumulation of full-length Ci (Ci155). In addition, high levels of Hh stimulate a distinct transcriptional activation activity of Ci155, which is required for the expression of Hh-responsive genes such as ptc (Jia, 2002 and references therein).

In both wing and leg discs, loss of sgg function in the A compartment either by using sgg mutations or by overexpressing a dominant negative form of GSK3 (DN-GSK3) causes the accumulation of high levels of Ci155 in a cell-autonomous fashion without affecting ci-lacZ expression. In wing discs, anterior sgg- cells or DN-GSK3-expressing cells located outside the wing pouch region ectopically express dpp, which is repressed by Ci75. However, anterior sgg- cells do not ectopically activate ptc, which is activated by Ci155. In leg discs, anterodorsal sgg- cells distant from the AP boundary ectopically express wg and low levels of dpp, a phenotype similar to that associated with sgg PKA double-mutant cells in which both Wg and Hh signaling pathways are ectopically activated. As in the case of wing discs, sgg- cells seem to transduce low levels of Hh signaling activity, because wg is not fully activated, and little, if any, ptc is expressed. One hypothesis that accounts for these observations is that loss of sgg function affects Ci processing to generate Ci75 but does not stimulate the activity of Ci155 (Jia, 2002).

The activity of Ci155 is regulated by several mechanisms including attenuation by Su(fu). Whereas loss of Su(fu) function does not cause any significant phenotypes, it dramatically enhances sgg- phenotypes. For example, anterior sgg;Su(fu) double-mutant clones organize wing duplication, whereas sgg single-mutant clones form only small outgrowths. In wing discs, anterior sgg;Su(fu) double-mutant cells activate low levels of ptc, which is not ectopically expressed in sgg single-mutant cells. In leg discs, anterior sgg;Su(fu) double-mutant cells express ptc and high levels of wg. Hence, Ci155 accumulated in sgg- cells is largely inactive and its activity is stimulated by removal of Su(fu) function (Jia, 2002).

Ectopic activation of the Wg pathway by overexpression of a constitutively active form of Armadillo (DeltaArm) does not cause the accumulation of high levels of Ci155 or activate any Hh-responsive genes. Hence, the constitutive Hh signaling activity in sgg- cells is not secondary to aberrant activation of the Wg pathway in these cells. Hh induces stabilization of Smoothened (Smo), a seven-transmembrane protein that transduces the Hh signal, but anterior sgg- cells do not stabilize Smo. In addition, sgg;smo double-mutant cells, like sgg single-mutant cells, accumulate high levels of Ci155, suggesting that Sgg acts downstream of Smo to regulate Ci processing (Jia, 2002).

The proteolytic processing of Ci requires the activities of several intracellular Hh signaling components, including PKA and the kinesin-related protein Costal2 (Cos2). Overexpressing either Cos2 or a constitutively active form of PKA (mC*) blocks the accumulation of Ci155 induced by Hh. In contrast, wing discs overexpressing mC* or Cos2 accumulate high levels of Ci155 after treatment with 50 mMLiCl, a specific inhibitor of GSK3 kinase activity. These observations suggest that Sgg acts downstream of, or in parallel with, PKA and Cos2 to regulate Ci processing (Jia, 2002).

PKA promotes Ci processing by directly phosphorylating it at multiple sites in its carboxy-terminal region. Whether Sgg/GSK3 also regulates Ci processing by direct phosphorylation was also investigated. The canonical GSK3-phosphorylation site consists of two Ser/Thr residues separated by three amino acids: (Ser/Thr 0)-X-X-X-(Ser/Thr +4). Phosphorylation of Ser/Thr at the +4 position by a priming kinase allows GSK3 to phosphorylate Ser/Thr at the 0 position. Examination of Ci sequence reveals three GSK3 consensus sites (Ser 852, Ser 884 and Ser 888) adjacent to two previously identified PKA phosphorylation sites (Ser 856 and Ser 892). To determine whether Ci can be a direct substrate of Sgg/GSK3, an in vitro kinase assay was carried out. Three glutathione S-transferase (GST)-Ci fusion proteins containing Ci fragments from amino acids 441-1,065 were generated: GST-Ci contains wild-type sequence; GST-Ci-3P has three PKA sites mutated (S838A, S856A and S892A); and GST-Cim3 has three GSK3 consensus sites mutated (S852A, S884A and S888A). GST-Ci is specifically phosphorylated by PKA but not by GSK3 without prior PKA treatment; however, it becomes a good substrate for GSK3 after primed phosphorylation by PKA. In contrast, neither GST-Ci-3P nor GST-Cim3 can be phosphorylated by GSK3 even after PKA treatment. These results suggest that primed phosphorylation by PKA at Ser 856 and Ser 892 allows GSK3 to phosphorylate Ci at Ser 852, Ser 884 and Ser 888 (Jia, 2002).

To determine whether Sgg/GSK3 phosphorylates Ci in vivo, Ci phosphorylation was examined in cl-8 cells treated with or without LiCl, which specifically blocks GSK3 kinase activity. Treating cl-8 cells with both a proteasome inhibitor, MG132, and a phosphatase inhibitor, okadaic acid (OA), results in the accumulation of hyperphosphorylated forms of Ci155, which exhibit much slower electrophoretic mobility on SDS polyacrylamide gel than unphosphorylated or hypophosphorylated forms. Treating cells with MG132 and OA in the presence of 50 mMLiCl results in hypophosphorylation of Ci because it eliminates the slowest-migrating forms of Ci155. These observations suggest that inhibition of Sgg/GSK3 kinase activity affects Ci phosphorylation in intact cells. The residual phosphorylation of Ci155 in the presence of LiCl is probably due to phosphorylation by PKA because LiCl does not inhibit PKA kinase activity (Jia, 2002).

If Ci processing is regulated by GSK3 phosphorylation, one would predict that mutating GSK3-phosphorylation sites in Ci should block its processing to generate the Ci75 repressor. To test this, UAS transgenes were generated containing hemagglutinin (HA)-tagged wild-type (UAS-HA-Ci) or mutant Ci (UAS-HA-Cim3); these were expressed in wing discs using the Gal4/UAS system. Whereas HA-Ci is partially processed into Ci75, HA-Cim3 does not give rise to detectable Ci75. The effect on the production of Ci75 of mutating GSK3-phosphorylation sites was also examined using an in vivo function assay. Either wild-type or mutant Ci was ectopically expressed in the P-compartment of wing discs carrying smo- clones, and hh-lacZ expression, which is inhibited by Ci75, was examined. P-compartment smo- cells expressing HA-Ci block hh-lacZ expression, indicating that wild-type Ci is processed to generate Ci75 in the absence of Hh signaling. In contrast, P-compartment smo- cells expressing HA-Cim3 do not repress hh-lacZ expression, indicating that HA-Cim3 does not produce Ci75 in vivo. Hence, mutating GSK3-phosphorylation sites in Ci affects its proteolytic processing to generate the repressor form (Jia, 2002).

To assess the importance of individual GSK3-phosphorylation sites for Ci processing, two additional mutant forms of Ci (HA-Cim1 and HA-Cim2) were examined using the in vivo function assay. P-compartment smo- cells expressing HA-Cim2 partially block hh-lacZ expression, suggesting that lack of phosphorylation at Ser 884 and Ser 888 attenuates Ci processing. P-compartment smo- cells expressing HA-Cim1 also partially inhibit hh-lacZ expression, however, to a lesser extent than those expressing HA-Cim2, suggesting that mutating Ser 852 greatly impedes, although does not completely abolish, Ci processing. Hence, efficient processing of Ci seems to require phosphorylation at all three GSK3 sites (Jia, 2002).

Taken together, these data suggest that Sgg/GSK3 acts in conjunction with PKA to promote hyperphosphorylation of Ci, which is essential for efficient Ci processing to generate its repressor form. The requirement for multiple phosphorylation seems to be a general mechanism to regulate proteolysis of regulatory proteins. The involvement of multiple phosphorylation may provide a way for Hh to differentially regulate Ci by controlling its level of phosphorylation. For example, low levels of Hh may cause partial dephosphorylation of Ci by opposing Sgg, resulting in an inhibition of Ci processing to generate Ci75 but leaving Ci155 inactive. In contrast, high levels of Hh may cause complete dephosphorylation of Ci by opposing PKA, which not only blocks Ci processing but also stimulates the activity of accumulated Ci155 (Jia, 2002).

GSK3 is involved in multiple signaling pathways, raising the question of how its activity is selectively regulated by individual pathways. An emerging theme is that GSK3 is present, together with its substrates, in distinct complexes that are regulated by different upstream stimuli. Future study will determine whether Sgg/GSK3 forms a complex with Cos2 or Ci and whether Hh regulates Sgg/ GSK3 within the complex. In vertebrates, three Gli proteins (Gli1, Gli2 and Gli3) are implicated in transducing Hh signals. Interestingly, all three Gli proteins contain multiple GSK3-phosphorylation consensus sites adjacent to PKA sites, raising the possibility that GSK3 may regulate Gli proteins in vertebrate Hh pathways. Hh and Wnt signaling pathways act in synergy in certain developmental contexts. The finding that GSK3 is involved in both Hh and Wnt pathways raises the possibility that these two pathways might converge at GSK3 in certain developmental processes (Jia, 2002).

PP4 and PP2A regulate Hedgehog signaling by controlling Smo and Ci phosphorylation

The seven-transmembrane protein Smoothened (Smo) and Zn-finger transcription factor Ci/Gli are crucial components in Hedgehog signal transduction that mediates a variety of processes in animal development. In Drosophila, multiple kinases have been identified to regulate Hh signaling by phosphorylating Smo and Ci; however, the phosphatase(s) involved remain obscured. Using an in vivo RNAi screen, PP4 and PP2A were identified as phosphatases that influence Hh signaling by regulating Smo and Ci, respectively. RNAi knockdown of PP4, but not of PP2A, elevates Smo phosphorylation and accumulation, leading to increased Hh signaling activity. Deletion of a PP4-interaction domain (amino acids 626-678) in Smo promotes Smo phosphorylation and signaling activity. It was further found that PP4 regulates the Hh-induced Smo cell-surface accumulation. Mechanistically, it was shown that Hh downregulates Smo-PP4 interaction that is mediated by Cos2. Evidence is provided that PP2A is a Ci phosphatase. Inactivating PP2A regulatory subunit Widerborst (Wdb) by RNAi or by loss-of-function mutation downregulates, whereas overexpressing regulatory subunit upregulates, the level and thus signaling activity of full-length Ci. Furthermore, Wdb counteracts kinases to prevent Ci phosphorylation. Finally, evidence was obtained that Wdb attenuates Ci processing probably by dephosphorylating Ci. Taken together, these results suggest that PP4 and PP2A are two phosphatases that act at different positions of the Hh signaling cascade (Jia, 2009).

The screen used to identify the phosphatases differs from previous screens because an in vivo assay was used to examine Smo expression levels, which is a more direct readout, and because knockdown of specific phosphatase gene(s) involved in Smo dephosphorylation might not affect the pathway activity in a significant way and such gene(s) could have been missed in the previous RNAi screens with cultured cells. This study identified PP4 as a novel Hh signaling component that regulates Smo phosphorylation. The study provides the first evidence for the physiological Smo and Ci phosphatases, and uncovers the underlying mechanism of Smo regulation by phosphatase (Jia, 2009).

This study identified PP4 and PP2A to be negative and positive regulators in the Hh pathway, and it was shown that they exert their roles through Smo and Ci, respectively. Are PP4 and PP2A the only phosphatases in the Hh pathway? Although the data suggest that PP4 is a phosphatase for Smo, the possibility of the involvement of other phosphatase(s) cannot be excluded. Hh induces extensive Smo phosphorylation at numerous Ser/Thr sites, and multiple kinases are involved in these phosphorylation events. It might be possible that multiple phosphatases could be involved. In addition, loss-of-function studies on PP2A regulating Ci are not based on null mutations. This was due to the fact that genetic null mutations of the catalytic and regulatory subunits cause cell lethality. Thus, the results might not be exclusive (Jia, 2009).

Removal of PP4 by RNAi in wing discs induced Smo accumulation in A-compartment cells both near and away from the AP boundary. In addition, PP4 RNAi induced the elevation and anterior expansion of Hh target gene expression. However, the accumulated Smo caused by PP4 RNAi did not ectopically activate Hh target genes in cells away from the AP boundary. In addition, although Smo phosphorylation was potentiated by knocking down PP4 or abolishing Smo-PP4 interaction, the elevated phosphorylation did not suffice to promote Smo cell-surface accumulation. These data suggest that the basal phosphorylation of Smo regulated by PP4 is not sufficient to activate Smo, and that de novo Smo activation still depends on Hh (Jia, 2009).

Previous studies have shown that PKA and CK1 are required for Hh-induced Smo accumulation and signaling activity. Phosphorylation-deficient forms of Smo (with PKA or CK1 sites mutated to Ala) are defective in Hh signaling, whereas SmoSD123, the phosphorylation-mimicking Smo, has potent signaling activity and high level of cell-surface accumulation. Thus, the PKA and CK1 sites are apparently crucial in mediating Smo phosphorylation and activation. Hh treatment may cause increased phosphorylation at these sites. In addition to PKA and CK1 sites, there are many other Ser/Thr residues that are phosphorylated upon Hh stimulation. Although phosphorylation-mimicking mutations at these sites alone did not have discernible effect on Smo, their phosphorylation could modulate the cell-surface accumulation and activity of Smo phosphorylated at the three PKA/CK1 sites, which may at least in part explain why cell-surface accumulation and activity of SmoSD123 is still regulated by Hh. This study found that removing PP4 alone promoted Smo phosphorylation but did not elevate the cell-surface accumulation of Smo. It is possible that high levels of basal Smo phosphorylation in the absence of PP4 do not reach the threshold for promoting Smo cell-surface accumulation. It is also possible that basal Smo phosphorylation mainly occurs at sites other than the crucial PKA/CK1 phosphorylation clusters. In support of this notion, it was found that knockdown PP4 by RNAi promoted SmoSD123 to further accumulate on the cell surface in the absence of Hh (Jia, 2009).

How is Smo phosphorylation regulated? Hh may regulate Smo phosphorylation by regulating the accessibility of its kinase and/or phosphatase. In this study, it was found that Smo interacts with PP4 through amino acids 626-678, a region previously mapped to be a Cos2-interacting domain. It was further found that Smo-PP4 association diminished when Cos2 was knocked down by RNAi. A previous study revealed that Cos2 impedes Hh-induced Smo phosphorylation by interacting with amino acids 626-678 of Smo and Hh-induced phosphorylation of Cos2 at Ser572 dissociates Cos2 from amino acids 626-678 of Smo, thereby alleviating its inhibition on Smo phosphorylation. This study found that Cos2 inhibits Smo phosphorylation by recruiting PP4 and Hh promotes Smo phosphorylation by preventing Cos2-PP4 complex from binding to amino acids 626-678 of Smo. SmoDelta626-678, when not interacting with PP4, could still interact with Cos2 via a Cos2-interaction domain near the Smo C terminus. The Cos2-binding Smo C terminus might not recruit PP4. Taken together, these findings suggest that Hh may promote Smo phosphorylation at least in part by reducing the accessibility of a phosphatase (Jia, 2009).

Phosphorylation of Ci/Gli controls the balance of its activator and repressor activity. This study has demonstrate a role of PP2A in dephosphorylating Ci and attenuating Ci processing. However, it is not known whether Hh regulates PP2A to dephosphorylate Ci. Previous studies have shown that Hh interferes with the Cos2-Ci-kinase protein complex. It is possibly that Hh also regulates Ci phosphatase, or the accessibility of the phosphatase. Future studies should determine whether PP2A interacts with Cos2-Ci and whether such interaction is regulated by Hh (Jia, 2009).

Many aspects of Smo and Ci/Gli regulation are conserved across species. For example, both Drosophila and mammalian Smo proteins undergo a conformational switch in response to Hh stimulation. Ci/Gli proteolysis is mediated by the same set of kinases and E3 ubiquitin ligases. In addition, it has been shown that PP2A is involved in vertebrate Hh signaling, probably by regulating Gli nuclear localization and activity. Therefore, it would be interesting to determine whether PP4 and PP2A play similar roles in regulating phosphorylation of vertebrate Smo and Gli (Jia, 2009).

Proteolysis of the Hedgehog signaling effector Cubitus interruptus requires phosphorylation by Glycogen Synthase Kinase 3 and Casein Kinase 1.

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 Shaggy/Glycogen synthase kinase 3 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).

How extensively must Ci be phosphorylated in order to be proteolyzed? Whether each potential PKA, GSK3, or CK1 phosphoacceptor site is essential for Ci proteolysis has not been tested in flies. Evidence obtained in tissue culture cell studies suggests that Ci proteolysis is largely inhibited by alteration of single PKA sites, and at least one PKA site (site 1) is critical in flies. Alteration of two GSK3 sites, adjacent to PKA sites 2 and 3, prevents Ci-155 proteolysis. Hence, the view is favored that each of the phosphorylation sites in this region of Ci contributes significantly to Ci-155 proteolysis (Price, 2002).

Inhibition of the 26S proteosome in clone 8 tissue culture cells leads to the accumulation of highly phosphorylated full-length Ci forms of lowered gel mobility, especially if phosphatase activity is also inhibited. Ci-155 from untreated clone 8 cells can be separated into about six isoforms by isoelectric focusing. The location of phosphorylated residues was not mapped in either of these studies. However, it appears that Ci phosphorylated on a small number of sites, perhaps largely PKA sites, is stable enough to visualize, whereas subsequent, perhaps cooperative, phosphorylation, most likely on adjacent GSK3 and CK1 sites, leads to rapid Ci-155 proteolysis and is therefore evident only if proteolysis is artificially inhibited (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).

Ci phosphorylation might create a binding site for a Ci partner other than Slimb. The apparent requirement for extensive phosphorylation of Ci could easily be rationalized if the binding partner presented an extensive surface for electrostatic interaction, such as the armadillo repeat region of ß-catenin. The binding of this ß-catenin domain to repeated serine/threonine-rich motifs of APC (Adenomatous Polyposis Coli) protein is stimulated by phosphorylation of APC. Thus, structures analogous to the ß-catenin armadillo repeats might accommodate phosphorylation-dependent binding of repeated motifs in Ci. Binding of ß-catenin itself to phosphorylated Ci would, of course, provide a high-affinity Slimb binding partner within the Ci complex and could explain the observation that Ci degradation is proteosome dependent but does not involve detectable ubiquitination of Ci (Price, 2002).

Loss of Sgg activity in wing disc clones induces Ci-155 to levels at least as high as the A/P border, but slightly lower than in PKA mutant clones. It is inferred that elevated Ci-155 levels result from inhibition of proteolysis to Ci-75 because ci RNA levels were unchanged and because Ci-75 repressor activity was largely absent from posterior smo sgg mutant clones in wing discs expressing Ci ubiquitously. In these clones there appears to be a low level of repressor, raising the possibility of a second GSK3 contributing in a minor way to the phosphorylation and proteolysis of Ci (Price, 2002).

Anterior sgg mutant clones induce some ectopic expression of Hh target genes but do not reproduce the strong phenotypes of PKA mutant clones, as assessed by Hh target gene expression, disc morphology, and adult morphology. Two possible explanations for the differences between PKA and sgg mutant clone phenotypes are offered. One possibility is that Ci lacking PKA site phosphorylation (and hence GSK3 and CK1 site phosphoserines) is more active than Ci lacking only GSK3 site phosphorylation. No significant differences have been found in the activity of Ci lacking three PKA sites (Ci3m), as compared to Ci lacking both GSK3 sites (CiNm) for the transgenic lines tested in wing discs or embryos. However, it was found that Ci lacking five consensus PKA sites (Ci5m) is more active than Ci3m. The fourth and fifth PKA sites are not flanked by GSK3 or CK1 sites. The phosphorylation of these PKA sites might therefore reduce the activity of Ci in sgg mutant clones relative to PKA mutant clones (Price, 2002).

A second possibility is that sgg mutations may prevent Hh target gene expression despite generating a phosphoform of Ci that is adequately activated and protected from proteolysis. This hypothesis was investigated by examining the phenotype of clones lacking both PKA and Sgg activities. Since GSK3 phosphorylation of Ci depends on priming by PKA phosphorylation, the absence of Sgg activity should not alter the phosphorylation state of Ci from that in PKA mutant clones. Nevertheless, the levels of Ptc protein induced in sgg PKA smo mutant clones are much lower than for PKA smo mutant clones in the wing pouch cells of the disc, and pattern duplication of wings normally associated with PKA mutant clones is suppressed by the additional inactivation of sgg. Thus, it is possible that a Sgg substrate in addition to Ci can affect Hh target gene expression, at least in anterior presumptive wing cells. This might contribute to the failure of sgg mutant clones in the wing pouch to induce ectopic Ptc expression (Price, 2002).

It is important to note that the positive input of Sgg on Hh target gene expression inferred above is evident only in the artificial circumstances of eliminating PKA and Sgg activities by genetic mutation. PKA and Sgg are normally active in anterior cells away from the A/P border, as manifested by the proteolysis of Ci-155, and any regulation of their activities by Hh at the A/P border is unlikely to be as dramatic as mutational inactivation. Indeed, there was no consistent reduction in the expression of ptc or dpp reporters in sgg mutant clones at the A/P border. Hence, the relevance of Sgg substrates other than Ci during normal development remains to be established (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).

Even though reduced GSK3 activity can stabilize Ci-155 and ß-catenin in wing discs, in wild-type discs, Ci-155 levels are not elevated in cells where Wg signals and ß-catenin is not stabilized by Hh signaling. These observations are reminiscent of the independent transmission of insulin and Wnt signals in vertebrate cells. Insulin stimulation leads to inactivation of GSK3 by phosphorylation at a specific PKB site, but GSK3 in complex with Wnt pathway components is spared from phosphorylation. Wnt signaling does not inactivate GSK3 by the same phosphorylation, although some reduction in total cellular GSK3 activity can be measured and Wnt signaling does reduce the phosphorylation of specific Wnt pathway components by GSK3. The relevant substrates for GSK3 in the insulin pathway are primed by prior phosphorylation, as is the case for Ci; however, axin, APC, and ß-catenin GSK3 sites do not appear to depend on priming. Thus, a combination of sequestration of GSK3 subpopulations through binding interactions and the use of different substrate sites insulate the Wnt pathway from the insulin pathway and may similarly segregate Wnt and Hh signaling pathways despite the common use of GSK3 (Price, 2002).

The involvement of GSK3 and CK1 in Ci-155 proteolysis raises the exciting possibility that Hh might regulate the activity of one or both of these kinases. Regulation of GSK3 activity is a particularly appealing mechanism because the key regulatory event in Wnt signaling is generally thought to be the inhibition of GSK3 activity. The mechanism by which Wnt signaling regulates GSK3 is still not clear but appears to involve several ancillary proteins, other kinases, and a phosphatase. Thus, similar complexity might be anticipated in the Hh signaling pathway and perhaps the participation of yet more proteins previously known for their involvement in Wnt signaling (Price, 2002).

Distinct protein degradation mechanisms mediated by Cul1 and Cul3 controlling Ci stability in Drosophila eye development

Cullins are the major components of a series of multimeric ubiquitin ligases that control the degradation of a broad range of proteins. The ubiquitin-like protein, Nedd8, covalently modifies members of the Cullin family. Nedd8 modifies Cullin 1 (Cul1, also known as Lin-19-like or simply Lin-19) in Drosophila. In mutants of Drosophila Nedd8 and Cul1, levels of the signal transduction effectors, Cubitus interruptus (Ci) and Armadillo, and the cell cycle regulator, Cyclin E (CycE), are unusually high, suggesting that the Cul1-based multimeric SCF ubiquitin ligase complex requires Nedd8 modification for the degradation processes of Ci, Arm, and CycE in vivo. Two distinct degradation mechanisms modulating Ci stability in the developing eye disc are separated by the morphogenetic furrow (MF) in which retinal differentiation is initiated. In cells anterior to the MF, Ci proteolytic processing promoted by PKA requires the activity of the Nedd8-modified Cul1-based SCFSlimb complex. In posterior cells, Ci degradation is controlled by a mechanism that requires the activity of Cul3, another member of the Cullin family. This posterior Ci degradation mechanism, which partially requires Nedd8 modification, is activated by Hedgehog (Hh) signaling and is PKA-independent (Ou, 2002).

The Hh pathway controls growth and pattern formation in many developmental processes in both vertebrates and invertebrates. The Hh signal is transmitted through a receptor complex consisting of Patched (Ptc) and Smoothened (Smo). In the absence of Hh, Ptc inhibits Smo activity, and the effector Cubitus interruptus (Ci) is phosphorylated by PKA, leading to the proteolysis of Ci, which is converted into Ci75 with the C terminus truncated. Ci75 functions as a transcriptional repressor in the Hh signaling pathway. Upon binding to Ptc, Hh relieves Smo from its repression state. Activated Smo mediates signaling to prohibit proteolytic processing of Ci. The intact full-length Ci (CiFL) functions as a transcriptional activator for expression of target genes of the Hh pathway (Ou, 2002).

In Drosophila, Hh signaling functions in patterning the A/P compartments in developing tissues such as embryonic segments and wing and leg imaginal discs. In development of the eye imaginal disc, Hh signaling is a major driving force of the retinal differentiation wave, the morphogenetic furrow (MF), which is caused by transient constriction in cell apical surface. The MF progresses anteriorly from the posterior margin of the eye disc during the third instar larval and early pupal stages. Anterior to the advancing MF, cells are proliferating, whereas posterior cells differentiate sequentially into photoreceptor, cone, or pigment cells. Transduction of Hh signaling in the MF is revealed by the accumulation of CiFL, which activates expression of target genes such as dpp and atonal in the MF. The induced MF cells soon differentiate and produce Hh proteins for further induction of more anterior cells, thus making the MF move forward (Ou, 2002).

The effect of neddylation on a broad spectrum of E3 ligases remains largely unknown. To investigate the role of neddylation in protein degradation control during developmental processes, Nedd8 and Cul1 mutants were identified and analyzed in Drosophila. The results suggest that neddylation is required for Cul1-mediated protein downregulation of the signaling pathway effectors Ci and Armadillo (Arm) and the cell cycle regulator CycE. Using the developing eye disc as a model system to study the regulation of CiFL stability, it was found that there is mechanistic difference in controlling CiFL stability between anterior and posterior cells separated by the MF. Whereas the Cul1-based SCFSlimb complex controls CiFL stability in anterior cells, a Cul3-dependent protein degradation mechanism controls CiFL stability in posterior cells. The differences between these two protein degradation mechanisms were further investigated (Ou, 2002).

In anterior cells of developing discs, CiFL proteolytic processing requires the activity of the Nedd8-modified, Cul1-based SCFSlimb complex. This CiFL proteolytic processing is inhibited by Smo signaling and promoted by PKA phosphorylation on CiFL. The mechanism by which CiFL is proteolyzed from CiFL to Ci75 is not clear. It is proposed that Nedd8 modifies and activates SCFSlimb for Ci ubiquitination and then proteolysis, as evidenced by Cul1 modification by Nedd8 and CiFL accumulation in Nedd8, Cul1, and slimb mutants. Consistently, proteolysis of CiFL depends on 26S proteasome activity. However, ubiquitinated Ci is not detected in cells treated with 26S proteasome inhibitors (Ou, 2002).

In the Hh signaling pathway, it is not clear how Smo signaling prevents CiFL from proteolysis. According to double mutant analysis, Nedd8 could be downstream or parallel to Smo and PKA signaling. Thus, it is possible that Hh signaling prevents CiFL from proteolysis through downregulating the level of Nedd8-modified Cul1. However, no change in the level of Nedd8-modified Cul1 could be detected in cell extracts prepared from the eye discs with ectopic Hh expression. It is therefore inferred that Hh may affect CiFL proteolysis through a Nedd8-independent mechanism (Ou, 2002).

Two modes of Ci downregulation in Drosophila eye development are proposed. In the undifferentiated cells anterior to the MF, Ci is phosphorylated by PKA constantly and processed by an SCFSlimb-dependent mechanism to generate the repressor form of Ci75. Upon binding to Hh, cells in the MF transduce Smo signaling to prevent this proteolytic processing. Thus, the transcriptional activator CiFL is preserved for activation of downstream genes in the MF (Ou, 2002).

In the posterior cells that are undergoing differentiation, a novel mechanism controls Ci degradation. Mutant analyses suggest that this mechanism is comprised of Smo signaling, Nedd8 modification, and Cul3 activity. The effect of Smo signaling in promoting Ci degradation in the posterior cells is in contrast to its effect on the anterior cells, in which Smo signaling prohibits CiFL processing. In addition to Smo signaling, Nedd8 modification activity also participates in this posterior Ci degradation. Further Cul1 mutant analysis suggests that Cullin proteins other than Cul1 are likely involved in this posterior degradation mechanism. This hypothesis has led to the identification of Cul3 as one candidate functioning in Ci degradation. More surprisingly, Cul3 activity is very restricted; Cul3 controls Ci degradation in the posterior, but not anterior, cells of the eye disc. CiFL accumulation may have an impact on proper differentiation of the posterior cells. In Cul3 mutants, cone cell differentiation is affected, probably due to the accumulation of CiFL (Ou, 2002).

Furthermore, the Ci degradation process is also distinct in posterior cells; Ci degradation is independent of PKA phosphorylation and proteolytic processing to the short form Ci-75. Based on these results, it is proposed that Smo signaling, acting in concert with the Nedd8 pathway, activates a Cul3-based ubiquitin ligase to degrade Ci in a PKA-independent mechanism in posterior cells of the eye disc (Ou, 2002).

It is not clear how Nedd8 modifies Cul3 in flies. Strong genetic interaction is observed between Nedd8 and Cul3 during eye and antennal development, suggesting that Nedd8 may also regulate Cul3. However, depletion of Nedd8 activity affects only posterior cells abutting the MF, in contrast to depletion of Cul3 activity, which increases the CiFL level in all posterior clones, indicating that some Cul3 activity is Nedd8-independent. It is possible that a basal Cul3 activity for Ci degradation is further enhanced by Nedd8 modification near the MF in which accumulated Ci may require efficient degradation for cells to enter proper differentiation (Ou, 2002).

Different protein-protein interactions may result in a switch between two Ci degradation mechanisms in eye discs. Ci is known to interact with Cos2, Fu, and Su(fu) to comprise a protein complex that promotes Ci degradation. Cos2, a motor-like protein with a kinesin motif, is required for tethering Ci in the cytosolic compartment and Ci proteolytic processing in the Drosophila developing wing. Similarly, Fu, a serine/threonine kinase, is also required for Ci processing. However, in Su(fu) mutants, levels of both long and short forms of Ci are reduced, suggesting that Su(fu) plays an additional role in Ci protein stability. Interestingly, the role of Su(fu) in controlling Ci stability seems modulated by Hh signaling. The results in this study indicate that, in contrast to the effect of Hh signaling in the anterior cells, Hh signaling downregulates the Ci level in the posterior cells of the eye disc. It is possible that the Ci protein complex is modulated by the sweep of the MF, and this change requires Hh signaling to expose Ci to the Cul3-based protein degradation machinery. Alternatively, additional factors may be activated by the sweeping of the MF and be required for Hh signaling to induce Cul3 activity that leads to constitutive Ci degradation (Ou, 2002).

Nedd8, the ubiquitin-like protein that covalently modifies members of the Cullin family, is highly conserved from yeast to mammals. Several Nedd8 alleles have been identifed in Drosophila, including two null alleles Nedd8AN015 and Nedd8AN024. The Nedd8 null mutants were growth-arrested in the first-instar larval stage and died within several days without further growth. Mutant clones were generated to analyze Nedd8 loss-of-function phenotypes; in the adult flies very few Nedd8AN015 mutant cells are identified, while in control experiments, large Nedd8+ clones are frequently recovered. Nedd8 mutant clones of small size, however, are present in the developing discs, suggesting that Nedd8 mutant cells are defective in proliferation and survival (Ou, 2002).

To study the relationship between Nedd8 and the F-box protein Slimb-mediated protein degradation, the protein stability for substrates of Slimb was studied in Nedd8 mutant cells. Nedd8 mutant cells in developing wing discs accumulate high levels of full-length Ci (CiFL) and Arm proteins, phenotypes identical to those observed in the slimb mutants. In Drosophila embryonic development, the signaling pathway mediated by the NFkappaB homolog Dorsal is required for patterning the dorsoventral identity. Accumulation of pIkappaBa/Cactus inhibits Dorsal activation, leading to repression of the downstream target gene, twist, an effect that has been observed in slimb mutants. twist expression was examined in embryos laid by Nedd8AN015/Nedd8203 females in which Nedd8203 is a hypomorphic allele. In such embryos, the twist expression domain is reduced along the dorsoventral axis and often found missing in many cells, revealing a requirement for Nedd8 in Dorsal signaling (Ou, 2002).

The Drosophila eye imaginal disc is an excellent model system for developmental study. Cells are undifferentiated and dividing randomly anterior to the MF, and cells posterior to the MF are differentiating into different types of cells. Thus, Nedd8 phenotypes can be observed in cells of different differentiation states in a single eye disc. The Hh pathway is the major signaling pathway in eye development, and the protein level of its effector Ci is tightly regulated in Drosophila. These studies focused on how Nedd8 regulates the CiFL level in the Hh pathway and the effects of Ci upregulation on eye development. It was found that in the Nedd8 clones that located anterior to the MF, CiFL accumulates to a level identical to that in the MF cells that transduce the Hh signaling pathway. Accumulation of CiFL also exists in posterior mutant cells that locate proximally but not distally to the MF. CiFL accumulation in Nedd8 mutant cells is not caused by an increase in the ci transcription level, because expression of ci-lacZ that recapitulates endogenous ci expression remains constant in Nedd8 mutant cells, indicating that posttranscriptional defects resulted in CiFL accumulation (Ou, 2002).

Elevated CiFL levels causes anterior Nedd8 mutant cells to adopt MF fate precociously. Nedd8 mutant cells are constricted on their apical surface, as revealed by the intensified phalloidin staining, and express the Hh-target gene, dpp, as detected by the expression of dpp-lacZ reporter gene. Furthermore, the early photoreceptor marker, Atonal, is induced. These phenotypes are observed only in mutant cells abutting the MF anteriorly, suggesting that accumulated CiFL in Nedd8 mutant cells is able to respond to Hh signaling (Ou, 2002).

CiFL accumulation in Nedd8 cells results from a defect in the machinery controlling CiFL protein processing. Ci protein processing is known to depend on the phosphorylation status of CiFL by PKA. The level of CiFL is downregulated when PKA is constitutively activated by the expression of its catalytic subunit. Therefore, the functional relationship between PKA activity and Nedd8 modification was examined. When the UAS-C* transgene was driven by eq-GAL4 for misexpression in the equator region of the eye disc, as visualized by the coexpressed GFP, the level of CiFL in the equator region was reduced, consistent with the observations that PKA phosphorylates Ci and promotes Ci proteolysis. Nedd8 mutant clones were then generated in the equator region where PKA is constitutively activated. In Nedd8 clones that overlap the eq-GAL4 expression domain, CiFL accumulates to a high level, identical to the level in the Nedd8 clone located externally to the eq-GAL4 expression domain. These results indicate that CiFL downregulation by PKA activity requires Nedd8 activity, and the effect of the Nedd8 pathway on CiFL processing is unlikely to be mediated through modulation of PKA activity (Ou, 2002).

Ci downregulation in the posterior cells of the eye disc requires Smo signaling and Nedd8 modification activity; CiFL degradation is mediated by a Cul3-dependent mechanism

The finding that CiFL accumulates in posterior smo3 clones indicates that Smo signaling contributes to the downregulation of CiFL in the posterior cells of the eye disc. This effect is in contrast to the smo role in the MF, where smo is required for CiFL activation. CiFL accumulation was also observed in the posterior Nedd8 mutant clones located proximally to the MF. In the smo3 Nedd8 double mutant clones, the level of CiFL is further enhanced, even in clones located distally to the MF, whereas no CiFL accumulation is detected in Nedd8 or smo3 clones, suggesting that Nedd8 and Smo function partially redundantly to downregulate Ci stability in the posterior cells of the eye disc (Ou, 2002).

The involvement of Nedd8 in controlling CiFL levels in the posterior cells of the eye disc suggests that Cullin proteins other than Cul1 may be involved in the posterior mechanism to control Ci stability. Among the mammalian Cullin family, Cul3 shares with the Cul1-based SCF complex the substrate CycE. To test whether Cul3 affects CiFL degradation in the eye disc, the available Drosophila Cul3 mutants were analyzed. CiFL accumulates in Cul3 mutant clones located posterior to the MF, with a higher level in nondifferentiating cells that surround differentiating photoreceptor clusters. In contrast, no CiFL accumulation is detected in anterior Cul3 mutant clones, indicating that Cul3 controls CiFL protein stability only in the posterior cells of the eye disc. Ci accumulation in posterior Cul3 mutant cells is controlled at the posttranscriptional level because ci expression is normal, as revealed by in situ hybridization. These results show that the CiFL degradation machinery in the posterior cells of the eye disc requires a Cul-3-mediated degradation mechanism. Ci accumulation is also detected in Cul3 mutant cells located in the A/P boundary of the wing disc. The level of Arm in Cul3 mutant clones in wing discs and the level of CycE in Cul3 mutant clones in eye discs remain constant, suggesting that Cul3 activity is specific to Ci (Ou, 2002).

In contrast to the Cul1-based SCFSlimb complex that controls CiFL processing only in the anterior cells of the eye disc, the Cul3-mediated Ci degradation mechanism is specific to the posterior cells. These specific activities in controlling Ci protein stability are not caused by differential gene expression of Cul1 and Cul3 in the eye disc. Ubiquitous mRNA expression patterns of both Cul1 and Cul3, and ubiquitous Cul1 protein expression are found all along the eye disc, suggesting that control of specificity is mediated by mechanisms other than regulation of Cul1 and Cul3 expression (Ou, 2002).

PKA phosphorylation promotes CiFL processing, and plays a role in the Hh signaling pathway for Ci activation. The requirement of PKA in CiFL degradation in the posterior cells of the eye disc was examined; CiFL downregulation is not regulated by PKA activity. Proteolytic processing of CiFL to the short form Ci75 is not a prerequisite for complete degradation in the posterior cells, in contrast to the proteolytic processing of the phosphorylated CiFL to the short form Ci75 in the anterior cells. To sum up, the results suggest that in the posterior cells of the eye disc, CiFL is degraded constitutively, and this degradation process is independent of PKA phosphorylation (Ou, 2002).

Drosophila Roc1a encodes a RING-H2 protein with a unique function in processing the Hh signal transducer Ci by the SCF E3 ubiquitin ligase

Substrate specificity of SCF E3 ubiquitin ligases is thought to be determined by the F box protein subunit. Another component of SCF complexes is provided by members of the Roc1/Rbx1/Hrt1 gene family, which encode RING-H2 proteins. Drosophila contains three members of this gene family. This study shows that Roc1a mutant cells fail to proliferate. Further, while the F box protein Slimb is required for Cubitus interruptus (Ci) and Armadillo/β-catenin (Arm) proteolysis, Roc1a mutant cells hyperaccumulate Ci but not Arm. This suggests that Slimb and Roc1a function in the same SCF complex to target Ci but that a different RING-H2 protein acts with Slimb to target Arm. Consequently, the identity of the Roc subunit may contribute to the selection of substrates by metazoan SCF complexes (Noureddine, 2002).

This study shows that the Drosophila Roc1a, Roc1b, and Roc2 genes encode RING-H2 proteins that stimulate E1- and E2-dependent ubiquitination in vitro and perform nonredundant roles in vivo. Roc1a mutant cells fail to proliferate normally, and consequently, loss of Roc1a function is lethal. The mechanistic basis for this proliferation defect is not known. S. cerevisiae Rbx1/Hrt1 mutants arrest in G1 phase because of a failure to proteolytically destroy Sic1p, an inhibitor of the Clb5,6/CDC28p kinases that are required for S phase. In contrast, Drosophila cells that lack Roc1a function do not appear to arrest at a specific point in the cell cycle, as determined by FACS analysis of mutant imaginal cells. This suggests that the block to cell proliferation is not a consequence of the failure to degrade a single regulator that plays a key role in one cell cycle transition. There are likely to be many substrates of Roc1a-containing SCF complexes, and phenotypic pleiotropy could mask a role for Roc1a in a specific cell cycle transition. No inappropriate accumulation of cyclin E, dE2F, or the p21/p27-like cdk inhibitor dacapo was detected in Roc1a mutant imaginal cells. While such negative results are difficult to interpret, these proteins are all known to be substrates for SCF-mediated ubiquitination and degradation in other systems (Noureddine, 2002).

Both Roc1a and slimb mutant cells inappropriately accumulate Ci155 protein. Since Roc1a and Slimb proteins are capable of interacting with each other and with other components of Drosophila SCF (e.g., Cul1) in vitro, the simplest interpretation of this result is that Roc1a and Slimb are part of a common SCF complex that targets Ci155 for ubiquitination and subsequent proteolysis. However, there are notable differences between the Roc1a and slimb mutant phenotypes. First, Roc1a-null mutant clones do not cause limb duplications, as do clones of cells homozygous for a hypomorphic allele of slimb. While differences in the spectrum of proteins affected by loss of each of these genes could very well explain this phenotypic difference, another simple explanation is that Roc1a-null mutant cells cannot proliferate. Indeed, null mutant clones of slimb are unable to proliferate extensively and do not cause large wing duplications. Second, whereas slimb mutant cells inappropriately activate dpp expression no matter where they arise in the anterior compartment of the wing disc, Roc1a mutant cells do not. Only when Roc1a mutant cells are found far from the A/P axis is dpp expression ectopically activated. One possible explanation for this result is that less Ci155 protein accumulates in Roc1a mutant cells than in slimb mutant cells. Although such differences were not readily apparent in antibody staining, this method does not provide very good quantitative measurements. The cells most distal from the A/P boundry are known to be more sensitive to misregulation of the Hh signaling pathway than cells closer to the A/P boundary. Moreover, regulatory events in addition to stabilization of Ci155, such as nuclear import, are also required for Ci155 to transduce the Hh signal. Consequently, the elevated amount of Ci155 protein in Roc1a mutant cells may not be sufficient, or the protein may not be appropriately activated, to stimulate dpp expression close to the compartment boundary. Why would less Ci155 accumulate in Roc1a mutant cells relative to slimb mutant cells if Ci155 were degraded by an SCF complex containing Slimb and Roc1a? There may be some redundancy among the different Roc proteins, such that some Ci155 is processed in the absence of Roc1a but that none is processed in the absence of slimb. Finally, slimb and Roc1a may affect the accumulation of Ci155 by completely independent mechanisms that affect other aspects of Ci regulation (Noureddine, 2002).

Perhaps the most revealing difference between the slimb and Roc1a mutant phenotypes is the differential effect on Arm destruction. While slimb-null mutant clones clearly accumulate cytoplasmic/nuclear Arm, no difference could be detected in the abundance of Arm protein between Roc1a+ and Roc1a mutant cells. Thus, mutation of Roc1a and slimb affect the steady-state levels of different sets of proteins. Similarly, no changes in cyclin E levels was detected in Roc1a mutant cells, mutations of Drosophila ago, which, like slimb, encodes an F box/WD-repeat protein, cause inappropriate accumulation of cyclin E and hyperplasia. If slimb and Roc1a function in vivo only in the context of an SCF complex, then SCF complexes containing both Slimb and Roc1a are not absolutely required for targeting Arm for ubiquitin-mediated proteolysis. The different effect that mutation of Roc1a has on Arm and Ci accumulation has important implications for an understanding of how SCF functions in animal cells. Two quite different models could explain the data. In the first model, the Roc genes encode proteins with fully redundant biochemical properties, and an SCF complex containing any one of them is capable of targeting Arm for ubiquitination. In this case, the loss of Roc1a would be compensated by another Roc protein. Indeed, RNAi inactivation of the apparent C. elegans Roc2 ortholog (R10A10.2) causes no phenotype, suggesting that Roc1 (ZK287.5) is sufficient to provide all SCF function in this organism. However, biochemical redundancy among the Drosophila Roc proteins can only explain the Ci phenotype if Ci is more sensitive to reductions of SCF activity than Arm. In this situation, the absence of Roc1a would reduce the total cellular complement of SCF activity (i.e., the sum of all Roc1a-, Roc1b-, and Roc2-containing complexes) below a critical threshold required for Ci, but not Arm, proteolysis. In the second model, the Roc genes encode proteins with independent biochemical properties, such that an SCF complex containing Slimb and Roc1a would be capable of targeting Ci for ubiquitination but incapable of targeting Arm. Since Slimb is required for Arm proteolysis, it would perform this role as part of an SCF complex containing a RING finger protein other than Roc1a (e.g., Roc1b or Roc2). Consequently, this model suggests that the F box protein Slimb does not by itself dictate the selection of a substrate by a particular SCF. Both models provide a possible explanation for the uniqueness of Roc1a function during development (Noureddine, 2002).

To begin testing these models genetically, attempts were made to rescue the Roc1a mutant by UAS-Roc1b overexpression with tubulin-GAL4 and by expression of the Roc1b coding sequence from the endogenous Roc1a promoter. In neither case was rescue of the Roc1a lethality observed, as occurs with the Roc1a transgene itself, suggesting that the Roc1a and Roc1b proteins are not functionally interchangeable. These data are more consistent with the model in which distinct Roc proteins confer certain biochemical properties to individual SCF complexes that affect the overall efficiency of ubiquitination of specific substrates in vivo. The identity of the Roc subunit could lead to differences in regulatory posttranslational modifications of SCF (e.g., cullin neddylation), recruitment of different E2s with different specificities to SCF, or SCF subcellular localization. Any of these properties could potentially affect how efficiently Ci and Arm or any other substrate is ubiquitinated in the context of a specific cellular Roc-SCFslimb complex (Noureddine, 2002).

Cullin-3 regulates pattern formation, external sensory organ development and cell survival during Drosophila development

Ubiquitin-mediated proteolysis regulates the steady-state abundance of proteins and controls cellular homoeostasis by abrupt elimination of key effector proteins. A multienzyme system targets proteins for destruction through the covalent attachment of a multiubiquitin chain. The specificity and timing of protein ubiquitination is controlled by ubiquitin ligases, such as the Skp1-Cullin-F box protein complex. Cullins are major components of SCF complexes, and have been implicated in degradation of key regulatory molecules including Cyclin E, beta-catenin and Cubitus interruptus. The Drosophila Cullin-3 homologue, Guftagu, has been genetically identified and molecularly characterized. Perturbation of Cullin-3 function has pleiotropic effects during development, including defects in external sensory organ development, pattern formation and cell growth and survival. Loss or overexpression of Cullin-3 causes an increase or decrease, respectively, in external sensory organ formation, implicating Cullin-3 function in regulating the commitment of cells to the neural fate. Cullin-3 function modulates Hedgehog signalling by regulating the stability of full-length Cubitus interruptus (Ci155). Loss of Cullin-3 function in eye discs but not other imaginal discs promotes cell-autonomous accumulation of Ci155. Conversely, overexpression of Cullin-3 results in a cell-autonomous stabilisation of Ci155 in wing, haltere and leg (but not eye), imaginal discs suggesting tissue-specific regulation of Cullin-3 function. The diverse nature of Cullin-3 phenotypes highlights the importance of targeted proteolysis during Drosophila development (Mistry, 2004).

Phosphorylation of Ci triggers its subsequent proteolysis. One mechanism that might couple phosphorylation with proteolysis is the ubiquitin-mediated degradation pathway regulated by ubiquitin ligases such as the SCF complex. The F-box-containing factor Slimb is required for the generation of Ci75, the repressor form of Ci. Using the developing eye disc as a model, Ou (2002) has shown that Ci155 stability is controlled differentially by dCul-1 and dCul-3. Slimb and dCul-1 function anterior to the morphogenetic furrow to target Ci155 for proteolysis, while dCul-3 functions posterior to the furrow to mediate the same event (Mistry, 2004).

The current study supports the link between Cullin/SCF function and Ci155 stability during imaginal disc development. Loss of dCul-3 function in posterior compartment cells of the wing disc immediately adjacent to the AP boundary results in a non-autonomous reduction in Ci155 accumulation in anterior compartment cells that abut dCul-3 mutant cells. Furthermore, overexpression of dCul-3 in the anterior but not posterior compartment of wing, haltere and leg imaginal discs leads to a cell-autonomous increase in Ci155 stability. Thus, dCul-1 and dCul-3 are required in distinct developmental contexts to regulate Ci155 stability and Hh signal transduction (Mistry, 2004).

Together with the results of Ou (2002), these data support the model that dCul-3 functions autonomously to regulate Ci155 stability in a region-specific manner. In the eye, dCul-3 likely acts in a complex to promote the cleavage of Ci155 into the Ci75 repressor form. dCul-3 could mediate this activity directly, by associating with a specific F-box protein that tethers Ci155 to an SCF complex containing dCul-3. Alternatively, dCul-3 could mediate this effect indirectly, by targeted degradation or modification of a protein involved in the regulation of Ci155 stability. In the wing, dCul-3 overexpression could lead to an autonomous accumulation of Ci155 either by titrating other SCF complex components that promote the limited proteolysis of Ci155 to Ci75 or by targeting a protein for degradation that is normally required to promote limited proteolysis of Ci155. At present these data do not distinguish clearly between these models, although the reciprocal phenotypes observed in dCul-3 mutant clones relative to tissues that overexpress dCul-3 suggest dCul-3 does not act solely in a dominant negative manner (Mistry, 2004).

The non-autonomous effect of dCul-3 loss on Ci155 stability suggests that dCul-3 can modulate Ci155 accumulation through multiple mechanisms. In this context, the simplest model is that dCul-3 function is required for the proper expression or transmission of the Hh signal. The apparent ability of dCul-3 to regulate Ci155 stability through at least two different mechanisms and the diversity of dCul-3 phenotypes, suggest that the composition of dCul-3-containing SCF complexes varies in a region- and stage-specific manner. Given this, a clear understanding of the molecular basis through which dCul-3 regulates Ci155 stability as well as the activity/levels of other proteins will require the identification of the direct targets of dCul-3/SCF complexes through biochemical and molecular genetic means (Mistry, 2004).

Debra-mediated ubiquitination and lysosomal degradation of Ci

Transcription factor Ci mediates Hedgehog (Hh) signaling to determine the anterior/posterior (A/P) compartment of Drosophila wing disc. While Hh-inducible genes are expressed in A compartment cells abutting the A/P border, it is unclear how the boundaries of this region are established. This study identified a Ci binding protein, Debra, that is expressed at relatively high levels in the band abutting the border of the Hh-responsive A compartment region. Debra mediates the polyubiquitination of full-length Ci, which then leads to its lysosomal degradation. Debra is localized in the multivesicular body, suggesting that the polyubiquitination of Ci directs its sorting into lysosome. Thus, Debra defines the border of the Hh-responsive region in the A compartment by inducing the lysosomal degradation of Ci (Dai, 2003).

Yeast two-hybrid screening was performed using the N-terminal repressor domain of Ci as bait. Of the over 100 clones isolated, 17 clones were derived from the same gene. The DNA sequence of isolated full-length cDNA clones was confirmed to be identical with that of a cDNA, identified by the Berkeley Drosophila Genome Project, that encodes a 1007 amino acid protein. This protein was designated as Debra (Dbr). Dbr has no obvious homology with any other known proteins, although it contains a Ser-rich region (amino acids 133-230) (Dai, 2003).

In vitro binding assays were performed using various forms of in vitro-translated Ci and the resin containing the full-length Dbr. Both the N-terminal repressor domain and the zinc finger region of Ci bind to Dbr. GST pull-down assays were performed using various forms of in vitro-translated Dbr and the GST-Ci fusion proteins (GST-Ci-R and GST-Ci-ZF) that contain either the repressor domain or the zinc finger region of Ci. The results indicated that the N-terminal 243 amino acids of Dbr bind to Ci (Dai, 2003).

Hh is thought to spread through A compartment cells, forming long-range concentration gradients that provide positional information. In contrast to this model, however, the two Hh target genes dpp and ptc are highly induced in a stripe of A compartment cells 9-10 cells away from the A/P border of the wing disc. Consistent with this, the form of Ci (Ci-155) that activates the Hh target genes is also present at high levels in this stripe. How the border of this stripe is determined was initially unclear, but in this study, it was found that Dbr may be involved, as it is highly expressed in an additional stripe of A compartment cells on the border of the region expressing dpp, ptc, and high levels of Ci-155. That Dbr plays a key role in determining the border of ptc gene expression was confirmed when it was noted that loss of Dbr in the clone of A compartment cells increased Ci-155 levels and the low levels of ectopic ptc expression. These low levels of ptc expression may be due to the fact that PKA suppresses Ci-155-dependent transactivation. In contrast to ptc, the ectopic expression of dpp was not observed in clones of dbrEP9 cells, although dpp expression on the A/P border is enhanced. This may be due to the presence of Ci-75, which suppresses dpp expression but not ptc expression. Thus, Dbr affects the levels, but not the boundary, of dpp expression (Dai, 2003).

Increased Ci-155 levels are evident in the dbr-deficient clones generated in the region, where Dbr protein levels are relatively high. In contrast, the degree of increase in Ci-155 levels is not so high in the clones far from the A/P boundary or the clones close to the A/P boundary. Thus, the expression pattern of Dbr is important for its biological role. Hh prevents the Dbr-induced ubiquitination of Ci-155. However, this is not due to the effect of Hh on Dbr protein levels. Ectopic expression by UAS-hh does not affect the Dbr protein levels. In addition, the levels of Dbr are not affected by smo mutations, suggesting that Hh does not regulate Dbr expression. PKA is required for the Dbr-induced ubquitination of Ci-155, while PKA and Hh act antagonistically. Therefore, Hh may block Ci-155 ubiquitination by inhibiting the PKA pathway (Dai, 2003).

The well-established function of ubiquitin is to target proteins for degradation by the 26S proteasome. In addition to this, ubiquitin is widely used as a sorting signal that determines the location and fate of proteins. For example, ubiquitination is required for the internalization of various membrane receptors, including several tyrosine kinase receptors. These proteins are monoubiquitinated at the cell surface by the ubiquitin ligase Cbl, leading to their internalization followed by delivery to the endosomal system. Ubiquitination is also necessary to regulate the sorting of cargo proteins into multivesicular body (MVB) vesicles. Carboxypeptidase S (CPS) is synthesized as an integral membrane precursor and then released from the membrane upon fusion of the multivesicular body/late endosome with the lysosome-like vacuole of yeast. Monoubiquitination of the short cytoplasmic tail of CPS is required for its sorting into MVB vesicles. The data published so far indicate that endocytosis requires only monoubiquitination, in contrast to proteasome-mediated degradation, which requires the formation of relatively long polyubiquitination chains. It is worthy to note that Dbr appears to induce the polyubiquitination of Ci to direct its sorting into lysosome (Dai, 2003).

Observations reveal that Ci proteins are localized in the early endosome and MVBs while Dbr occurs only in the MVBs. This suggests that Ci is first sorted into the early endosome without Dbr, indicating that ubiquitination is not required for this step. It is well known that microtubules and the microtubule motor protein kinesin are required for efficient transcytosis and delivery of proteins to late endosomes and lysosomes. Since Ci associates with microtubles via direct binding to the kinesin-like molecule Costal-2, the incorporation of Ci into the early endosome may occur on the microtubles. Once Ci is in MVBs, it binds to Dbr, which induces its ubiquitination. MVBs are formed when the limiting membrane of the endosome invaginates and buds into its lumen. The ubiquitin moiety of Ci may act as a signal for its budding into the MVB lumen. The ubiquitinated Ci is then delivered into the lumen of the lysosome upon the fusion of the MVBs with this organelle, after which the resident proteases degrade both the vesicles and the ubiquitinated Ci (Dai, 2003).

Both the PKA phosphorylation sites and the processing site of the Ci protein are required for its Dbr-induced degradation. In addition, these sites are required for the proteasome-dependent processing of Ci-155, which also involves Slimb. Thus, Ci-155 levels are regulated by two separate degradative processes. That both processes share common regulatory elements suggests that it is likely that the events leading to the lysosomal degradation and proteasome processing of Ci-155 occur in parallel. Since Slimb contains an F box/WD40 repeat, and its vertebrate homolog is a component of the SCF ubiquitin ligase complex, Slimb is likely to act as an E3 ligase in transferring the ubiquitin moiety to Ci. In the absence of Dbr, Slimb induces the proteolytic processing of Ci-155 to Ci-75 via the proteasome, possibly by mediating limited Ci-155 ubiquitination that then serves as a proteolytic processing signal. When Dbr exists, Slimb cooperates to induce the full ubiquitination of Ci-155 that targets it for lysosomal degradation via MVBs. Dbr does not induce Ci-75 degradation. Slimb binds to both the N- and C-terminal regions of Ci-155. It may be that the binding of Slimb to Ci-75, which lacks the C-terminal region of Ci-155, is too weak to induce the ubiquitination of Ci-75, resulting in the maintenance of this form of Ci in the cell (Dai, 2003).

At present, it is not clear how Dbr enhances Ci ubiquitination. The C-terminal half of Dbr shares weak homology with the C-terminal half of the Sec12 protein from the yeast Pichia pastoris (37% similarity, 177 amino acids out of 466 amino acids). The yeast Saccharomyces cerevisiae Sec12 protein, which is an integral membrane glycoprotein and has guanine-nucleotide-exchange activity, is required for the formation of transport vesicles generated from the endoplasmic reticulum. However, only Pichia Sec12 contains the region homologous with Dbr. This C-terminal region may be required for the interaction with some components within vesicles. Thus, while Dbr does not have a membrane-spanning region, its C-terminal region could interact with some components inside the MVBs (Dai, 2003).

Drosophila Cand1 regulates Cullin3-dependent E3 ligases by affecting the neddylation of Cullin3 and by controlling the stability of Cullin3 and adaptor protein

Cullin-RING ubiquitin ligases (CRLs), which comprise the largest class of E3 ligases, regulate diverse cellular processes by targeting numerous proteins. Conjugation of the ubiquitin-like protein Nedd8 with Cullin activates CRLs. Cullin-associated and neddylation-dissociated 1 (Cand1) is known to negatively regulate CRL activity by sequestering unneddylated Cullin1 (Cul1) in biochemical studies. However, genetic studies of Arabidopsis have shown that Cand1 is required for optimal CRL activity. To elucidate the regulation of CRLs by Cand1, a Cand1 mutant was analyzed in Drosophila. Loss of Cand1 causes accumulation of neddylated Cullin3 (Cul3) and stabilizes the Cul3 adaptor protein HIB. In addition, the Cand1 mutation stimulates protein degradation of Cubitus interruptus (Ci), suggesting that Cul3-RING ligase activity is enhanced by the loss of Cand1. However, the loss of Cand1 fails to repress the accumulation of Ci in Nedd8(AN015) or CSN5(null) mutant clones. Although Cand1 is able to bind both Cul1 and Cul3, mutation of Cand1 suppresses only the accumulation of Cul3 induced by the dAPP-BP1 mutation defective in the neddylation pathway, and this effect is attenuated by inhibition of proteasome function. Furthermore, overexpression of Cand1 stabilizes the Cul3 protein when the neddylation pathway is partially suppressed. These data indicate that Cand1 stabilizes unneddylated Cul3 by preventing proteasomal degradation. This study proposes that binding of Cand1 to unneddylated Cul3 causes a shift in the equilibrium away from the neddylation of Cul3 that is required for the degradation of substrate by CRLs, and protects unneddylated Cul3 from proteasomal degradation. Cand1 regulates Cul3-mediated E3 ligase activity not only by acting on the neddylation of Cul3, but also by controlling the stability of the adaptor protein and unneddylated Cul3 (Kim, 2010).

The neddylation pathway is highly conserved in many organisms, and the neddylation step is essential for Cullin-mediated E3 ubiquitin ligase activation. Cand1 is a highly conserved protein that binds to unneddylated Cullins and sequesters Cul1 from the CRL complex. It has been suggested that Cand1 inhibits CRL activity in vitro. However, studies from Arabidopsis have shown that loss of Cand1 leads to decreased CRL activity, indicating that Cand1 is required for efficient CRL function. Drosophila was used as a model system to elucidate this paradoxical effect of Cand1. First, it was found that loss of Cand1 increases the ratio of Nedd8 modified to unmodified Cul3 and the level of Cul3 adaptor HIB/rdx, causing enhanced degradation of CiFL, despite little effect on Cul1. Although Cand1 has been reported to negatively regulate CRL activity by binding to unneddylated Cul1 and dissociating the CRL complex in vitro, accumulations of neddylated Cullin and adaptor protein have never been observed in studies of Cand1 depletion. These provide a better understanding of the role of Cand1 in vivo, suggesting that the regulations of Cul3 neddylation and adaptor stability are important for Cand1 to control CRL activity. Unlike the results of Arabidopsis studies, in which Cand1 is required for optimal CRL activity, this study demonstrates that the Cand1 mutation of Drosophila stimulates the degradation of CiFL by enhancing Cul3-RING ligase activity. In addition, a novel insight is provided into the role of Cand1 by which Cand1 is involved in the stabilization of unneddylated Cul3. Evidence is presented that Cand1 protects unneddylated Cul3 from proteasomal degradation (Kim, 2010).

The absence of Cand1 increased the level of neddylated Cul3, and it suggests that Cand1 could inhibit the neddylation of Cul3. However, the overexpression of Cand1 had no effect on Cul3 neddylation. The amount of Cand1 seems to be sufficient to prevent Cul3 neddylation in the wild-type background. However, neddylation of Cul3 was decreased when Cand1 was expressed in the Cand1 mutant background, indicating that Cand1 can suppress Cul3 neddylation (Kim, 2010).

CiFL is processed by two different Cullins, Cul1 and Cul3, in the eye disc of Drosophila. In the posterior area of the eye imaginal disc, CiFL is degraded by Cul3-mediated E3 activity, where loss of Cand1 affects the stability of CiFL. Because it was observed that mutation of Cand1 decreases the level of CiFL, the levels of adaptor proteins of Cul1 and Cul3 were further investigated. It was found that the level of the Cul3 adaptor protein HIB/rdx is also increased in the Cand1 mutant, whereas the levels of Slimb, the F-box protein of the Cul1 RING ligase, remain constant. It suggests that Cand1 could regulate Cul3-based E3 ligase activity by suppressing the level of HIB/rdx. Several adaptor proteins are destabilized by autoubiquitination of CRL activity. CSN also maintains adaptor stability by deneddylating Cullin and recruiting deubiquitination enzymes. Interestingly, it has recently been observed that the CSN-associated deubiquitinating enzyme Ubp12 maintains the stability of the Cul3 adaptor, but not the F-box, Cul1 adaptor. This provides a possible clue that Cand1 may regulate the stability of HIB/rdx through deubiquitinating enzymes by working with CSN. Direct interaction of Cand1 with HIB/rdx suggests another possibility that Cand1 might suppress the level of HIB/rdx through a direct association with HIB/rdx. Taken together, the evidence presented in this study indicates that Cul3-dependent E3 ubiquitin ligase activity is increased by the loss of Cand1 function (Kim, 2010).

It has been suggested that Nedd8 covalent conjugation to Cullin causes instability of the Cullin protein. However, the current results show that the neddylated form of Cul3 has maintained protein stability in the Cand1 mutant, albeit at a slightly reduced Cul3 protein level. This observation could be related to the function of CSN because there is a significant decrease in the total amount of Cullins in CSN mutant cells. Both CSN and Cand1 proteins have been proposed to be involved in the cycle of assembly and disassembly of the CRL complex. This model explains how Cand1 and CSN have paradoxical effects on CRL activity and insists that Cand1-mediated cycling is required for optimal CRL activity. However, the data do not support this cycling model, in which loss of Cand1 enhances the degradation of CiFL as a result of increased activity of CRLs. The double-mutant analyses suggest that regulation of the neddylation pathway is a major mechanism for CiFL degradation. Loss of Cand1 failed to suppress accumulation of CiFL protein in Nedd8AN015 or CSN5null mutant clones. The functions of Nedd8 and CSN with regard to Cullin seem to play a more dominant role in regulating CRLs than that of regulation by Cand1. This could explain why overexpression of Cand1 in CSN5null mutant causes an increase only in the neddylated forms of Cullin, although Cand1 stabilizes unneddylated Cullin (Kim, 2010).

Inhibition of proteasome function by overexpressing a dominant-negative form of a proteasome subunit causes accumulation of unneddylated Cul3. The neddylation defective dAPP-BP1 mutant also exhibits elevated levels of unneddylated Cul3, but repressed proteasomal activity in the dAPP-BP1null mutant fails to causes Cul3 accumulation. These results support the theory that unneddylated Cul3 is degraded by the proteasome, but this degradation effect is inhibited by mutation of the Nedd8 E1-activating enzyme, dAPP-BP1. Accumulation of Cul3 in the dAPP-BP1 mutant is suppressed by loss of Cand1, and decreased Cul3 in the dAPP-BP1, Cand1 double-mutant is again accumulated by reducing proteasome activity. This shows that Cand1 is responsible for the accumulation of unneddylated Cul3 in the dAPP-BP1 mutant as a result of inhibition of proteasome-mediated degradation. Repression of proteasome function in the Cand1 mutant induces accumulation of unneddylated Cul3, showing that neddylated Cul3 is destabilized by the proteasome in the absence of Cand1 (Kim, 2010).

Recent reports indicate that supplementation of substrate and adaptor to Cullin-RING ligases promotes Cullin neddylation and dissociation of the Cullin-Cand1 complex. In agreement with the previous reports, the current data also suggest that the neddylation process might regulate the dissociation of Cul3 from Cand1. If Cand1 is dissociated from Cullin by neddylation, a defect in the neddylation process might promote the interaction of Cand1 with unneddylated Cul3. This could explain why the level of Cul3 was not affected by overexpression of Cand1 in the dAPP-BP1 null mutant, even if Cand1 overexpression increases Cul3 protein levels in the dAPP-BP1null heterozygote background (Kim, 2010).

Although Cand1 affects mostly the Cul3 protein, it also influences the Cul1 protein. Cand1 can bind to Cul1 and the overexpression of Cand1 induces the stabilization of Cul1 as well as Cul3. However, the effect of Cand1 on Cullins seems to differ depending on the type of tissue. Immunoblot analysis of Cand116 extracts from third-instar brain lobes and eye discs showed no distinguishable effect on the ratio of neddylated Cul1, but loss of Cand1 caused a reduction of CiFL protein in the anterior region of the wing disc, where the Cul1-dependent E3 ligase degrades CiFL protein (Kim, 2010).

It is proposed that Cand1 contributes to the fine-tuning of Cul3-mediated E3 ligase activity by acting on the neddylation state as well as on the stability of unneddylated Cul3 and adaptor protein. Binding of Cand1 to unneddylated Cul3 would shift the equilibrium away from the neddylation of Cul3 that is required for substrate degradation and then cause sequestration of unneddylated Cul3 from proteasomal degradation. Moreover, Cand1 could be involved in the suppression of Cul3 adaptor protein, HIB/rdx, to regulate CRL activity. Loss of Cand1 shifts the equilibrium toward the neddylated form of Cul3 and increases the level of Cul3 adaptor HIB/rdx, which leads to enhanced degradation of CiFL, a substrate of CRLs. Neddylation of Cul3 is essential for CRL activity, so the mutation of Cand1 fails to down-regulate accumulation of CiFL in Nedd8 or CSN5 mutants. In the absence of dAPP-BP1, unneddylated Cul3 would tend to bind to Cand1, which protects unneddylated Cul3 from proteasomal degradation and induces accumulation of Cul3 (Kim, 2010).

The mechanisms underlying the Cullin neddylation pathway are closely conserved in Drosophila and in mammals. Consequently, the study of Drosophila Cand1 and Cullin provides a novel insight into the regulation of Cullin based E3 ligases by Cand1 (Kim, 2010).

Sxl is in a complex that contains all of the known Hh cytoplasmic components: Hh promotes the entry of Sxl into the nucleus in the wing disc

The sex determination master switch, Sex-lethal (Sxl), controls sexual development as a splicing and translational regulator. Hedgehog (Hh) is a secreted protein that specifies cell fate during development. Sxl is in a complex that contains all of the known Hh cytoplasmic components, including Cubitus interruptus (Ci) the only known target of Hh signaling. Hh promotes the entry of Sxl into the nucleus in the wing disc. In the anterior compartment, the Hh receptor Patched (Ptc) is required for this effect, revealing Ptc as a positive effector of Hh. Some of the downstream components of the Hh signaling pathway also alter the rate of Sxl nuclear entry. Mutations in Suppressor of Fused or Fused with altered ability to anchor Ci are also impaired in anchoring Sxl in the cytoplasm. The levels, and consequently, the ability of Sxl to translationally repress downstream targets in the sex determination pathway, can also be adversely affected by mutations in Hh signaling genes. Conversely, overexpression of Sxl in the domain that Hh patterns negatively affects wing patterning. These data suggest that the Hh pathway impacts on the sex determination process and vice versa and that the pathway may serve more functions than the regulation of Ci (Horabin, 2003).

Sxl co-immunoprecipitates with Cos2 and Fu in the female germline. Since Ci is not expressed in germ cells, it is probable that a different Hh cytoplasmic complex might exist in germ cells. In somatic cells, Sxl is expressed in all female cells while Ci is expressed in only a subset. To test whether the Hh pathway differentiates between the two proteins in somatic cells, Sxl was immunoprecipitated from embryonic extracts and the immunoprecipitates probed for the various Hh cytoplasmic components. The immunoprecipitates showed that Cos2, Fu and Ci are complexed with Sxl. The specificity of this association of Sxl with the Hh pathway components was verified using antibodies to either Ci or Su(fu), and testing the immunoprecipitates for the presence of Sxl. Both co-immunoprecipitated with Sxl. The Ci immunoprecipitate was also tested for another Hh cytoplasmic component, Fu, which was present as expected. These interactions are maintained in a Su(fu)LP background (protein null allele). An IP of Ci from Su(fu)LP embryos brought down Sxl, as well as Fu and Cos2. Taken together, these data suggest that cells that express Ci and Sxl have both proteins in the same complex with the known cytoplasmic components of the Hh signaling pathway (Horabin, 2003).

Previous work on the germline has suggested that the Hh signaling pathway affects the intracellular trafficking Sxl. The cross talk between these two developmental pathways has been analyzed in tissues where both Hh targets can be present in the same cell. While analysis of embryos only uncovered an effect of Cos2 on Sxl, analysis of wing discs allowed several specific effects to be uncovered. At least three new functional aspects of the Hh pathway are suggested:

  1. More than one 'target' protein can exist in the Hh cytoplasmic complex.
    Immunoprecipitation experiments using extracts from embryos indicate that Sex-lethal and the known Hh signaling target Ci are in the same complex. The two proteins can co-immunoprecipitate each other as well as other known members of the Hh cytoplasmic complex. Even when Su(fu), the cytoplasmic component that most strongly anchors Sxl in the cytoplasm, is removed, Sxl can still be co-immunoprecipitated with both Ci and Fu. As a whole, these results suggest that at least some proportion of the two Hh 'target' proteins are in a common complex within the cell. Additionally, the wing defects produced when Sxl is overexpressed in the Hh signaling region suggest that their relative concentrations are important for their normal functioning (Horabin, 2003).
  2. The Hh targets can be affected differentially.
    The presence of two 'targets' within the Hh cytoplasmic complex, raises the question of how they can be differentially affected. The data show that the various members of the Hh pathway do not affect Sx1 and Ci similarly. Smo appears to be dispensable for the transmission of the Hh signal in promoting Sx1 nuclear entry, while Smo is critical for the activation of Ci. Conversely, while Ptc is essential for the effect of Hh on Sxl, it is dispensable for the activation of Ci. The Fu kinase (fumH63 background) also appears to have no role in Hh signaling with respect to Sxl, while it is critical for the activation of Ci. By contrast, both Su(fu) and the Fu regulatory domain act similarly on Sxl and Ci, serving to anchor them in the cytoplasm (Horabin, 2003).

    Taken together, these data suggest that the presence of Hh can be relayed to the cytoplasmic components differentially and, while the data do not address the point, suggest how different outcomes might be achieved. Ptc has been proposed to be a transmembrane transporter protein that functions catalytically in the inhibition of Smo via a diffusible small molecule. The stimulation of Sxl nuclear entry by the binding of Hh to Ptc might also involve a change in the internal cell milieu, but in this case the Hh cytoplasmic complex may be affected independently, not requiring a change in the activity of Smo or the Fu kinase (Horabin, 2003).

  3. Ptc can signal the presence of the Hh ligand in a positive manner.
    Several experiments indicate that Hh bound to Ptc enhances the nuclear entry of Sxl. That Smo has no role in transmitting the Hh signal is most clearly demonstrated by expressing the PtcD584 protein in both the anterior and posterior compartments of the dorsal half of the wing disc. PtcD584 acts as a dominant negative and so activates Ci in the anterior compartment, but it fails to enhance the levels of nuclear Sxl in the anterior because it sequesters Hh in the posterior compartment. The double mutant condition of ptc clones in a hhMRT background clearly places Ptc downstream of Hh, while showing Ptc can act positively in transmitting the Hh signal (Horabin, 2003).

A positive role for Ptc, but in this case in conjunction with Smo, in promoting cell proliferation during head development has recently been reported. In this situation, however, Hh acts negatively on both Ptc and Smo in their activation of the Activin type I receptor, suggesting an even greater variance from the canonical Hh signaling process (Horabin, 2003).

While the effects on Sxl in the anterior compartment show a dependence on the known Hh signaling components, it is not clear what promotes the rapid nuclear entry of Sxl in the posterior compartment. Su(fu) is expressed uniformly across the disc so it does not appear to be responsible for the AP differences, and ptc clones have no effect (and Ptc RNA and protein are not detected in the posterior compartment). Removal of Hh, however, reduces the nuclear entry rate of Sxl in both compartments. In this regard, the parallel between Hh pathway activation and Sxl nuclear entry in the posterior compartment is worth noting. Fu is also activated in the posterior compartment in a Hh-dependent manner, even though Ptc is not present. It is not clear what mediates between Hh and Fu (Horabin, 2003).

The data also suggest that the Hh cytoplasmic complex may have slightly different compositions in different tissues and/or at developmental stages. In the female germline and in embryos, the absence of Cos2 leads to a severe reduction in Sxl levels. However, in wing discs when mutant clones are made using the same cos2 allele, there is no effect on Sxl. It is suggested that between the third instar larval stage and eclosion, the composition of the Hh cytoplasmic complex may change again to make Sxl more sensitive to Cos2. This would explain why removal of Cos2 can produce sex transformations of the foreleg even though mutant clones in wing discs (and also leg discs) show no alterations in Sxl levels (Horabin, 2003).

A similar argument might apply to the weak sex transformations of forelegs produced by PKA clones. Alternatively, PKA may have a very weak effect but the assay on wing discs is not sufficiently sensitive to allow detection of small effects; PKA was found to have a modest effect on Sxl nuclear entry in the germline. Sxl is sufficiently small (38-40 kDa) to freely diffuse into the nucleus, or the protein may enter the nucleus as a complex with splicing components. This may account for the limited sex transformations caused by removal of Hh pathway components (Horabin, 2003).

Removal of several of the Hh pathway components, such as smo, gives the same weak sex transformation phenotype, even though smo has no effect on Sxl nuclear entry. Additionally, there is no correlation between a positive and a negative Hh signaling component and whether there is a resulting phenotype. Changing the dynamics of the activation state of the Hh cytoplasmic complex may perturb the normal functioning of Sxl, since Sxl appears to be in the same complex as Ci. For example, if the Hh pathway is fully activated because of a mutant condition, the relative amounts of Sxl in the cytoplasm versus nucleus at any given time, may be different from the wild-type condition. Perturbing the usual cytoplasmic-nuclear balance could compromise the various processes that Sxl protein regulates. Sxl acts both positively and negatively on its own expression through splicing and translation control and, additionally, regulates the downstream sex differentiation targets. The latter could also be responsible for the weak sex transformations seen, in view of the recent demonstration that doublesex affects the AP organizer and sex-specific growth in the genital disc (Horabin, 2003).

With the exception of Cos2, which can produce relatively substantial effects on Sxl levels in embryos as well as sex transformations in the foreleg, the effects of removal of any of the other Hh pathway components are generally not large. The strong effects of Cos2 on Sxl could be because it affects the stability of Sxl. However, Sxl depends on an autoregulatory splicing feedback loop for its maintenance making the protein susceptible to a variety of regulatory breakdowns. If Cos2 altered the nuclear entry of Sxl, for example, its removal could compromise the female-specific splicing of Sxl transcripts by reducing the amounts of nuclear Sxl. Splicing of Sxl transcripts would progressively fall into the male mode to eventually result in a loss of Sxl protein (Horabin, 2003).

Cos2 and Fu have been reported to shuttle into and out of the nucleus, and their rate of nuclear entry is not dependent on the Hh signal. That Ci and Sxl are complexed with the same Hh pathway cytoplasmic components, and share and yet have unique intracellular trafficking responses to mutations in the pathway, makes it tempting to speculate that the Hh cytoplasmic components may have had a functional origin related to intracellular trafficking that preceded the two proteins. Whether this reflects a more expanded role in regulated nuclear entry remains to be determined (Horabin, 2003).

Differential regulation of Hedgehog target gene transcription by Costal2 and Suppressor of Fused: Regulation of Ci activity

The mechanism by which the secreted signaling molecule Hedgehog (Hh) elicits concentration-dependent transcriptional responses from cells is not well understood. In the Drosophila wing imaginal disc, Hh signaling differentially regulates the transcription of target genes decapentaplegic (dpp), patched (ptc) and engrailed (en) in a dose-responsive manner. Two key components of the Hh signal transduction machinery are the kinesin-related protein Costal2 (Cos2) and the nuclear protein trafficking regulator Suppressor of Fused [Su(fu)]. Both proteins regulate the activity of the transcription factor Cubitus interruptus (Ci) in response to the Hh signal. This study analyzed the activities of mutant forms of Cos2 in vivo and found effects on differential target gene transcription. A point mutation in the motor domain of Cos2 results in a dominant-negative form of the protein that derepresses dpp but not ptc. Repression of ptc in the presence of the dominant-negative form of Cos2 requires Su(fu), which is phosphorylated in response to Hh in vivo. Overexpression of wild-type or dominant-negative cos2 represses en. These results indicate that differential Hh target gene regulation can be accomplished by differential sensitivity of Cos2 and Su(Fu) to Hh (Ho, 2005).

The data suggest that the activities of Cos2 and Su(fu) are independently regulated by different concentrations of Hh along the gradient that forms from posterior to anterior. In the anterior cells distant from the AP boundary, little or no Hh is received and target genes are silent. In these cells, Cos2 is required for proteolytic processing of Ci into its repressor form and possibly for the delivery of CiFL for lysosomal degradation. The data suggest that Cos2 requires an intact P-loop for its role in these events. Cos2 ATPase activity may be inhibited in cells receiving very low levels of Hh, preventing Ci proteolysis and stabilizing CiFL. The stabilization of CiFL results in the activation of dpp. Nearer the AP border, where higher levels of Hh are received, Su(fu) becomes phosphorylated, inactivating its negative regulatory hold on Ci, while inhibition of the ATPase activity of Cos2 continues to allow stabilization of Ci. In this situation, ptc and dpp are transcribed. Finally, at the highest levels of Hh signaling adjacent to the AP border, Cos2 is required for activation of the pathway and the expression of en. S182N expression, or cos2 over-expression, inhibits the induction of en by endogenous Hh in these cells. The elements of this model are addressed below (Ho, 2005).

Ci plays a central role in determining which genes are repressed or activated in response to different concentrations of Hh. In order to activate target genes such as dpp or ptc, Ci must be stabilized in its full-length form. In wild-type discs, Hh stabilizes Ci by antagonizing molecular events that reduce the concentration of nuclear CiFL. In addition to the constitutive nuclear export of Ci, there are two ways CiFL concentration is reduced: full-length Ci is proteolytically processed into a repressor form; and CiFL is degraded by a lysosome-mediated process involving a novel protein called Debra. In these experiments, the stabilization of CiFL was accomplished by expressing S182N in responsive cells, which antagonizes Cos2 repressor activity and results in the accumulation of high levels of CiFL, with minimal effects on the levels of CiR. This same type of differential effect on CiR and CiFL is accomplished by Debra, which causes the lysosomal degradation of CiFL without affecting the production of CiR. Cos2 and Debra may act in concert to destabilize CiFL, while Cos2 may also aid in the production of CiR via a Debra-independent mechanism. This would involve presenting Ci to the kinases, PKA, CKI and GSKß (Shaggy) for phosphorylation and processing by the proteasome. Since Debra regulates Ci stability in limited areas of the wing disc but S182N can stabilize Ci throughout the anterior compartment, it is likely that S182N interferes with both Debra-dependent and Debra-independent mechanisms of Ci stability to achieve the observed effect: cell-autonomous stabilization of CiFL leading to derepression of dpp (Ho, 2005).

These results suggest that Cos2 may use its ATPase activity to transport Ci to a location where it becomes phosphorylated in preparation for processing, or to the site of processing itself. Alternatively, the ATPase activity may be important for regulating the conformation of Cos2 and its binding to partners such as Smo, Su(fu), Fu and Ci, which would be a novel role for the P-loop in a kinesin-related protein. The S182N mutation may lock Cos2 in a conformation that changes association with binding partners. For example, S182N may decrease the ability of Cos2 to bind Ci, releasing Ci from the cytoplasm, resulting in an increased level of CiFL in the nucleus and the activation of dpp (Ho, 2005).

The human ortholog of Suppressor of fused is a tumor suppressor gene. Su(fu) can associate with Ci, and with the mammalian homologs of Ci, the Gli proteins, through specific protein-protein interactions. Through these interactions, Su(fu) controls the nuclear shuttling of Ci and Gli, as well as the protein stability of CiFL and CiR. Flies homozygous for Su(fu) loss-of-function mutations are normal, so the importance of Su(fu) becomes evident only when other gene functions are thrown out of balance, as in a fu mutant background, with extra or diminished Hh signaling caused by ptc, slimb and protein kinase A mutations or when altered Cos2 is produced (Ho, 2005).

To activate ptc transcription in the wing disc, two conditions have to be met simultaneously: CiFL must be stabilized, and the activity of Su(fu) must be reduced. Removal of Su(fu) changes S182N from a ptc repressor into a ptc activator. Removal of Su(fu) may result in the modification, activation or relocalization of CiFL, or in further sensitizing the system to stabilized CiFL. In Su(fu) homozygous animals, the quantity of CiFL and CiR proteins is greatly diminished, and Su(fu) mutant cells are more sensitized to the Hh signal. The lower levels of both CiFL and CiR in mutant Su(fu) cells may contribute to the sensitivity of these cells to Hh, since a small Hh-driven change in the absolute concentration of either form of Ci would result in a significant change in the ratio between the two proteins. Both CiFL and CiR bind the same enhancer sites, so their relative ratio is likely to be important in determining target gene expression. S182N expression tips the ratio of CiFL to CiR toward CiFL, and reducing the absolute quantities of both Ci isoforms by removing Su(fu) will enhance this effect. Furthermore, Su(fu) binds Ci and sequesters it in the cytoplasm in a stoichiometric manner Reducing the amount of Su(fu) should release more CiFL to the nucleus to activate ptc (Ho, 2005).

The activity of Su(fu) must be regulated or overcome so that target genes can be activated at the right times and places in response to Hh. The regulation of Su(fu) activity may occur by Hh-dependent phosphorylation. A phosphoisoform of Su(fu), Su(fu)-P, was detected in discs where GAL4 was used to drive extra Hh expression. At high concentrations of Hh, the phosphorylation of Su(fu) is not antagonized by overexpression of cos2 or either of the cos2 mutants, suggesting that phosphorylation of Su(fu) occurs independently of Cos2 function. One kinase involved in the phosphorylation of Su(fu) is the Ser/Thr kinase Fused, a well-established component of Hh signal transduction. It is not known whether the phosphorylation of Su(fu) by Fu is direct or indirect (Ho, 2005).

The phosphorylation state of Su(fu) may be an important factor in determining Hh target gene activity. Phosphorylation of an increasing number of Su(fu) molecules with increasing Hh signal may gradually release Ci from all of the known modes of Su(fu)-dependent inhibition, such as nuclear export and recruitment of repressors to nuclear Ci, leading to higher levels of CiFL in the nucleus and the activation of Hh target genes such as ptc (Ho, 2005).

Anterior en expression was used as an in vivo reporter of high levels of Hh signaling. cos2 mutant cells at the AP boundary fail to activate en, suggesting that Cos2 plays a positive regulatory role in en regulation. S182N, S182T and Cos2 overexpression mimics the cos2 loss-of-function condition with respect to en: en remains off in these cells. One interpretation of these data is that all the Cos2 proteins are able to associate with another pathway component, such as Smo, and overproduction of any of them inactivates some of the Smo in non-productive complexes not capable of activating en (Ho, 2005).

In contrast to the activity of all the other mutations generated, deletion of the C terminal domain creates a protein (Cos2DeltaC) that represses normal dpp, ptc and en expression in the wing disc. In this in vivo assay, Cos2DeltaC acts just like wild-type Cos2. A similar deletion has been shown to retain function in cell culture assays. This mutant, expressed under the control of its endogenous promoter, rescues the lethality and wing duplication phenotypes of a cos2 loss-of-function allele over a cos2 deficiency. The results of the rescue experiment bring up a new possibility: that the C-terminal domain of Cos2, and the Cos2-Smo interaction via the C terminus of Cos2, is not necessary for repressor activities of Cos2. Alternatively, Cos2DeltaC could complement or boost the activity of the hypomorphic allele cos211, which was used for the rescue experiment (Ho, 2005).

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).

Modulation of the Suppressor of fused protein regulates the Hedgehog signaling pathway in Drosophila embryo and imaginal discs; Modulation of the different Ci states

The Suppressor of fused (Su(fu)) protein is known to be a negative regulator of Hedgehog (Hh) signal transduction in Drosophila imaginal discs and embryonic development. It is antagonized by the kinase Fused (Fu) since Su(fu) null mutations fully suppress the lack of Fu kinase activity. In this study, the Su(fu) gene was overexpressed in imaginal discs and opposing effects were observed depending on the position of the cells, namely a repression of Hh target genes in cells receiving Hh and their ectopic expression in cells not receiving Hh. These effects were all enhanced in a fu mutant context and were suppressed by cubitus interruptus (Ci) overexpression. The Su(fu) protein is poly-phosphorylated during embryonic development and these phosphorylation events are altered in fu mutants. This study thus reveals an unexpected role for Su(fu) as an activator of Hh target gene expression in absence of Hh signal. Both negative and positive roles of Su(fu) are antagonized by Fused. Based on these results, a model is proposed in which Su(fu) protein levels and isoforms are crucial for the modulation of the different Ci states that control Hh target gene expression (Dussillol-Godar, 2006).

Su(fu) plays a negative role in Hh signalization since it participates both in the cytoplasmic retention of Ci and in the inhibition of the activation of Ci155. This study analyzed the effects of Su(fu) overexpression on appendage development and on the expression of several Hh target genes in the corresponding discs. In parallel, its accumulation and post-translational modifications were examined during embryonic development in fu+ and fu mutant backgrounds (Dussillol-Godar, 2006).

The effects of Su(fu) overexpression on the Hh pathway were assessed by examining both the adult appendage development and the transcription of well characterized Hh targets (such as dpp and ptc) and accumulation of full-length Ci (Ci155) in the corresponding discs. No effect was detected in the posterior compartment, but two apparently opposite effects were observed in the anterior compartment depending on the distance from the source of Hh.

(1) At the A/P border, there was a decrease in the response to low and high levels of Hh signaling. Indeed, dpp and, to a lesser extent, ptc gene expression was reduced. This result is in agreement with the known inhibitory role of the Su(fu) protein in cells transducing the Hh signal (Dussillol-Godar, 2006).

(2) More anteriorly, in cells which do not receive the Hh signal, overexpression of Su(fu) led to anterior duplications in adult appendages. This was correlated with an ectopic expression of dpp in the wing disc or dpp and wg in the leg disc, associated with an accumulation of Ci155. Ectopic ptc expression was also seen but at a much lower level. These effects phenocopy those of cos2 loss of function mutants or of ectopic hh expression. They can be interpreted as a constitutive activation of the pathway. However, the fact that only low levels of ectopic ptc expression are induced shows that the highest levels of Ci activation are not attained (Dussillol-Godar, 2006).

High Ptc protein levels at the boundary are known to sequester the Hh. Thus, the anterior ectopic dpp expression observed in this study in discs overexpressing Su(fu) could be secondary to the deregulation of the Hh pathway at the A/P border: the initial decrease of Ptc at the A/P boundary would result in a further diffusion of Hh to the neighboring cells in which Ci cleavage would be inhibited, allowing hh and dpp expression. So, step by step, a partial activation of the pathway could be propagated up to the anterior region of the wing pouch. Alternatively, the anterior effects of Su(fu) overexpression could occur independently of events at the A/P border. This latter hypothesis is favored for two reasons: (1) induction of Su(fu) overexpression in the A region, outside the A/P border (using either the vgBE-GAL4 driver or clonal analysis), showed that the ectopic activation of dpp can occur independently of Su(fu) overexpression at the A/P border, (2) no significant ectopic hh expression could be detected (Dussillol-Godar, 2006).

At least three Ci states have been postulated to exist, depending on the Hh signal gradient: (1) a fully active Ci (Ciact) responsible for high ptc expression in a stripe 4–5 cells wide close to the A/P border, (2) a full-length Ci (Ci155) sufficient for dpp expression 10–15 cell diameters away from the A/P border, (3) a cleaved Ci form (Ci75) in anterior cells not receiving Hh which represses hh and dpp expression. The balance between these forms of Ci depends on the regulation of non-exclusive processes such as cytoplasmic tethering, protein stability, nuclear shuttling and cleavage. At least two complexes that contain Ci have been identified: a tetrameric Su(fu)–Ci–Fu–Cos2 complex (complex A) probably present in cells receiving a high level of Hh and a trimeric Ci–Fu–Cos2 complex (complex B) which is devoid of Su(fu) and bound to microtubules in the absence of Hh. At the molecular level, Su(fu) binds to N-terminal Ci and thus has the capacity to bind both Ci155 and Ci75. Su(fu) was shown to sequester Ci in the cytoplasm thus controlling the nuclear shuttling of Ci. It was also shown to be involved in the stability of Ci155 and Ci75 (Dussillol-Godar, 2006).

This study shows that overexpression of Su(fu) differentially affects the expression of Hh target genes in Hh-receiving and non-receiving cells and that these effects are all reversed by overexpression of Ci. Moreover, the resulting anterior ectopic activation of dpp is associated with an important accumulation of Ci155. To account for these data, it is hypothesized that Su(fu) overexpression disturbs the balance between the different Ci complexes and thus between the different Ci states. A model is proposed for Hh signaling in imaginal discs in which the effects of Su(fu) over-expression result mainly from the cytoplasmic retention of Ci155. At the A/P boundary in Hh-receiving cells, Ci155 is normally present in a tetrameric complex with Su(fu), Fu and Cos2 (complex A). In these cells, Hh signaling via the activation of Fu blocks Cos2 and Su(fu) negative effects in the tetrameric complex, thus preventing Ci cleavage and cytoplasmic retention and favoring the release of Ci, its activation and nuclear access. Su(fu) overexpression could lead to the recruitment of a significant fraction of endogenous Ci155 into complexes in which Su(fu) is no longer inhibited by Fu. A fraction of Ci is thus sequestered in the cytoplasm as an inactive full-length form. Co-overexpression of Ci along with Su(fu) would provide enough Ci to buffer the excess of Su(fu), leading to the formation of active Ci155. In the anterior region where Hh is absent, Ci is present in a microtubule-bound trimeric complex (complex B) containing Fu and Cos2 but not Su(fu), leading to Ci cytoplasmic tethering and favoring its cleavage in the Ci75 repressive form. This complex would be in equilibrium with a Fu–Su(fu)–Ci complex. In this complex, Su(fu) would act as a safety lock for the cytoplasmic retention of an uncleaved fraction of Ci155 potentially able to yield some active forms of Ci. When Su(fu) is overexpressed, extra Su(fu) would bind Ci155, preventing it from joining the microtubule-bound complex. Ci would not be effectively processed, leading to the accumulation of uncleaved Ci155. The reduction in the amount of Ci75 would be sufficient to allow the expression of dpp but not that of hh, which has been reported to be more sensitive to Ci75 repression than dpp. There would be an enrichment in the other complex but only a few active Ci forms would be produced in agreement with the almost total absence of ectopic ptc expression (Dussillol-Godar, 2006).

The present data show that all the effects induced by overexpression of Su(fu) were enhanced in fu mutants, namely pupal lethality, ectopic anterior expression of dpp and ptc genes and their decrease at the antero-posterior border (Dussillol-Godar, 2006).

At the A/P border, Fu is normally required to antagonize the negative effect of Su(fu) in Hh receiving cells. In fu mutant discs overexpressing Su(fu), the negative effects that Su(fu) exerts on Ci155 cytoplasmic retention in the tetrameric complex would no longer be counteracted by Fu. The shifting of the equilibrium towards the inactive Su(fu)–Ci complex is increased. Less active Ci is available and the reduction in dpp and ptc expression is aggravated (Dussillol-Godar, 2006).

The anterior ectopic activation of the pathway seen in discs overexpressing Su(fu) was greatly enhanced in fu mutants. These unexpected results provide evidence for an inhibitory role of Fu on Ci155 in the absence of the Hh signal. In the absence of Hh, Fu activity could favor the normal restrictive effect of Su(fu) on Ci155 in the Fu–Su(fu)–Ci complex. In fu mutants, the negative effect of Su(fu) on the trapped fraction of Ci155 would be weakened and enough Ci155 would be active to induce transcription of dpp and of ptc (Dussillol-Godar, 2006).

Strikingly, unlike Su(fu) loss of function mutations, Su(fu) overexpression failed to distinguish between the two classes of fu alleles. Since the regulatory domain is probably necessary for Fu kinase activity, the effects seen are probably all mostly due to a loss of Fu kinase activity which would reduce the level of phosphorylation of Su(fu). As shown here and in several recent reports, the Su(fu) protein is phosphorylated in the embryo. Multiple levels of phosphorylation were detected, with hyperphosphorylated forms that accumulate at a period in embryonic development when Fu is activated by the Hh signal and that are significantly reduced in fu mutants. Thus, Fu could modulate Su(fu) activity by controlling, directly or indirectly, its phosphorylation. In the absence of Hh signaling, a low level of Su(fu) phosphorylation by Fu would reinforce the negative effect of Su(fu), whereas a higher phosphorylation level would inactivate Su(fu) in Hh responding cells at the A/P border (Dussillol-Godar, 2006).

Nevertheless, phosphorylated isoforms were not totally abolished in fu mutants, suggesting that other kinase(s) can phosphorylate Su(fu). In agreement with this point, numerous putative phosphorylation sites for kinases such as Caseine kinase II or PKC, but not PKA, are present in the Su(fu) protein. However, the biological implications of the Su(fu) isoforms and their modulation by the Hh transduction signal remain to be demonstrated (Dussillol-Godar, 2006).

Smoothened regulates activator and repressor functions of Hedgehog signaling via two distinct mechanisms

The secreted protein Hedgehog (Hh) plays an important role in metazoan development and as a survival factor for many human tumors. In both cases, Hh signaling proceeds through the activation of the seven-transmembrane protein Smoothened (Smo), which is thought to convert the Gli family of transcription factors from transcriptional repressors to transcriptional activators. This study provides evidence that Smo signals to the Hh signaling complex, which consists of the kinesin-related protein Costal2 (Cos2), the protein kinase Fused (Fu), and the Drosophila Gli homolog cubitus interruptus (Ci), in two distinct manners. Many of the commonly observed molecular events following Hh signaling are not transmitted in a linear fashion but instead are activated through two signals that bifurcate at Smo to independently affect activator and repressor pools of Ci (Ogden, 2006).

This work demonstrates that targeting the association between Smo and the Cos2 cargo domain functionally separates the known molecular markers of the Hh pathway into two distinct categories: those events dependent on a direct association between the Cos2 cargo domain and Smo and those not dependent on this direct association. The Hh-induced readouts requiring direct Smo-Cos2 association include Smo phosphorylation, stabilization, and translocation to the plasma membrane, which facilitate intermediate to high level activation of Ci. Hh-induced Fu and Cos2 hyperphosphorylation, Hedgehog signaling complex relocalization from vesicular membranes to the cytoplasm, and Ci stabilization do not appear to require a direct Smo-Cos2 cargo domain association. Thus, although Smo is necessary for all aspects of Hh signaling, only the molecular events grouped with Ci activation appear to require direct association between Cos2 and Smo. In vivo, carboxyl-terminal Smo binding domain expression is also capable of attenuating Hh signaling. This observation is consistent with in vitro observation that carboxyl-terminal Smo binding domain inhibits critical requirement(s) for pathway activation (Ogden, 2006).

A model has been proposed suggesting the existence of two independently regulated pools of the Hedghog signaling complex (HSC), one involved in pathway repression (HSC-R), and one involved in activation (HSC-A). HSC-R is dedicated to priming Ci for processing into the Ci75 transcriptional repressor, whereas HSC-A is dedicated to activation of stabilized Ci155 in response to Hh. this study provides evidence that the effects of these two HSCs can be functionally separated by specifically targeting the interaction between Smo and the Cos2 cargo domain. Moreover, distinct molecular markers were identified for each HSC. It is proposed that in HSC-R, the membrane vesicle tethered Cos2 functions as a scaffold to recruit protein kinase A, glycogen synthase kinase 3ß, and casein kinase I, which in turn phosphorylate Ci. Hyperphosphorylated Ci is then targeted to the proteasome by the F-box protein supernumerary limbs (Slimb), where it is converted into Ci75. In response to Hh, Fu and Cos2 are phosphorylated and dissociate from vesicular membranes and microtubules, which is suggested to result in the attenuation of HSC-R function. This allows for the subsequent accumulation of full-length Ci. The mechanism by which HSC-R function is inhibited by Hh-activated Smo is not clear but appears to require the carboxyl-terminal tail of Smo and, by this analysis, appears to occur independently of a direct Smo-Cos2 cargo domain association. However, the direct Cos2-Smo association is critical for regulation of HSC-A. In the absence of Hh, HSC-A is tethered to vesicular membranes, through Smo, where it is kept in an inactive state. In the presence of Hh, Cos2 bound directly to Smo acts as a scaffold for the phosphorylation of Smo by protein kinase A, glycogen synthase kinase 3ß, and casein kinase I. Phosphorylation of Smo triggers its stabilization and relocalization to the plasma membrane with HSC-A, where Ci is proposed to be activated. Thus, Cos2 plays a similar role in both HSC-R and HSC-A. In the former case, coupling protein kinase A, glycogen synthase kinase 3ß, and casein kinase I with Ci and, in the latter case, coupling the same protein kinases with the carboxyl-terminal tail of Smo (Ogden, 2006).

An alternative interpretation of these data is that disruption of the Cos2 cargo domain-Smo association separates high and low level Hh signaling. It has been suggested that a second, low affinity Smo binding domain may reside within the coiled-coil domain of Cos2. Thus, high level signaling, where all aspects of the Hh pathway are activated may require both Cos2 interaction domains to be directly bound to Smo. In either scenario, HSC-R function would be regulated independently of HSC-A function (Ogden, 2006).

It is concluded that targeted disruption of Cos2 cargo domain-Smo binding by CSBD is able to functionally separate the activities ascribed to the two HSC model. This two-switch system is amenable to the formation of a gradient of Hh signaling activity across a field of cells, in that the relative activity of HSC-R to HSC-A is directly proportional to the level of Hh stimulation a cell receives. The opposing functional effects of the two complexes can then establish unique ratios of Ci75 to activated Ci, resulting in distinct levels of pathway activation on a per cell basis (Ogden, 2006).

The contributions of protein kinase A and smoothened phosphorylation to hedgehog signal transduction in Drosophila

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 (aee Double-time) 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).

Phosphorylation of the atypical kinesin Costal2 by the kinase Fused induces the partial disassembly of the Smoothened-Fused-Costal2-Cubitus interruptus complex in Hedgehog signalling

The Hedgehog (Hh) family of secreted proteins is involved both in developmental and tumorigenic processes. Although many members of this important pathway are known, the mechanism of Hh signal transduction is still poorly understood. In this study, the regulation of the kinesin-like protein Costal2 (Cos2) by Hh was analyzed. A residue on Cos2, serine 572 (Ser572), is necessary for normal transduction of the Hh signal from the transmembrane protein Smoothened (Smo) to the transcriptional mediator Cubitus interruptus (Ci). This residue is located in the serine/threonine kinase Fused (Fu)-binding domain and is phosphorylated as a consequence of Fu activation. Although Ser572 does not overlap with known Smo- or Ci-binding domains, the expression of a Cos2 variant mimicking constitutive phosphorylation and the use of a specific antibody to phosphorylated Ser572 showed a reduction in the association of phosphorylated Cos2 with Smo and Ci, both in vitro and in vivo. Moreover, Cos2 proteins with an Ala or Asp substitution of Ser572 were impaired in their regulation of Ci activity. It is proposed that, after activation of Smo, the Fu kinase induces a conformational change in Cos2 that allows the disassembly of the Smo-Fu-Cos2-Ci complex and consequent activation of Hh target genes. This study provides new insight into the mechanistic regulation of the protein complex that mediates Hh signalling and a unique antibody tool for directly monitoring Hh receptor activity in all activated cells (Reul, 2007).

These data show that phosphorylation of Cos2 residue Ser572 is necessary for the full activation of Hh signalling, and that this phosphorylation is dependent on the kinase Fu. It is likely that Fu directly phosphorylates Cos2 on Ser572, but it was not possible to purify an activated Fu kinase to confirm this. The phosphorylation of this residue strongly decreased the association of Cos2 with both Ci and Smo, an important step in the regulation of the cytoplasmic anchoring of Ci. By contrast, Cos2-572A, a Cos2 mutant that cannot be phosphorylated at Ser 572, remained associated with Smo and Ci but was much less sensitive to Hh regulation; this is because both its restraining activity on Ci and its association with Ci were only minimally sensitive to the presence of Fu and to the activation of Hh signalling. Phosphorylation of Ser572 of Cos2 induces the partial disassembly of the protein complex (Reul, 2007).

The data show that Cos2 phosphorylated on Ser572 does not bind Smo. However, previous studies have shown that Cos2 is phosphorylated and is pulled down by Smo in response to Hh stimulation. How can these data be reconciled? First, it is possible that not all Cos2 proteins that bind to Smo are phosphorylated. Indeed, only a limited fraction of Cos2 and Fu are sensitive to Hh activation. This is clearly observed with Fu (only 50% of the protein undergoes an electromobility shift upon Hh activation), but is more difficult to quantify with Cos2 because of its very small and diffused electromobility shift. Nevertheless, if Cos2 behaves similarly to Fu, it would mean that 50% of the total Cos2 (corresponding to the non-modified protein in Hh-treated cells) should be able to bring enough Smo down to be detectable in immunoprecipitates. Second, it is possible that Smo still binds to phosphorylated Cos2 on Ser572, but with much less affinity. Third, phosphorylation on Ser572 is not responsible for all Cos2 mobility shift, because Cos2-572A still shifts upon OA treatment, suggesting that other phosphorylated sites are present. Therefore, some phosphorylated isoforms that are not phosphorylated on Ser572 might also be associated with Smo. It is thus possible that this study has revealed only one of a series of sequential phosphorylation events on Cos2 that ultimately lead to the complete dissociation of Cos2 from Smo. Finally, it is worth mentioning that more Smo is present in the Cos2 IP from Hh-treated cells than in non-Hh treated cells. This is thought to simply reflect an increased level of Smo resulting from Hh signalling activation, and not the Hh-dependent regulation of the efficiency of the interaction of Smo with Cos2 (Reul, 2007).

The role of the Cos2 protein in the complex is to serve as a platform to allow both positive and negative regulators to be brought into close proximity with Smo and Ci. Thus, the role of Cos2 in transmitting a response can be masked by the role of Cos2 in limiting pathway activity in the absence of Hh. At low concentrations, it is able to stimulate Hh reporter activity in vitro and engrailed expression in vivo. But in Cos2-572A-expressing cells, engrailed expression was lower than in wild-type discs, and the in vitro stimulation of Hh signalling could not be potentiated by Fu activity. Moreover, the restraining activity of Cos2-572A on Ci could not be counteracted by Hh or Fu in vitro. Therefore, it is proposed that the Ser572 to Ala substitution on Cos2 rendered Cos2 less sensitive to Hh and Fu regulation. Because Cos2-572A still binds to its partners, it could bring Fu into proximity with its other targets. Indeed, it is likely that Fu activation leads not only to the direct phosphorylation of Cos2 but also to direct changes in Ci and/or other partners, such as Sufu. This explains why Cos2-572A is still able to stimulate Hh signalling, albeit not to its highest level (Reul, 2007).

From the Cos2-572A results, one could wonder why Cos2-572D did not constitutively activate the pathway. Because the Cos2-572D form is in a 'frozen' state compared with the wild-type form, cycles of phosphorylation/dephosphorylation are blocked and thus Cos-572D cannot participate in the Hh complex signalling anymore. The data show that constitutively phosphorylated Cos2 and endogenous phospho-Cos2 are bound to Fu but are dissociated from Smo and Ci. Therefore, Fu bound to phosphorylated Cos2 would be absent from the complex, preventing the release of all the cytoplasmic anchors from Ci (Reul, 2007).

Because the Cos2 Ser572 residue is not part of the Ci- or Smo-binding domains, but phosphorylation of this site nevertheless leads to the dissociation of these two proteins from Cos2, it is proposed that the Fu-mediated modification of Cos2 induces the protein to undergo a conformational change that leads to the disassembly of the complex. The disassembly is partial because phosphorylated Cos2 and Fu are still associated. Interestingly, it has been proposed that the binding of Cos2, Sufu and Fu to Ci masks a nuclear localisation site on Ci (Ci-NLS). A conformational change that supports this idea: that disassembly of the complex is necessary to expose the Ci-NLS and for consequent nuclear translocation (Reul, 2007).

Regulation of Ci-SCFSlimb binding, Ci proteolysis, and hedgehog pathway activity by Ci phosphorylation

Hedgehog (Hh) proteins signal by inhibiting the proteolytic processing of Ci/Gli family transcription factors and by increasing Ci/Gli-specific activity. When Hh is absent, phosphorylation of Ci/Gli triggers binding to SCF ubiquitin ligase complexes and consequent proteolysis. This study shows that multiple successively phosphorylated CK1 sites on Ci create an atypical extended binding site for the SCF substrate recognition component Slimb. GSK3 enhances binding primarily through a nearby region of Ci, which might contact an SCF component other than Slimb. Studies of Ci variants with altered CK1 and GSK3 sites suggest that the large number of phosphorylation sites that direct SCFSlimb binding confers a sensitive and graded proteolytic response to Hh, which collaborates with changes in Ci-specific activity to elicit a morphogenetic response. When Ci proteolysis is compromised, its specific activity is limited principally by Su(fu), and not by Cos2 cytoplasmic tethering or PKA phosphorylation (Smelkinson, 2007).

The central task of Hh signal transduction in Drosophila is to regulate the activity of the transcription factor Ci. This is accomplished by regulating the levels of Ci-155 activator and Ci-75 repressor and the specific activity of Ci-155. To examine the regulation of Ci-155-specific activity in isolation, a Ci variant (Ci-S849A) was developed that escapes PKA-dependent proteolysis but has a minimally altered pattern of PKA-initiated phosphorylation. At 29°C, Ci-S849A was expressed in wing discs (via C765-GAL4) at roughly the same level as endogenous Ci, but at 20°C, the levels of Ci-S849A were distinctly lower. At 29°C, Ci-S849A induced the Hh target gene reporter ptc-lacZ in A cells that are not stimulated by Hh. Since ptc induction requires Ci-155 activator and is not accomplished simply by loss of Ci-75 repressor, it is concluded that elevated levels of Ci-155 can suffice to confer some activator function. At 20°C, Ci-S849A induced ptc-lacZ in A cells only when Su(fu) was removed. By contrast, Ci-S849A activity was not detectably increased by loss of either PKA activity or Cos2 activity in P smo mutant clones, where Hh signaling is blocked. Thus, Su(fu) is the principal component that limits Ci-155-specific activity when Ci-155 is protected from PKA-dependent proteolysis, whereas PKA and Cos2 normally limit Ci-155 activity simply by promoting its proteolysis (Smelkinson, 2007).

The prior assertion that PKA limits Ci-155-specific activity by direct phosphorylation was based on the use of an inappropriate reagent (Ci-U), which was mistakenly thought to be inert to PKA-dependent proteolysis. A similar role for Cos2 was previously inferred principally from the observation that Ci-155 accumulated more rapidly in the nuclei of anterior Leptomycin B-treated wing disc cells when those cells lack . It appears that the inferred cytoplasmic retention of Ci-155 by Cos2 contributes very little quantitatively to limiting the activity of stabilized Ci-155. That conclusion is supported by the observation that loss of Cos2 function did not enhance the weak induction of En seen in pka mutant clones of otherwise WT wing discs. The remaining, long-standing observations suggesting roles for PKA and Cos2 in limiting Ci-155-specific activity are the different degrees of Hh target gene induction in pka (strongest), cos2 (intermediate), and slimb (weakest) mutant wing disc clones. It is suggested that this might result from different degrees of disruption of PKA-dependent proteolysis in these clones. This suggestion is consistent with the proposed role of Cos2 in facilitating Ci-155 phosphorylation and with the observation that ptc-lacZ can be induced in slimb mutant clones when PKA activity is halved (Smelkinson, 2007).

Both GST-Ci association with SCFSlimb and Ci-155 proteolysis depend on two phosphorylated regions of Ci. The first region provides an essential Slimb binding site that can be created by five successive CK1 phosphorylations primed initially by PKA site 1. Of the four phosphorylated residues within the motif (844SpTpYYGSpMQSp852) that interacts directly with Slimb, at least one (S849) is essential for binding, and two others (S844 and S852) enhance binding (T845 is essential, but priming and binding functions have not been separated). The second critical phosphorylated region of Ci includes two GSK3 sites (S884 and S888) that are primed by PKA site 3. This region enhances, but is not sufficient for, binding to SCFSlimb (Smelkinson, 2007).

The requirement for multiple successive phosphorylations by PKA, CK1, and GSK3 to create a high-affinity SCFSlimb binding domain on Ci-155 has two important consequences. First, it demands a special mechanism for facilitating Ci phosphorylation that is met by Cos2. Second, it provides a mechanism through which a small change in Ci-155 phosphorylation, induced for example by limited dissociation of protein kinases from Cos2, can be translated into a substantial inhibition of Ci-155 proteolysis. The sharp increase in Ci-155 levels at the anterior limit of Hh signaling territory shows that a low dose of Hh does indeed severely curtail PKA-dependent Ci-155 proteolysis. Hh could inhibit proteolytic processing of Ci variants driven by either PKA-primed GSK3 sites (Ci-SL) or PKA-primed CK1 sites (Ci-G2,3E and Ci-Y846G), but complete inhibition was observed only for the latter pair. This suggests that the sensitive response of Ci proteolysis to Hh depends principally on CK1 (Smelkinson, 2007).

How does inhibition of Ci-155 proteolysis affect Ci-155 activity? Previously, the properties of Ci-U and slimb mutant clones were taken as evidence that inhibition of proteolysis does not suffice for Ci activation. Since Ci-U is subject to PKA-dependent proteolysis and PKA does not affect the specific activity of Ci-155, instead the properties of Ci-S849A and pka mutant clones were relied upon to conclude that complete inhibition of PKA-dependent proteolysis does suffice to induce the Hh target gene ptc. This, in turn, suggests that the high Ci-155 levels anterior to the stripe of elevated ptc expression at the AP border of wing discs result from substantial, but incomplete, inhibition of Ci-155 proteolysis (Smelkinson, 2007).

It has generally been assumed that inhibition of Ci-155 proteolysis is uniformly strong throughout the AP border and that the activation of ptc and en in nested domains is due solely to changes in Ci-155-specific activity elicited by increasing levels of Hh. However, several factors suggest that there may also be a significant gradient of residual PKA-dependent proteolysis at the AP border that contributes to morphogen action (Smelkinson, 2007).

First, the precise degree of substantially inhibited Ci-155 proteolysis can determine whether Hh target genes are induced or not. This is evident from differences in ptc-lacZ induction among proteolytically impaired Ci variants and between pka and slimb mutant clones (Smelkinson, 2007).

Second, Su(fu) is the principal regulator of Ci-155 activity when Ci-155 levels are elevated, yet Hh instructs an almost unchanged morphogenetic response in the absence of Su(fu). It is likely that a proteolytic gradient is critical under these conditions, although it is also possible that Cos2 assumes a more significant role in regulating Ci-155-specific activity when Su(fu) is absent (Smelkinson, 2007).

Third, it was found that the loss of any one of four phosphoserines that contribute to Ci-Slimb binding (S844, S852, S884, and S888) diminishes, but does not abolish, Slimb binding. For S888A (G3A) this results in elevated Ci-155 levels and an increased activity, but residual proteolysis is clearly evident from the generation of sufficient Ci-75 to repress hh-lacZ. Thus, dispersion of direct Slimb binding determinants among several phosphorylatable residues provides a mechanism for Hh to elicit graded inhibition of Ci-155 proteolysis. It is speculated that in response to high levels of Hh, most Ci-155 molecules will not bind to SCFSlimb at all because they lack at least two of the six key phosphorylated residues, whereas a large proportion of Ci-155 molecules may bind SCFSlimb with intermediate affinity in response to low or intermediate Hh levels because they lack only one critical phosphoserine (Smelkinson, 2007).

Fourth, regulation of Ci-155-specific activity depends on Ci-155 levels. Thus, Ci-155 is only activated by loss of Su(fu) when Ci-155 levels are elevated by Hh or appropriate mutations, presumably because other stoichiometric binding partners such as Cos2 act redundantly with Su(fu) when their Ci-155 binding capacity is not saturated. It would therefore be expected that the release of Ci-155 from repressive partners is progressively facilitated as the relative levels of Ci-155 increase. This would allow increasing Hh levels to enhance Ci-155 activity through synergistic effects on Ci-155 levels and Ci-155-specific activity (Smelkinson, 2007).

The archetypal β-TRCP/Slimb substrates, β-catenin and IKB, contain a single, dually phosphorylated, high-affinity binding site (DSpGxxSp) that triggers rapid substrate proteolysis. The primary direct Slimb binding motif that has been defined (SpTpYYGSpMQSp) in Ci differs notably by the presence of Tyr instead of Gly at the third position, by the inclusion of a fourth electronegative residue at its C terminus, and by binding with lower affinity, permitting additional influences on Ci-SCFSlimb association. The fourth electronegative residue (pS852) likely interacts with at least one of two positively charged Slimb surface residues (R333 and R353) based on their potential proximity and reduced GST-Ci binding to the R333A/R353A Slimb variant (Smelkinson, 2007).

Most known β-TRCP substrates include phosphorylated or acidic residues that are two to four residues C-terminal to the standard six amino acid binding motif (DSpGXXSp), but their contribution to binding has not generally been assessed. Even β-catenin includes such phosphorylated residues that are known to have an essential priming role but have not been tested rigorously for direct interactions with β-TRCP. The variant β-TRCP binding motif (EEGFGSpSSp) of mammalian Wee1A presents a notable exception, in which a β-TRCP Arg residue equivalent to R353 of Slimb interacts with the phosphoserines at position 6 and 8 of this motif. This suggests that positive surface residues of β-TRCP/Slimb may commonly stabilize association with extended binding motifs. It is speculated that extended β-TRCP/Slimb binding motifs are likely to be especially important and prevalent in substrates lacking Gly at the third position because it was found that R333 or R353 (or both) of Slimb promotes binding to Ci-WT, but not to Ci-SL, and pS852 contributes significantly to Slimb binding in Ci-WT, but not in Ci-Y846G (Smelkinson, 2007).

Vertebrate Gli homologs of Ci also have a residue other than Gly at the third position (generally Ala) and a potentially phosphorylated Ser at the C terminus of a putative extended β-TRCP binding motif of 9 to 11 residues (SSAYx(x)SRRSS). Both a second Wee1A binding motif (DSAFQEPDS) and a β-TRCP binding motif of the p100 precursor of NFKB p52 (DSAYGSQSVE) also lack a Gly residue at the third position and include residues beyond the six amino acid core motif that might, by analogy to Ci, potentiate binding (Smelkinson, 2007).

Studies of Ci provide a clear precedent for the use of an extended β-TRCP/Slimb binding motif to translate regulated substrate phosphorylation into regulated proteolysis. However, it was also found that Slimb binding cannot be predicted by focusing on only the interactions of charged residues. Thus, fully phosphorylated Ci includes two sequences with a distribution of charged residues similar to that of the primary SCFSlimb binding site (837DSpQNSpTpASpTp and 858SpSpQVSpSpIPTp compared with 844SpTpYYGSpMQSp), but those sites neither suffice for Slimb binding (in Ci-S849A) nor enhance binding significantly in vitro (as revealed by D837A, T842A, S858A, S859A, and Ds2 variants). This probably reflects significant binding contributions of nonpolar residues in positions 3-5 of an extended Slimb/β-TRCP binding motif, as suggested by the presence of Tyr or Phe at position 4 of the functional motifs of Ci, Gli, p100, and Wee1A (Smelkinson, 2007).

Several F-box proteins that use WD40 repeats to bind substrate (FBW proteins) also include a dimerization domain that directs assembly of higher-order SCF complexes. Some substrates of these SCF complexes (for example, Cyclin E for Fbw7 and Wee1A for β-TRCP) contain more than one phosphorylated region capable of interacting with the same WD40 binding surface of the FBW protein. This raises the question of whether the cooperative function of two or more such regions depends on SCF dimerization and simultaneous interaction with two FBW subunits of a dimeric complex (Smelkinson, 2007).

This study found that Ci also contains two phosphorylated regions that contribute to SCF association, and Slimb molecules can bind to each other within higher-order functional SCF complexes. It was also found that Slimb self-association enhanced binding to GST-Ci relative to GST-Ci-SL, which contains a single DSGxxS motif; however, it was not required for both phosphorylated regions of GST-Ci to stimulate binding. Hence, simultaneous binding to separate Slimb monomers within a larger complex can be excluded as a requisite mechanism for cooperativity between the two phosphorylated regions of Ci. This is consistent with recent structural studies that predict a wider separation of WD40 binding surfaces within an SCF dimer than can be spanned readily by the two critical phosphorylated regions of a single Ci molecule (Smelkinson, 2007).

Does the region preceding PKA site 3 of Ci bind directly to the FBW component (Slimb) of an SCF complex as for Cyclin E and Wee1A? That model was proposed for Gli-2/3 proteins, which include a recognizable, potentially extended, variant Slimb-binding motif (DSYDPISTDAS). The analogously positioned sequence in Drosophila (SFYDPISPGCS) retains the YDPIS sequence and the two GSK3 sites at position 7 and 11 (underlined) but lacks a Ser at position 2 (italics), which is required in Gli-2/3 for normal β-TRCP association and proteolysis. Also, Ala substitution of the first Ser in the Drosophila motif (together with three other Ser residues) had only a minor effect on Slimb binding in vitro and Ci-155 proteolysis in vivo. Thus, this region of Ci does not have a clearly recognizable and demonstrably functional, conventional β-TRCP/Slimb binding site. It is, nevertheless, conceivable that the conserved elements of the putative β-TRCP binding motif of Gli-2/3 might provide a very weak direct interaction with the WD40 domain of Slimb that is sufficient to enhance SCFSlimb association (Smelkinson, 2007).

However, this study also found that the GSK3 enhancement of Ci-Slimb binding conferred by the GSK3 sites preceding PKA site 3 was lost if Slimb lacked an F-box domain and consequent direct association with SkpA and its SCF complex partners. This result is interpreted with some caution because Slimb-ΔF also bound less well than wild-type Slimb to a canonical β-catenin substrate and to GST-Ci that was phosphorylated only at its primary Slimb binding site. Nevertheless, the result suggests that the region of Ci immediately preceding PKA site 3 might augment SCF association by binding directly to an SCF component other than Slimb (Smelkinson, 2007).

Whether GSK3 stimulates Ci binding to SCFSlimb via a direct interaction with Slimb, an unprecedented interaction with another SCF component, or a conformational effect on the primary Slimb binding site of Ci, the Ci-G3A transgene reveals that the stimulation conferred by GSK3 phosphorylation is critical for efficient Ci-155 proteolysis and for Hh pathway silencing. Since Slimb self-association enhanced GST-Ci, but not GST-Ci-SL, binding in vitro, it is suspected that this may also be important for Ci-155 proteolysis in vivo. It is not known if SCFSlimb dimerization (or oligomerization) is regulated, but the different modes of association of SCFSlimb with Ci and β-catenin certainly provide several opportunities for SCF regulatory mechanisms or mutations to affect the Hh pathway without altering the Wnt/β-catenin pathway (Smelkinson, 2007).

G protein Galphai functions immediately downstream of Smoothened in Hedgehog signalling

The Hedgehog (Hh) signalling pathway has an evolutionarily conserved role in patterning fields of cells during metazoan development, and is inappropriately activated in cancer. Hh pathway activity is absolutely dependent on signalling by the seven-transmembrane protein smoothened (Smo), which is regulated by the Hh receptor patched (Ptc). Smo signals to an intracellular multi-protein complex containing the Kinesin related protein Costal2 (Cos2), the protein kinase Fused (Fu) and the transcription factor Cubitus interruptus (Ci). In the absence of Hh, this complex regulates the cleavage of full-length Ci to a truncated repressor protein, Ci75, in a process that is dependent on the proteasome and priming phosphorylations by Protein kinase A (PKA). Binding of Hh to Ptc blocks Ptc-mediated Smo inhibition, allowing Smo to signal to the intracellular components to attenuate Ci cleavage. Because of its homology with the Frizzled family of G-protein-coupled receptors (GPCR), a likely candidate for an immediate Smo effector would be a heterotrimeric G protein. However, the role that G proteins may have in Hh signal transduction is unclear and quite controversial, which has led to widespread speculation that Smo signals through a variety of novel G-protein-independent mechanisms. This study presents in vitro and in vivo evidence in Drosophila that Smo activates a G protein to modulate intracellular cyclic AMP levels in response to Hh. The results demonstrate that Smo functions as a canonical GPCR, which signals through Gαi to regulate Hh pathway activation (Ogden, 2008).

To examine whether a G protein is involved in Hh signalling, a series of G proteins was targetted by double-stranded RNA (dsRNA)-mediated knockdown. Drosophila clone-8 (Cl8) cells were treated with control or Gα-subunit-specific dsRNA and assayed for changes in Hh-mediated induction of a ptc-luciferase reporter construct. Whereas s (also called G-sα60A) and o (also called G-oα47A) dsRNAs do not significantly alter Hh-induced reporter activation knockdown is able to trigger a decrease in Hh-dependent reporter gene expression. Although not as effective as Smo knockdown in silencing Hh reporter gene activation, i (also called G-iα65A) dsRNA specific to the coding sequence, or 3' untranslated region (UTR), reduces Hh-induced reporter activity by approximately 70%, supporting a role for Gαi in the Hh pathway. To confirm the specificity of i dsRNA effects attempts were made to rescue reporter activity through ectopic expression of wild-type i or constitutively active GαiQ205L. Hh-stimulated reporter activity can be restored by both wild-type and constitutively active Gαi, confirming the specificity of the i dsRNA-mediated effects. Western blot analyses of Cl8 lysates reveal that cells treated with i dsRNA show attenuated stabilization of Ci and decreased Fu phosphorylation in response to Hh. Hh-induced Smo phosphorylation is maintained in the presence of i dsRNA, suggesting that Gαi functions downstream of Smo and upstream of Fu and Ci (Ogden, 2008).

To determine whether Gαi can modulate Hh pathway activity in vivo, i constructs were expressed in wing imaginal discs using MS1096-Gal4 or C765-Gal4. Expression of an inactive i mutant (iG204A) or wild-type i has little effect on wing vein patterning. However, expression of constitutively active iQ205L results in widening of longitudinal vein LV3-LV4 spacing and ectopic vein material on LV2 and LV3. The severity of this phenotype is dose-dependent, as higher-level expression of UAS-GαiQ205L triggers more severe ectopic vein material anterior to LV3, and further widening of LV3-LV4 spacing. Expression of iQ205L in wing imaginal discs also results in over-growth of the wing pouch, along with expansion of full-length Ci. This Ci expansion triggers ectopic expression of the Hh target gene decapentaplegic (dpp) in the wing pouch, as shown by a dpp-lacZ reporter gene. Gαi-mediated ectopic expression of dpp is consistent with the ectopic veins observed in wings expressing iQ205L. Taken together, these results support a role for activation of Gαi in regulating the stability of Ci, and link Gαi to regulation of a known Hh target gene (Ogden, 2008).

To determine whether Gαi functions downstream of Smo in vivo, the ability of Gαi to modulate Hh pathway activity was analysed in a smo sensitized background. As previously demonstrated, expression of a dominant-negative smo transgene, UAS-Smo5A, results in severe disruption of LV3-LV4 wing patterning. Expression of wild-type Gαi in this smo sensitized background allows for partial rescue of wing vein structures in the LV3/LV4 zone. Expression of constitutively active iQ205L results in a more complete rescue of the Hh loss-of-function phenotype, allowing for near total restoration of LV3/LV4 patterning. As a control, UAS-GFP was co-expressed with Smo5A, and found to have no effect on the Smo5A-induced phenotype (Ogden, 2008).

To examine the ability of GαiQ205L to modulate Ci stability and Hh target gene activation in the smo sensitized background, wing imaginal discs were immunostained with antibodies that recognize full-length Ci and the target gene product Ptc. UAS-Smo5A expression results in decreased ptc expression and disruption of the Ci gradient. Expression of constitutively active iQ205L in this smo sensitized background results in partial restoration of the Ci gradient and a near-complete rescue of ptc expression at the anterior/posterior border. These results support the model that Gαi contributes to the regulation of Hh target gene expression and Ci stability. Furthermore, the fact that this regulation occurs when Smo function is compromised suggests that Gαi affects Hh signalling at a level downstream of Smo (Ogden, 2008).

To determine whether Gαi is required for Hh signalling in vivo, Hh target gene expression was examined in clones of cells homozygous for i mutation. The null allele iP20 removes the entire coding region of the i gene, and is homozygous lethal. iP8 is a putative hypomorph, which removes the bulk of exons 1 and 2, but leaves the transcriptional start site intact and produces a transcript. Flies that are homozygous for the iP8 mutation are viable, but weak. Mosaic analysis reveals that expression of the Hh target gene dpp is decreased in both iP20 and iP8 mutant clones, supporting a role for Gαi in activation of Hh target genes in vivo. To confirm that the effects on dpp expression are due to loss of i, attempts were made to rescue iP20 null clones with UAS-i. Ectopic expression of i is able to rescue dpp reporter gene expression in iP20 clones, consistent with decreased dpp expression resulting from disruption of i (Ogden, 2008).

To determine whether compromised Gαi activity alters Hh-dependent patterning, the viable mutant allele iP8 and an additional viable allele described to be a null or strong hypomorph, i57 were used. Whereas homozygous iP8 and i57 mutants do not have vein fusions that are typical of strong Hh loss of function, their wings are smaller than wild-type wings. Small wing size might result from altered dpp expression in anterior cells of the wing pouch, as Dpp regulates wing blade size. Additionally, both iP8 and i57 mutant flies demonstrate varying degrees of incomplete thorax closure, as shown by mild to severe thoracic clefts. This phenotype is also consistent with decreased dpp expression, in that Dpp, in conjunction with JNK signalling, controls spreading of the anterior edge of wing imaginal discs to initiate thorax closure. To confirm that this phenotype results from decreased Hh signalling, ptc was expressed in the notum and dorsal compartment of the wing imaginal disc. ptc expression triggers the formation of a thoracic cleft when expressed under control of pannier and apterous promoters, suggesting that the thoracic phenotype observed in i flies results from compromised Hh signalling. Because iP20 null mutant animals are not viable, their wings or thoraces could not be examined. However, attenuation of Hh signalling by expressing dominant-negative Smo5A is enhanced in iP20 heterozygotes, as shown by disruption of LV3 (Ogden, 2008).

in vitro and in vivo data suggest that loss of Gαi might compromise Ci stabilization in Hh-receiving cells. When Ci and Smo levels were examined in i mutant clones, both appeared to be increased in a cell-autonomous manner. These results are consistent with the modest stabilization of Smo and Ci on in vitroi knockdown in non-Hh-treated cells. Although these results are unexpected, as Gαi loss is predicted to increase PKA activity and Ci degradation, previous studies have demonstrated that PKA functions to regulate Hh signalling both positively and negatively. Phosphorylation of Smo by PKA has a positive role in pathway activation, and might account for the modest stabilization of Ci that was observed (Ogden, 2008).

If Smo signals through Gαi it should be able to induce Gαi activation rapidly in response to Hh stimulation. To assay for Hh-mediated activation of Gαi, Cl8 cells were treated with conditioned media containing the amino-terminal Hh signalling molecule (HhN) or control conditioned media, then assayed for Hh-induced changes in intracellular cAMP. Within 5-10 min, HhN treatment reduces the basal intracellular cAMP concentration by approximately 50%. To confirm that the Hh-induced decrease in intracellular cAMP is dependent on Hh signalling through Smo and Gαi, cells were treated with smo, i or control dsRNA, then assayed for a Hh-induced decrease in cAMP. Whereas cells transfected with control dsRNA maintain the ability to decrease intracellular cAMP in response to HhN, cells transfected with either smo or i dsRNA are attenuated in their ability to do so. Taken together, these results support the idea that i is activated rapidly, in a Smo-dependent manner, in order to modulate cAMP levels in response to Hh (Ogden, 2008).

To determine whether modulation of cAMP can alter Hh signalling in vivo, a hypomorphic mutant allele of the cAMP-specific phosphodiesterase dunce (dnc1) was used to raise intracellular cAMP levels in a Hh-independent manner. Hemizygous dnc1 animals are viable with no obvious Hh defects. However, introduction of the dnc1 mutation into a smo sensitized background enhances the Smo loss-of-function phenotype, resulting in wings with near complete elimination of wing vein patterning. This enhanced Hh loss-of-function phenotype is similar to the phenotype obtained on decreasing smo gene dosage by one-half in the same smo sensitized background. Along with in vitro cAMP assays, these results indicate that Hh activates Smo to modulate intracellular cAMP, via Gαi, and that this function is important for proper pathway activity in vivo (Ogden, 2008).

Cos2 associates with membranes, microtubules, PKA, Smo, Fu and Ci. To determine whether Cos2 facilitates the coupling of Gαi with these Hh signalling components, lysates were prepared from cells expressing HA-Gαi, and then immunoprecipitated Cos2. It was found that Gαi associates with the Cos2 complex, and that this association is enriched in response to Hh. The binding of Fu to Cos2 is not altered by Hh, suggesting that the recruitment of Gαi to this protein complex is regulated. This result suggests that Cos2 facilitates the coupling of Smo with Gαi and additional downstream effectors necessary to transduce the Hh signal (Ogden, 2008).

This study has shown a requirement for Gαi in the Hh signalling pathway. Hh-mediated recruitment and activation of Gαi results in decreased intracellular cAMP, indicating that Hh may regulate PKA through modulation of the intracellular cAMP concentration. It was also demonstrated that Gαi can modulate Hh pathway activity in vitro and in vivo, and seems to do so at a level downstream of Smo. Furthermore, loss of Gαi alters Hh signalling in vivo, supporting the idea that Gαi is a requisite member of the Hh pathway (Ogden, 2008).

Interaction between Ataxin-2 Binding Protein 1 and Cubitus-interruptus during wing development in Drosophila

Animal growth and development is dependent on reiterative use of key signaling pathways such as Hedgehog (Hh) pathway. It is widely believed that Cubitus-interruptus (Ci) mediates all functions of Hh pathway. This study reports that CG32062 (Ataxin-2 binding protein 1), the Drosophila homologue of Ataxin-2 Binding Protein 1, functions as a cofactor of Ci to specify intervein region between L3 and L4 veins of the adult wing. CG32062 was identified in an enhancer-trap screen to identify genes that show differential expression between wing and haltere discs. Ci-mediated transactivation of knot/collier (kn) in the developing wing imaginal disc is dependent on dA2BP1 function. Protein interaction studies and chromatin-immunoprecipiation experiments suggest that Ci helps dA2BP1 to bind kn promoter, which in turn may help Ci to activate kn expression. These results suggest a mechanism by which Ci may activate targets such as kn, which do not have classical Ci/Gli-binding sites (Usha, 2010).

Hedgehog (Hh) is an important signaling molecule regulating a variety of events throughout development as well as adult life of all higher eukaryotes. All functions of Hh are mediated by Ci. This raises the question of how spatio-temporal diversity in the output of Hh pathway is established. It is generally assumed that tissue-specific factors act to regulate Ci function. So far, the only report of such factors is of Teashirt (Tsh) and Armadillo (Arm). It is believed that these two proteins complex with Ci to specify naked cuticle at distinct positions along the antero-posterior axis of Drosophila embryos. However, it is still unclear how they might influence Ci function (Usha, 2010).

This report demonstrates a role for dA2BP1 as a co-factor of Ci necessary for regulating kn expression during wing development. This study identified dA2BP1, an ortholog of Ataxin2 Binding Protein or Fox-1, in a previous study involving identification of genes with differential expression in wing and haltere discs. Loss of dA2BP1 leads to loss of intervein region between L3-L4 veins of adult wings- a phenotype similar to kn mutants. Furthermore, down regulation of dA2BP1 leads to a complete loss of kn RNA expression (Usha, 2010).

The role of Hh in patterning the vein and intervein regions is well established. kn has been shown to be a direct target of Hh signaling in patterning the intervein region between veins 3 and 4. While over-expression of Ci leads to upregulation of kn, down-regulation of dA2BP1 can suppress this effect, suggesting that dA2BP1 is necessary for Ci-mediated kn expression. Immunoprecipitation experiments suggest interactions at the protein levels (which could be either direct or indirect) between dA2BP1 and the 155 kDa activator form of Ci. In-vitro, Ci and dA2BP1 can independently bind to upstream sequence of kn; ChIP experiments done using truncated versions of Ci suggest that in-vivo binding of dA2BP1 on kn enhancer is dependent on Ci but not vice-versa. Thus, dA2BP1 appears to be a factor recruited by Ci to bring about transcription of kn. This appears to be context (or promoter-) dependent since, in the same cells that express kn, Ci is also known to regulate the expression of dpp and, this function is independent of dA2BP1 (Usha, 2010).

In vivo genetic interaction studies followed by immunoprecipitation experiments with different forms of Ci suggest interactions between dA2BP1 and repressor forms of Ci too. However, it is likely that interaction between dA2BP1 and full-length activator form of Ci is important during wing morphogenesis (Usha, 2010).

The promoter-specific role of dA2BP1 leads to questions on the nature of interactions between dA2BP1 and Ci to activate kn, but not for the activation of dpp. It is possible that dA2BP1 may enhance the stability of binding and transcriptional activator function of Ci on kn promoter. In this context, it is to be noted that dA2BP1 binds to the Zinc finger domain of Ci, which is the main DNA binding domain. As dA2BP1 has DNA-binding properties (based on in vitro evidence), it is suggested that once bound to Ci, dA2BP1 may anchor on DNA and this helps stabilizing binding of Ci to kn promoter. As binding of dA2BP1 on kn-promoter is also sequence dependent (based on the results of in vitro electromobility shift experiments: dA2BP1 binds sequences from kn promoter and not those of dpp), this property may also help in better stabilizing Ci-dA2BP1 complex on kn promoter, which does not have classical Ci/Gli-binding sites. Alternatively, when both Ci and dA2BP1 together bind to kn sequences, dA2BP1 may recruit additional factors on to the chromatin. This local chromatin modulation may help in regulating kn expression. Identification of other proteins, if any, that are part of dA2BP1-Ci complex may help in understanding precise molecular mechanism by which dA2BP1 modulates Ci function (Usha, 2010).

dA2BP1 appears to be a complex protein with a predicted RNA-binding motif and poly-glutamine domains. RNAi-mediated knockdown of dA2BP1 using ubiquitous GAL4 drivers such as tubulin-GAL4, hs-GAL4 etc were early larval lethal, suggesting that dA2BP1 has vital function during development. Its dynamic expression pattern too indicates the same. This study analyzed just one part of its function. It is likely that it interacts with different proteins/RNA in different tissues generating diversity in its functions (Usha, 2010).

Regulation of mammalian Gli proteins by Costal 2 and PKA in Drosophila reveals Hedgehog pathway conservation

Hedgehog (Hh) signaling activates full-length Ci/Gli family transcription factors and prevents Ci/Gli proteolytic processing to repressor forms. In the absence of Hh, Ci/Gli processing is initiated by direct Pka phosphorylation. Despite those fundamental similarities between Drosophila and mammalian Hh pathways, the differential reliance on cilia and some key signal transduction components had suggested a major divergence in the mechanisms that regulate Ci/Gli protein activities, including the role of the kinesin-family protein Costal 2 (Cos2), which directs Ci processing in Drosophila. This study shows that Cos2 binds to three regions of Gli1, just as for Ci, and that Cos2 functions to silence mammalian Gli1 in Drosophila in a Hh-regulated manner. Cos2 and the mammalian kinesin Kif7 can also direct Gli3 and Ci processing in fly, underscoring a fundamental conserved role for Cos2 family proteins in Hh signaling. Direct PKA phosphorylation regulates the activity, rather than the proteolysis of Gli in Drosophilia, and evidence is provided for an analogous action of PKA on Ci (Marks, 2011).

There are clearly many conserved features of Hh signaling between Drosophila and mammals, and some explicit differences, most obviously with regard to the employment of cilia. However, incomplete understanding can lead to premature conclusions as to exactly what is conserved. When studies of Gli activity were initiated in Drosophila, the absence of key fly Smo activation and Cos2-binding domains from mammalian Smo, coupled to studies showing no effect of Kif7 and Kif27 RNAi on the Hh pathway in tissue culture cells, had led to the suggestion that a Cos2-like molecule was not important in regulating Gli activity in mammals. This study has shown that Cos2 is essential for Gli3 processing into a repressor form in Drosophila, that Cos2 prevents constitutive Gli1 activity while permitting Gli1 activation by Hh in Drosophila and that Cos2 binds to three distinct regions of Gli1, as observed for Ci. These results strongly indicated that a Cos2-like molecule must regulate the activity of Gli proteins in mammals. Indeed, genetic elimination of Kif7 in mouse was subsequently found to reduce processing of Gli3 and was also inferred to result in partial activation of Gli2 in the absence of a Hh ligand. The fundamental demonstration of conservation of Cos2 function focuses attention on more precise questions about how Cos2-family proteins regulate Ci/Gli protein function. Some of these questions have been addressed by comparing the regulation of Ci and Gli proteins in Drosophila (Marks, 2011).

Although physiological regulation of Gli1 protein activity by Shh was demonstrated by knocking Gli1 coding sequence into the mouse Gli2 locus, those mice have not been extensively characterized further to test which specific factors regulate Gli1 activity in mouse. In mice with wild-type Gli genes, the dependence of Gli1 transcription on Hh pathway activity coupled with the ubiquitous presence of Gli2 as a major sensor of pathway activity has prevented an assessment of post-transcriptional regulation of Gli1. For example, genetic loss of mouse suppressor of fused (Sufu) leads to substantial activation of Shh-target genes and transcriptional induction of Gli1, leading only to the conclusion that Sufu normally limits Gli2 activity. Similarly, the effects of mutations affecting cilia or Kif7 have been interpreted only with respect to Gli3 and Gli2 activities (Marks, 2011).

This study found that silencing Gli1 in the absence of Hh signaling requires PKA, PKA sites S544 and S560, Cos2 and the Cos2-binding region equivalent to CORD in Ci, but that T374 and the DSGVEM motif, which has been previously implicated in Gli1 regulation, are not required for Gli1 silencing or activation by Hh. Although the known dependence of Ci phosphorylation on Cos2 suggests that Cos2 might be required to promote Gli1 phosphorylation by PKA, the greater activity of Gli1 lacking the CORD region compared with Gli1 lacking PKA sites in anterior wing disc cells suggests that Cos2 may do more than simply promote Gli1 phosphorylation (Marks, 2011).

How is Gli1 silenced by PKA phosphorylation in Drosophila? Ci silencing involves equivalent PKA sites (in position and immediate sequence context) and creates a Slimb-binding site essential for Ci-155 processing. However, additional PKA and PKA-primed phosphorylation sites important for efficient Ci and Gli3 proteolysis are absent from Gli1. Thus, it would be difficult to predict a priori whether PKA and Hh alter Gli1 activity by regulating its proteolysis (Marks, 2011).

Direct assessment of Gli1 protein levels (using a C-terminal HA tag) in wing disc cells indicated that neither Hh stimulation nor PKA inhibition increased Gli1 levels. However, Ci-155 levels can be a deceptive indicator of PKA-dependent proteolysis because Ci-155 can also be degraded by an activation-dependent mechanism that is independent of PKA. Hence, Ci-155 levels are elevated at intermediate levels of Hh signaling, but in cells exposed to the highest levels of Hh, where PKA-dependent processing is strongly inhibited, Ci-155 levels are very similar to those in anterior cells with no Hh exposure. The observation that Gli1 levels may actually be slightly reduced in posterior cells relative to anterior cells, and in anterior clones deficient for PKA, suggests that activation may also de-stabilize Gli1 and could disguise any putative, opposite contribution from PKA-dependent proteolysis. Consequently, evidence of PKA-dependent proteolysis was sought by creating a Ci-Gli1 fusion protein for which PKA-dependent proteasome digestion would be expected to be arrested and produce Ci-75 repressor. The activity of the Ci-Gli1 fusion protein was regulated by PKA, Cos2 and Hh, as for Gli1 and Ci, but no repressor was formed. Hence, no evidence was found of PKA-dependent proteolysis of Gli1. Weak induction of ptc-lacZ expression in anterior cells was found to require much higher levels of Gli1 protein than evident for equivalent (or greater) ptc-lacZ induction by Gli1 lacking PKA sites 544 and 560, or by wild-type Gli1 in response to Hh. Thus, both PKA inhibition and Hh clearly increase the specific activity of Gli1 and do not appear to stabilize Gli1 protein (Marks, 2011). If PKA principally regulates the specific activity of Gli1 in Drosophila, it might be expected that the same mechanism can regulate Ci-155 activation. Direct evidence for such a role was not observed previously when comparing the activity of low levels of proteolysis-resistant Ci (Ci-S849A) in posterior smo and smo pka mutant clones, even though it is clear that endogenous Ci-155 is activated more strongly by loss of PKA than by loss of Slimb. Likewise, loss of Cos2 activates Ci-155 more strongly than loss of Slimb but did not detectably increase the activity of Ci S849A in posterior smo mutant clones. Su(fu) also has the potential to limit Ci-155 activity. By eliminating Su(fu), this study revealed a repressive influence of both PKA and Cos2 on Ci-S849A in posterior smo mutant clones. It is concluded that the potentially redundant effects of PKA, Cos2 and Su(fu) limiting Ci-155 activity can each be exposed in the absence of Ci-155 processing but are probably most readily seen at high Ci-155 levels. The experiments with Ci do not reveal the mechanism by which PKA and Cos2 can limit Ci-155 activity. An equivalent regulatory influence was much clearer for Gli1; it involves direct Cos2 binding and phosphorylation of defined PKA sites, providing an important starting point for further investigation (Marks, 2011).

Kif7 is known to promote the proteolysis of Gli2 and Gli3 in mouse. This study has shown that Kif7 can also promote Ci-155 and Gli3 processing in Drosophila. Cos2 promotes Gli3 processing and limits Gli1 activity in Drosophila. In all of these cases, PKA and conserved PKA sites have analogous effects to Cos2, consistent with the common hypothesis that Cos2 and Kif7 can promote phosphorylation of Ci-155 and Gli proteins at specific PKA and PKA-primed sites (Marks, 2011).

This study has found that Cos2 effects on Gli1 required the CORD-equivalent region of Gli1. Regulation of Ci-155 by Cos2 does not require the CORD region of Ci. In fact, Ci-155 can be processed and silenced in the absence of both CDN and CORD domains, provided a third Cos2 interaction domain within its zinc fingers is present. Gli1 shares all three Cos2 interaction domains. Moreover, Kif7 can promote processing of Ci lacking CDN and CORD regions, implying that a single Ci/Gli interface with a Cos2/Kif7 protein can silence Ci-155 but not Gli1. It is possible that a single Cos2/Ci-155 interface suffices because it is stabilized by additional common binding partners, Fu and Su(fu), and that these proteins do not contribute efficiently to Cos2/Gli1 binding (Marks, 2011).

Most importantly, Hh inhibits the Cos2-dependent processing of Gli3 and Cos2-dependent silencing of Gli1 in Drosophila. The mechanisms by which Hh opposes Cos2 actions on Ci-155 are not yet fully resolved. One potential mechanism, the dissociation of PKA, CK1 and GSK3 from Cos2, would be expected to influence both Ci and Gli protein activities. However, there is probably also a role for Hh-dependent dissociation of Cos2 from Ci, implying that the interactions between Cos2 (or Kif7) and Gli proteins may be regulated by an analogous mechanism (Marks, 2011).

Essential roles of the Tap42-regulated protein phosphatase 2A (PP2A) family in wing imaginal disc development of Drosophila melanogaster

Protein ser/thr phosphatase 2A family members (PP2A, PP4, and PP6) are implicated in the control of numerous biological processes, but understanding of the in vivo function and regulation of these enzymes is limited. This study investigated the role of Tap42, a common regulatory subunit for all three PP2A family members, in the development of Drosophila wing imaginal discs. RNAi-mediated silencing of Tap42 using the binary Gal4/UAS system and two disc drivers, pnr- and ap-Gal4, not only decreased survival rates but also hampered the development of wing discs, resulting in a remarkable thorax cleft and defective wings in adults. Silencing of Tap42 also altered multiple signaling pathways (HH, JNK and DPP) and triggered apoptosis in wing imaginal discs. The Tap42RNAi-induced defects were the direct result of loss of regulation of Drosophila PP2A family members (MTS, PP4, and PPV), as enforced expression of wild type Tap42, but not a phosphatase binding defective Tap42 mutant, rescued fly survivorship and defects. The experimental platform described in this study identifies crucial roles for Tap42 phosphatase complexes in governing imaginal disc and fly development (Wang, 2012).

Understanding about the in vivo function of α4/Tap42, especially in development, is limited in part because global knockout of this gene in mice and flies leads to early embryonic death (see Cygnar, 2005 and Kong, 2004). Cellular studies have also revealed that depletion of α4/Tap42 causes death in embryonic stem cells, mouse embryonic fibroblasts, adipocytes, hepatocytes, B and T cells of the spleen and thymus, and Drosophila S2 cells (Bielinski, 2007; Kong, 2004; Yamashita, 2006). Although studies of a conditional (Cre-LoxP) α4 knockout in mouse hepatocytes and a mosaic assay of Tap42 in Drosophila wing disc have provided insights into the cellular biology of α4 and Tap42 (Cygnar, 2005; Kong, 2004), the impact of these gene products on the development of tissues and host have not yet been described. This report utilized Tap42-targeted RNAi and the Gal4/UAS system to investigate the biological effects of silencing Tap42 expression in specific Drosophila tissues. Suppressing the Tap42 gene using two tissue-specific drivers (pnr-Gal4 and ap-Gal4) led to a pleiotropic fly phenotype, which included major deformities in the adult thorax and wings as well as decreased survival rates. The experimental platform described in this study has allowed exploration of the role of Tap42 and Tap42-regulated phosphatases in the control of cellular signaling, tissue development, and Drosophila viability (Wang, 2012).

Analyses of Tap42RNAi wing discs revealed significant alterations in multiple signal transduction pathways including JNK, DPP, and HH. Marked increases in p-JNK signals were found in ap-Gal4>Tap42RNAi wing discs. This observation, together with previous studies showing increased c-Jun phosphorylation in α4-null mouse embryonic fibroblasts (Kong, 2004) and activated JNK in Tap42-depleted clones of fly wing discs (Cygnar, 2005), indicate that α4/Tap42 likely plays a negative role in regulation of JNK signaling. Silencing the Tap42 gene in the ap gene domain also changed DPP and HH signaling in the wing discs. Although ap-Gal4-mediated silencing of Tap42 had a profound effect on JNK, DPP, and HH signaling, these pathways were unaffected in pnr-Gal4>Tap42RNAi wing discs, thus demonstrating that the thorax cleft phenotype seen in the pnr-Gal4>Tap42RNAi flies is not due to alterations in these pathways. Collectively, these findings indicate that Tap42 plays a crucial role in the modulation of JNK, DPP, and HH signaling, but the effects of Tap42 on these pathways appear to play a minimal role in normal thorax development (Wang, 2012).

The HH pathway is one of the major guiding signals for imaginal disc development. Recent investigations have revealed that the phosphorylation state of Ci and Smo, two components of the HH signaling pathway, are controlled by Drosophila PP2A (Mts) and PP4 (Jia, 2009). Additional studies implicate a role for specific Mts complexes in the control of HH signaling, whereby holoenzyme forms of Mts containing the Wdb and Tws regulatory B subunits act at the level of Smo and Ci, respectively (Su, 2011). Together, these findings point to key roles for Mts and PP4 in HH signaling and suggest that a common subunit of these phosphatases, namely Tap42, may also be involved in HH signaling. Indeed, the current data clearly show that Tap42 plays an important regulatory role in this pathway as silencing of Tap42 within the wing discs leads to an elimination of both Smo and Ci expression. Although the precise role(s) of Tap42 in the control of HH signaling remains unclear, it likely involves Tap42-dependent regulation of one or more phosphatase catalytic subunits (e.g., Mts, PP4, and possibly PPV) or specific holoenzymes forms of these phosphatases (e.g., Wdb/Mts, Tws/Mts). The pleiotropic effects of Tap42RNAi on JNK, DPP, and HH signaling could be due to loss of Tap42's regulation of phosphatase activity, cellular levels, holoenzyme assembly, or subcellular localization (Wang, 2012).

Depletion of α4 in mouse embryonic fibroblasts caused an increase in phosphorylation of a variety of established PP2A substrates, which was attributed to a 'generalized defect in PP2A activity.' Instead of the expected unidirectional increase in protein phosphorylation, the current findings demonstrate a dual role for Tap42 in the control of JNK activation as hyperphosphorylation and hypophosphorylation of JNK were observed in the dorsal and ventral sides of the Tap42RNAi wing disc, respectively, relative to control wing discs. Silencing of Tap42 in the ap domain also impacted DPP in a bi-directional fashion; these flies exhibited significantly decreased DPP expression in the scutellum but augmented expression around the wing blade. Consistent with previous studies showing that PP2A functions at different levels within the Ras1 and HH pathways, the current data indicate that Tap42-regulated phosphatases likely target multiple substrates within the JNK and DPP pathways in different regions of wing discs (Wang, 2012).

Close examination of the PE cells in the wing disc revealed that Tap42 expression occurs in only a fraction of these cells. It is noteworthy that the majority of Tap42 localized in rows of cells delineating the PE/DP (peripodial epithelium/disc proper) boundary. These cells are commonly referred to as 'medial edge' cells, which represent a subpopulation of PE cells that play a crucial role in thorax closure during metamorphosis. Interestingly, α4-PP2A complexes appear to play a major role in the control of cell spreading, migration, and cytoskeletal architecture, presumably via their ability to modulate the activity of the small G-protein Rac. Yeast Tap42 has also been implicated in the cell cycle-dependent and polarized distribution of actin via a Rho GTPase-dependent mechanism. Therefore, it is hypothesized that the wing disc structural deformities and thorax cleft phenotype of Tap42RNAi flies are a result of unregulated phosphatases leading to defective spreading and migration of the medial edge cells during metamorphosis. The thorax cleft phenotype provides an opportunity to delineate the precise roles of Tap42-phosphatase complexes in processes controlling thoracic closure (e.g., cell spreading and migration) (Wang, 2012).

α4/Tap42 appears to function as an essential anti-apoptotic factor as cells lacking this common regulatory subunit of PP2A family members undergo rapid death. These studies implicate a role for α4/Tap42-dependent regulation of PP2A-like enzymes, and presumably the phosphorylation state of multiple pro- and anti-apoptotic proteins, in the maintenance of cell survival. The current findings reveal that silencing Tap42 in wing discs triggers apoptosis, thus providing supportive in vivo evidence that depletion of Tap42 (α4) leads to deregulated phosphatase action, which switches these enzymes from pro-survival to pro-apoptotic mediators. Because JNK activation is a hallmark feature of apoptosis, the overlap of apoptotic cells and hyperphosphorylated JNK indicates that the Tap42RNAi-induced apoptosis may be dependent on JNK activation (Wang, 2012).

Since α4 is required for maintaining the normal function of PP2A, PP4, and PP6, it is suspected that misregulation of these phosphatases could be responsible for the pleiotrophic phenotypes observed in Tap42RNAi flies. Consistent with this idea, introduction of the mtsXE2258 heterozygous allele into ap-Gal4>UAS-Tap42RNAi flies partially rescued the thorax and wing defects, and significantly improved fly survival rates. The partial rescue by mtsXE2258 suggests that the defects seen in the Tap42RNAi flies are due, in part, to unregulated Mts activity, possibly as a result of increased Mts levels or enzymatic activity. Indeed, previous studies have demonstrated an accumulation of Mts in Tap42-depleted clones of the fly wing disc. Thus, mtsXE2258 appears to function as a mild mutant that partially restores misregulated Mts function following depletion of Tap42. However, given the biochemical findings showing that Tap42 also interacts with PP4 and PPV, additional studies will be needed to determine the relative contribution of these phosphatases to the Tap42RNAi-induced defects (Wang, 2012).

The phenotypes observed in flies expressing Tap42RNAi could also be attributed to loss of a phosphatase-independent function(s) of Tap42 that controls normal fly development. However, introduction of a phosphatase binding-defective mutant of Tap42 (Tap42ED) into the Tap42RNAi background failed to rescue the phenotypes and lethality associated with Tap42 depletion. In contrast to Tap42ED, introduction of Tap42WT fully rescued the phenotypes and lethality of the Tap42RNAi flies. These findings indicate that the Tap42RNAi-induced phenotypes are entirely due to the impaired interactions between Tap42 and PP2A family members, and provide compelling support for the hypothesis that Tap42-dependent regulation of the functions of these enzymes is crucial for normal wing disc development and Drosophila viability (Wang, 2012).

Although understanding the exact molecular mechanisms underlying Tap42's regulation of PP2A family members is still incomplete, these studies clearly demonstrate that Tap42-phosphatase interactions play crucial roles in the control of multiple signaling pathways governing cell growth and survival. The experimental platform described in this report will undoubtedly serve as a valuable system to further explore the in vivo function and regulation of Tap42-phosphatase complexes. Furthermore, given the remarkable phenotypes seen in the Tap42RNAi flies (e.g., thorax cleft and deformed wings), it is anticipated that this model system will drive future studies (e.g., phenotype-based suppressor/enhancer screens) aimed at identifying direct targets of Tap42-regulated phosphatases, as well as additional pathways under the control of these phosphatase complexes (Wang, 2012).

Kto-Skd complex can regulate ptc expression by interacting with Ci in Hedgehog signaling pathway

Hedgehog (Hh) signaling pathway plays a very important role in metazoan development by controlling pattern formation. Drosophila imaginal discs are subdivided into anterior and posterior compartments which derive from adjacent cell populations. The anterior/posterior (A/P) boundaries which are critical to maintain the position of organizers are established by a complex mechanism involving the Hh signaling. This study uncovered the regulation of ptc in Hh signaling pathway which is contributive to A/P boundary formation by two subunits of mediator complex, Kto and Skd, which can also regulate boundary location. Collectively, further evidence is provided that Kto-Skd affects the A/P-axial development of the whole wing disc. And Kto can interact with Cubitus interruptus (Ci) and bind to the Ci-binding region on the ptc promoter. Both subunits are regulated by Hh signals to downregulate ptc expression (Mao, 2014).

Ter94 ATPase complex targets k11-linked ubiquitinated Ci to proteasomes for partial degradation

The Cubitus interruptus (Ci)/Gli family of transcription factors can be degraded either completely or partially from a full-length form [Ci155/GliFL] to a truncated repressor (Ci75/Gli(R)) by proteasomes to mediate Hedgehog (Hh) signaling. The mechanism by which proteasomes distinguish ubiquitinated Ci/Gli to carry out complete versus partial degradation is not known. This study shows that Ter94 ATPase and its mammalian counterpart, p97, are involved in processing Ci and Gli3 into Ci75 and Gli3R, respectively. Ter94 regulates the partial degradation of ubiquitinated Ci by Cul1-Slimb-based E3 ligase through its adaptors Ufd1-like and dNpl4. Cul1-Slimb-based E3 ligase, but not Cul3-Rdx-based E3 ligase, modifies Ci by efficient addition of K11-linked ubiquitin chains. Ter94Ufd1-like/dNpl4 complex interacts directly with Cul1-Slimb, and, intriguingly, it prefers K11-linked ubiquitinated Ci. Thus, Ter94 ATPase and K11-linked ubiquitination in Ci contribute to the selectivity by proteasomes for partial degradation (Zhang, Z., Lv, X., et al., 2013).

Hh signaling plays important roles in metazoan development, and its malfunction is implicated in numerous human congenital disorders and cancers. Secreted Hh proteins bind Patched (Ptc)-iHog coreceptors to relieve an inhibitory effect of Ptc on Smoothened (Smo), which leads to activation of the Ci/Gli family of zinc finger transcription factors. Biochemical and genetic studies in Drosophila have revealed several important steps in the regulation of Ci/Gli activity. In the absence of Hh, full-length Ci, Ci155, is sequentially phosphorylated by protein kinase A (PKA), glycogen synthase kinase 3 (GSK3), and casein kinase I (CKI) and then ubiquitinated by Cullin 1 (Cul1)-Supernumerary limbs (Slimb, known also as β-TrCP)-based E3 ligase. This results in partial degradation by proteasomes, leaving the N terminus of Ci intact (Ci75) as a transcriptional repressor. In the presence of Hh, unphosphorylated Ci155 accumulates and enters nucleus to activate Hh target genes. As a feedback control of the pathway, active Ci155 induces the expression of roadkill (rdx)/Hib to form Cul3-Rdx-based E3 ligase and promotes complete proteasomal degradation of Ci155 (Zhang, Z., Lv, X., et al., 2013).

Although it is well established that Ci is ubiquitinated by Cul1-Slimb and Cul3-Rdx-based E3 ligases under different conditions, it remains unknown how proteasomes distinguish ubiquitinated Ci for partial versus complete degradation. As ubiquitinated proteins are transferred to proteasomes by different pathways, it is hypothesized that some specific components are involved in the recruitment of ubiquitinated Ci for partial degradation. Transitional elements of the endoplasmic reticulum 94 kDa (Ter94) was identified as the Drosophila homolog of yeast CDC48, which is a member of the ATPase associated with various cellular activities (AAA) family. In mammals, the CDC48/Ter94 homolog p97 (also known as VCP) mainly functions in endoplasmic reticulum-associated degradation (ERAD). Proteomic analysis revealed that p97 might play a broad role in regulating the turnover of ubiquitin proteasome system (UPS) substrates. This study has shown that Ter94 is a component of the Ci processing machinery and is critical for Ci75 formation (Zhang, Z., Lv, X., et al., 2013).

The control of partial versus complete proteasomal degradation of Ci and Gli3 is a major regulatory step in Hh signal transduction. How proteasomes distinguish ubiquitinated Ci to carry out either partial or complete degradation is not known. Based on the current findings, the following model is proposed. In the absence of Hh, Ci155 is phosphorylated and ubiquitinated by Cul1-Slimb-based E3 ligase to generate Ci75. In this process, K11-linked ubiquitin chains are added onto Ci155. Ter94Ufd1-like/dNpl4 forms a complex with Cul1-Slimb-based E3 ligase through Ufd1-like and Roc1a, and another component dNpl4 is bound to the K11-linked ubiquitin chains on Ci155. Through ATP hydrolysis, Ter94Ufd1-like/dNpl4 facilitates the delivery of ubiquitinated Ci155 to the proteasomes for processing (Zhang, Z., Lv, X., et al., 2013).

Besides Ci and Gli3, the best example of partial degradation is the processing of human nuclear factor-κB (NF-κB) and its yeast homologs, SPT23 and MGA2. Previous studies have suggested that some common features of processing determinant domain (PDD) are involved in the partial proteasomal degradation of Ci and NF-κB. However, Ci also undergoes complete proteasomal degradation by Cul3-Rdx-based E3 ligase. Why such 'degradation stop signals' fail to work in such instances and how proteasomes make the decision for partial or complete degradation are unknown. Based on the current results and previous studies, it is proposed that Ter94/p97 complex may specifically target 'partial-degradation-proteins' to proteasomes through K11-linked ubiquitin chains. Further investigation is needed to provide direct evidence to support this hypothesis. As many similarities are shared between Ci and NF-κB precursors in partial degradation, it will be interesting to test whether p97 and K11-linked ubiquitination are also involved in the partial degradation and/or maturation of p100 in NF-κB signaling (Zhang, Z., Lv, X., et al., 2013).

This study found that K11-linked chains are added onto Ci by Cul1-Slimb-based E3 ligase in the absence of Hh pathway activity, whereas Cul3-Rdx-based E3 ligase mainly adds K48-linked chains on Ci when the pathway is active. This illustrates a phenomenon that the same protein can be modified with different types of ubiquitin chains by distinct E3 ligases. Although K11-linked chains added on APC substrates lead to complete degradation, the data demonstrate that K11-linked chains are involved in the partial degradation of Ci. These findings also raise the interesting possibility that the topology of ubiquitin chains may be recognized as a selective signal for proteasomal degradation. As mixed or heterologous ubiquitin chains may exist, further investigation is essential to determine whether mixed ubiquitin chains are formed by Cul1-Slimb-based E3 ligase on Ci (Zhang, Z., Lv, X., et al., 2013).

Hedgehog-regulated atypical PKC promotes phosphorylation and activation of Smoothened and Cubitus interruptus in Drosophila

Smoothened (Smo) is essential for transduction of the Hedgehog (Hh) signal in both insects and vertebrates. Cell surface/cilium accumulation of Smo is thought to play an important role in Hh signaling, but how the localization of Smo is controlled remains poorly understood. This study demonstrates that atypical PKC (aPKC) regulates Smo phosphorylation and basolateral accumulation in Drosophila wings. Inactivation of aPKC by either RNAi or a mutation inhibits Smo basolateral accumulation and attenuates Hh target gene expression. In contrast, expression of constitutively active aPKC elevates basolateral accumulation of Smo and promotes Hh signaling. The aPKC-mediated phosphorylation of Smo at Ser680 promotes Ser683 phosphorylation by casein kinase 1 (CK1), and these phosphorylation events elevate Smo activity in vivo. Moreover, aPKC has an additional positive role in Hh signaling by regulating the activity of Cubitus interruptus (Ci) through phosphorylation of the Zn finger DNA-binding domain. Finally, the expression of aPKC is up-regulated by Hh signaling in a Ci-dependent manner. These findings indicate a direct involvement of aPKC in Hh signaling beyond its role in cell polarity (Jiang, 2014).

back to: Cubitus interuptus Protein Interactions part 1/2

cubitus interruptus continued:

Biological Overview | Evolutionary Homologs | Regulation | Targets of Activity | Developmental Biology | Effects of Mutation | References

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