fused


REGULATION

Targets of Activity

Direct Wg autoregulation (autocrine signaling) is masked by its paracrine role in maintaining hh, which in turn maintains wg. zeste-white3 (shaggy) and patched (ptc) mutant backgrounds have been used to genetically uncouple this positive-feedback loop and to study autocrine wg signaling. Direct wg autoregulation differs from wg signaling to adjacent cells in the importance of fused, smoothened and cubitus interruptus, relative to zw3 and armadillo. wg autoregulation during this early HH-dependent phase differs from later wg autoregulation in its lack of gooseberry participation (Hooper, 1994).

wg transcription is one of the targets of hh activity. It has been suggested that the spatial control of wg expression depends on the limited range of the hh signal and the differential competence of responding cells. Ubiquitous expression of the hh gene causes the ectopic activation of wg in only a subset of the cells of each parasegment. Competence of cells to express wg is independent of their ability to receive the hh signal. wg activation requires the function of fused, suggesting that the putative hh signal is transduced by the serine/threonine kinase that fused encodes (Inghram, 1993).

fused is required at least until germ band shortening for correct spatial expresssion of engrailed. The distribution of the CI protein is altered in fused, hedgehog and wingless mutants, suggesting Fused may regulate CI protein levels while CI in turn, regulates wingless (Motzny, 1995).

dpp is a target of the hh signal acting through Fused. fu mutations rescue the phenotype due to ectopic expression of hh or to the lack of patched activity. fu is also required for the activation of engrailed caused when hh is ectopically activated in the wing disk. Although fu, cos-2 and ci probably form part of the same pathway that controls dpp expression, Protein kinase A probably controls dpp expression by a different pathway (Sánchez-Herrero, 1996).

Two signaling proteins, Wingless and Hedgehog, play fundamental roles in patterning cells within each metamere of the Drosophila embryo. Within the ventral ectoderm, Hedgehog signals both to the anterior and posterior directions: anterior flanking cells express the wingless and patched Hedgehog target genes whereas posterior flanking cells express only patched. Furthermore, Hedgehog acts as a morphogen to pattern the dorsal cuticle, on the posterior side of cells where it is produced. Thus responsive embryonic cells appear to react according to their position relative to the Hedgehog source. The molecular basis of these differences is still largely unknown. This paper shows that one component of the Hedgehog pathway, the kinase Fused accumulates preferentially in cells that could respond to Hedgehog but that Fused concentration is not a limiting step in the Hedgehog signaling. Direct evidence is presented that Fused is required autonomously in anterior cells neighboring Hedgehog in order to maintain patched and wingless expression, while in turn, Wingless maintains engrailed and hedgehog expression. By expressing different components of the Hedgehog pathway only in anterior, wingless-expressing cells, it could shown that the Hedgehog signaling components Smoothened and Cubitus interruptus are required in cells posterior to Hedgehog domain to maintain patched expression, whereas Fused is not necessary in these cells. This result suggests that Hedgehog responsive ventral cells in embryos can be divided into two distinct types depending on their requirement for Fused activity. In addition, the morphogen Hedgehog can pattern the dorsal cuticle independent of Fused. In order to account for these differences in Fused requirements, the existence of position-specific modulators of the Hedgehog response is proposed (Thérond, 1999).

These results clearly support the involvement of the Fu serine/threonine kinase in Hh signal transduction in the embryonic segment within cells producing Wg. Although Fu is present in all embryonic cells, expression of Fu only in the wg-expressing cells of the anterior compartment in a fu mutant context is sufficient to restore a wild-type transcription pattern of both wg and en and a normal cuticular pattern. In contrast, Fu expression in the posterior compartment -- the en-expressing compartment -- has apparently no effect, either on the transcription of wg and en or at the phenotypic level. Together these data show that Fu is not necessary in the majority of cells where it is expressed and suggest that its activity could be induced in wg-expressing cells in response to Hh. Fu protein is evenly distributed in the embryo until stage 9 when it begins to accumulate in the anterior compartment (Thérond, 1999).

What is regulating Fu accumulation? Because FU mRNA distribution is uniform at least until stage 10 it is hypothesized that the localization is due to post-translational regulation. Another component of the Hh pathway, Cos2, also accumulates in the anterior compartment at this stage independent of the level of the Hh signal. As for Cos2, the uniform level of Fu in the anterior compartment seems to be constitutive to anterior cells and independent of Hh signal. Indeed, this regulation is observed during the time when local signaling by Wg and Hh stabilize each other’s expression. At this stage anterior cells at the A/P border are receiving Hh and responding to it. Other anterior cells distal to the A/P border do not receive Hh but have the potential to respond to it. Thus, these results are inconsistent with Hh regulating Fu and Cos2 accumulation in the entire anterior compartment. Fu accumulation could be related to its association with Ci and Cos2 within the same protein complex. Nevertheless, since fu expression is only required in the wg-expressing cells for proper patterning, the higher Fu protein levels in the whole of the anterior compartment do not seem to have any functional significance (Thérond, 1999).

Control of antagonistic components of the Hedgehog signaling pathway by microRNAs in Drosophila

Hedgehog (Hh) signaling is critical for many developmental processes and for the genesis of diverse cancers. Hh signaling comprises a series of negative regulatory steps, from Hh reception to gene transcription output. Stability of antagonistic regulatory proteins, including the coreceptor Smoothened (Smo), the kinesin-like Costal-2 (Cos2), and the kinase Fused (Fu), is affected by Hh signaling activation. This study shows that the level of these three proteins is also regulated by a microRNA cluster. Indeed, the overexpression of this cluster and resulting microRNA regulation of the 3'-UTRs of smo, cos2, and fu mRNA decreases the levels of the three proteins and activates the pathway. Further, the loss of the microRNA cluster or of Dicer function modifies the 3'-UTR regulation of smo and cos2 mRNA, confirming that the mRNAs encoding the different Hh components are physiological targets of microRNAs. Nevertheless, an absence of neither the microRNA cluster nor of Dicer activity creates an hh-like phenotype, possibly due to dose compensation between the different antagonistic targets. This study reveals that a single signaling pathway can be targeted at multiple levels by the same microRNAs (Friggi-Grelin, 2009).

cos2, fu, and smo mRNA can be regulated by a cluster of microRNAs, including miR-12 and miR-283, in Drosophila wing disc. The overexpression of this cluster decreases the levels of Smo, Cos2, and Fu proteins and activates the Hh pathway, as evidenced by the induction of dpp expression in the wing imaginal discs and by the adult wing outgrowth. The experiments presented in this study with the 3'-UTR sensors of smo, fu, or cos2 are in favor of a direct binding. To constitute a real proof of a direct effect, further experiments as direct biochemical binding assay or compensatory mutation between the 3'-UTR and the miRNAs will be necessary to perform (Friggi-Grelin, 2009).

Programs that have been created to genomewide predictions of Drosophila miRNA targets provide lists of presumptive miR-12, and miR-283 regulated genes. In addition to the current in vivo validations, miR-12 binding sites are predicted on the 3'-UTR of ci and no sites were found on the 3'-UTR of the Su(fu) gene. No decrease was observed in either of these two proteins in the microRNA cluster overexpressing clones. It is interesting to note that Su(fu) mRNA, encoding another negative regulator of Hedgehog signaling, has been shown to be targeted by miR-214 in zebrafish. Absence of miR-214 results in the reduction of muscle cell types, the specification of which is dependent on Hh pathway activity. Nevertheless, the current study shows that in Drosophila wing discs an absence of microRNA does not modify the Hh pathway, raising the question of what the role of microRNAs in Drosophila Hh pathway regulation is (Friggi-Grelin, 2009).

Could the microRNAs overexpression phenotype that was identified be artifactual and simply the result of forced overexpression of the microRNA cluster in a tissue in which it should be silent? It is thought that the answer is no, because Northern blot analysis and the increase of miR-sensor in the dcr-1 mutant clones showed that the microRNA cluster is indeed expressed in this tissue. This suggests that the cluster likely has a role in this tissue in which it is normally present. Is the microRNA cluster regulation of the cos2 and smo 3'-UTRs physiological? It is thought so, because an absence of either the microRNA cluster or of Dicer in the wing imaginal disc induces an increase in the Cos2- and Smo-sensor lines. This signifies that the microRNAs expressed from the cluster regulate the cos2 and smo 3'-UTRs and thus display some functionality in the disc during larval development. Altogether, these data clearly show that an artifactual situation in which the microRNA cluster is expressed in a tissue in which it should not be present has not been created. The miRs overexpression was also tested on embryonic patterning but it did not lead to any phenotype, suggesting that the miR cluster regulation on the Hh pathway is specific to larval tissues (Friggi-Grelin, 2009).

As miR-12 and miR-283, and likely redundant miRs, are present in every cell of the wing disc, one possibility is that their normal roles are to dampen down the levels of Hh pathway components, particularly Cos2 and Smo, to prevent the accidental activation or downregulation of the pathway. Indeed, expressing both the microRNA cluster and its targets in the same tissue could provide a means of 'buffering stochastic fluctuations' in mRNA levels or in protein translation rates within the Hh signaling pathway, as has been proposed for other processes (Friggi-Grelin, 2009).

The data possibly indicate that miRNAs are able to regulate two antagonistic components of the pathway, Cos2 and Smo. It has been shown that the stability of these two proteins is 'interdependent': an increased level of Cos2 in the wing imaginal disc lowers the level of Smo, and, in the opposite direction, increased Smo decreases the level of Cos2. It is proposed that the interregulation of Cos2/Smo levels is independent of their relative activities because Cos2 effect on Smo levels is observed in posterior cells in which Cos2 activity is strongly inhibited by the constitutive activation of the pathway. Therefore, eliminating the miRNA-mediated inhibition of Cos2 and Smo in Delta3miR or dcr-1 mutant cells likely initially increased the levels of both proteins, but then the resulting higher levels of each protein presumably downregulated the other; the net variation of Cos2 and Smo levels would therefore be null. This hypothesis is favored because the independent Smo- and Cos2-sensor lines, which are unaffected by this Cos2/Smo interregulation, showed increased levels of GFP staining in Delta3miR and dcr-1 mutant animals. This suggests that the levels of both Cos2 and Smo are increased in the mutant animals but, because of the downregulation of each protein by the other, no ultimate alterations in the levels of the proteins are observed. If so, an Hh phenotype would not be expected to be seen in the miR mutant (Friggi-Grelin, 2009).

The screen created a situation in which the expression of the microRNA cluster is deregulated, ultimately destabilizing Cos2 protein levels and thereby activating Ci and Hh target gene expression. Importantly, a similar situation might be encountered during tumoral development. Aberrant Hh signaling activity is known to trigger the development of diverse cancers. While several of these tumors have been linked to mutations in Hh signaling components, not all of them have, leaving open the possibility that they are caused by other factors such as microRNA misexpression. Interestingly, more than half of the known human microRNA genes are located near chromosomal breakpoints associated with cancer, and in some documented cases the microRNAs are amplified, leading to overexpression. Some upregulated microRNAs are possibly able to bind mRNAs encoding negative regulators of Hh signaling, such as Su(fu) or Ptc, and could thus induce the misactivation of the Hh pathway, as is observed in some cancers. Therefore, a fine analysis of microRNA expression levels and the levels of known Hh components should be considered in studies of Hh pathway-related cancers (Friggi-Grelin, 2009).

What does this study add to the current knowledge about miRNA regulation? The study shows that a cluster of three microRNAs can target several antagonistic components of the same pathway in vivo. This is novel and unexpected. This raises the question of how to interpret the miRNA expression signatures observed in human tumors. Indeed, as stated above, it has been proposed that miRNAs are differentially expressed in human cancers and contribute to cancer development. The working hypothesis in the cancer/miRNAs field is that key cancer genes are regulated by aberrant expression of miRNAs. The identification of a specific miRNA:mRNA interactor pair is generally accepted as being of biological importance when the mRNA encodes a tumor suppressor or an oncogene whose expression is modified in the tumor. This study shows indirectly that this is an oversimplified view, because identifying an oncogene or tumor suppressor as a target of a miRNA may not provide a full explanation for tumor development if the same miRNA hits other antagonistic components of the same pathway that nullify the effect of the identified miRNA:mRNA interactor pair (Friggi-Grelin, 2009).

Protein Interactions

Phosphorylation of Fused occurs in response to Hedgehog and cannot be blocked by activation of Protein kinase A, which is thought to be an antagonist of signaling from hedgehog. This suggests that Fused and Protein kinase A function downstream of Hedgehog but in parallel pathways that eventually converge downstream of Fused (Thérond, 1996b).

The Costa (also known as Costal2 or Cos2) protein can be coimmunoprecipitated with antibodies to Fused. Both Fused and a hyperphosphorylated isoform of Fused designated FU-P are found in this conplex. FU-P predominates over Fused when precipitates are prepared with Cos2 antisera. Antisera against either Fused or Cos2 precipitate Cubitus interruptus protein as well. Fractionated extracts of cultured cells have two complexes larger than Fused itself, a population of about 40 million Da, and a population of greater than 700,000 Da. The 700 kDa fraction is by far the most abundant. Fused, Cos2 and Ci are enriched in microtubules formed from repolymerized tubulin. Binding of Cos2 and Fused to microtubules is barely detectable in Hedgehog treated cultured cells. These findings sustain the hypothesis that signaling from Hh releases the complexes from microtubules, which would in turn facilitate translocation of Ci to the nucleus (Robbins, 1997).

Labeling with radioactive phosphate reveals that Fused and Costal2 are phosphorylated in both cultured S2 cells and Hedgehog treated S2 cells. The phosphorylations of Fused and Cos2 is on serine. Cos2 coimmunoprecipates with kinase dead Fused mutant proteins. Thus, functional Fused kinase is probably not necessary for Fused and Cos2 to associate. There is no evidence for binding of Cos2 to the products of truncated Fused protein lacing the C-terminal domain of Fused (Robbins, 1997).

The discovery of a multiprotein complex in the cytoplasm provides some of the explanation for regulation in the Hedgehog pathway, but the dynamic roles of Costa and Fused are not yet well understood and the fine details are still obscure. Stimulation of cells with Hh leads to an additional serine phosphorylation for both Fused and Cos2. The protein kinase(s) responsible for these phosphorylations have not been identified. The Hh-induced phosphorylation of Fused appears as long as 30 minutes after induction, suggesting that it represents a feedback device rather than an event in initial signal transduction. This leads in turn to the possibliity that Fused is not autophosphorylating, even though the phosphoryation can be abolished by mutations in the catalytic domain of Fused. Similarly, Fused is apparently not directly responsible for the phosphorylation of Cos2, which occurs even when inactivating mutations are present in the kinase domain of Fused (Robbins, 1997).

Suppressor of fused, isolated as a mutation suppressing the fused phenotype, interacts with fused in the fused segment polarity function. Fused and Suppressor of fused could act through a competitive posttranslational modification of a common target in the hedgehog signaling pathway. The amorphic Su(fu) mutation is viable, shows a maternal effect and displays no phenotype by itself. The Su(fu) gene encodes a 53-kD protein, contains a PEST sequence and shows no significant homologies with known proteins. Proper development requires a fine tuning of the genetic doses of fu and Su(fu), both maternally and zygotically (Pham, 1995).

Su(fu) enhances a cos-2 phenotype and cos-2 mutations interact with fu in a way similar to Su(fu). A close relationship might exist between fu, Su(fu) and cos-2 throughout development. The Fu+ kinase might be a posterior inhibitor of Costal-2+ while Su(fu)+acts as an activator of Costal-2+. The expression pattern of wingless and engrailed in fu and fused-Su(fu) mutant embryos supports this interpretation (Preat, 1993).

The Hedgehog (Hh) family of signaling proteins mediates inductive interactions either directly or by controlling the transcription of other secreted proteins through the action of Gli transcription factors, such as Cubitus interruptus (Ci). In Drosophila, the transcription of Hh targets requires the activation of the protein kinase Fused (Fu) and the inactivation of both Suppressor of fused [Su(fu)] and Costal-2 (Cos-2). Fu is required for Hh signaling in the embryo and in the wing imaginal disc and acts also as an antitumorigen in ovaries. All fu- phenotypes are suppressed by the loss of function of Su(fu). Fu, Cos-2 and Ci are co-associated in vivo in large complexes which are bound to microtubules in an Hh-dependent manner. The role of Su(fu) in the intracellular part of the Hh signaling pathway has been investigated. Using the yeast two-hybrid method and an in vitro binding assay, Su(fu), Ci and Fu are shown to interact directly to form a trimolecular complex, with Su(fu) binding to both its partners simultaneously. Su(fu) and Ci also co-immunoprecipitate from embryo extracts. It is proposed that, in the absence of Hh signaling, Su(fu) inhibits Ci by binding to it and that, upon reception of the Hh signal, Fu is activated and counteracts Su(fu), leading to the activation of Ci (Monnier, 1998).

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).

It is proposed that, in the absence of Hh signaling, Su(fu) inhibits Ci by binding to it and that, upon reception of the Hh signal, Fu is activated and counteracts Su(fu), leading to the activation of Ci (Monnier, 1998). Since the Hedgehog signal transduction pathway is complex, involving multiple inputs to create the repressor and activator forms of ci, Ci[rep] and Ci[act] respectively, the components of the pathway must be manipulated independently in order to discover the role of Su(fu). In particular, neutralization of the effects of the kinase PKA is necessary in order to unravel the effects of the Fused kinase, and its interacting protein Su(fu). The absence of Cos2 or PKA activity prevents formation of the cleaved form of Ci, namely Ci[rep]. However mere prevention of Ci cleavage does not suffice for Ci[act] formation (Methot, 1999). To test whether, in these mutant contexts, prevention of Ci[rep] formation is linked to the generation of Ci[act], PKA or Cos2 were removed Ci[act] activity was assessed. While the repression of hh transcription is indicative for the presence of Ci[rep], the upregulation of ptc transcription serves as an indicator for the presence of Ci[act] (Methot, 1999). ptc expression was examined in A cells mutant for either cos2 or PKA; in both cases, ptc is upregulated. These results indicate that A cells mutant for cos2 or PKA generate Ci[act], and suggest that neutralization of the activities or effects of either Cos2 or PKA is an important step for the formation of Ci[act]. Interestingly, although the C terminus of Fu is required for Ci[rep] formation, its absence does not lead to ectopic ptc-lacZ expression and Ci[act] formation (Methot, 2000).

Therefore, Ci[act] is generated in cells that lack PKA activity. An equivalent situation can be created by mutating the PKA phosphorylation sites of Ci. One (CiPKA1) or four (CiPKA4) PKA phosphorylation sites were mutated and the ability of these mutants to activate ptc-lacZ in wing imaginal discs was compared. Ubiquitous weak expression of wild-type Ci leads to ptc-lacZ activation only in Hh-exposed cells. This indicates that under physiological conditions, transcriptional activity of Ci is under the control of Hh. In contrast, CiPKA1 activates ptc-lacZ in all cells, regardless of their exposure to Hh. Thus, CiPKA1 is constitutively active in vivo. Identical results have recently been described by several groups, and are taken as indication of a role for PKA in Ci activation. Interestingly, closer examination of discs ubiquitously expressing CiPKA1 reveals that P cells express ptc-lacZ at higher levels than A cells. This suggests that the transcriptional activity of CiPKA1 can be further enhanced by Hh signaling. Similar results were obtained with CiPKA4. To test whether it is the reception of the Hh signal that stimulates the basal activity of CiPKA, the function of Smo was removed from a subset of P cells expressing CiPKA4. Such cells transcribe ptc-lacZ at lower levels than their smo+ neighbors, levels that are similar to those found in anterior smo+ CiPKA cells. Thus Ci protein that is constitutively active due to mutated PKA phosphorylation sites is further stimulated in its transcriptional activity by the Hh signal (Methot, 2000).

It is possible that the reduction of ptc-lacZ expression in posterior smo-;CiPKA4cells is due to formation of some Ci[rep], which could compete with the Ci[act] activity of CiPKA. The repressor assay described above was used to test whether CiPKA4 can be converted to a transcriptional repressor; hh transcription in smo- posterior cells is essentially unaffected. This indicates that CiPKA4 cannot be converted to Ci[rep] and that the reduction of ptc expression in smo- CiPKA4 cells is not due to the presence of Ci[rep] (Methot, 2000).

Components of the Hh pathway that could stimulate the basal activity of CiPKA were then sought. Fu is one of the few proteins that have a positive input on Hh signaling. ptc-lacZ levels were examined in wing imaginal discs that ubiquitously express CiPKA1, in either a wild-type or fu1 background. ptc-lacZ expression is clearly reduced in P cells of fu1 discs. Similar results have also been obtained with CiPKA4. Slightly elevated ptc-lacZ can still be seen near the AP compartment boundary in fu1 discs, and may be the result of cumulative activities of endogenous Ci[act] and CiPKA. It is concluded that Fu kinase enhances the basal activity of CiPKA (Methot, 2000).

Beyond this basal activity, Fu stimulates CiPKA by inhibiting Su(fu) activity. fu is tightly linked to Su(fu), both genetically and biochemically. To test whether the modulation of CiPKA activity involves Su(fu), CiPKA4 was ubiquitously expressed together with myc-tagged Su(fu) or GFP as a negative control. CiPKA4 (in the presence of GFP) induces ptc-lacZ expression everywhere in the wing imaginal disc but at higher levels in the P compartment. Co-expression with mycSu(fu) abolishes ptc-lacZ expression in the A compartment and reduces ptc-lacZ levels in P cells. Thus Su(fu) inhibits the activity of CiPKA4. This result is strengthened by the converse experiment, where the absence of Su(fu) [in Su(fu)LP homozygous animals] reduces the difference in ptc-lacZ levels between A and P CiPKA1-expressing cells. To determine whether Su(fu) negatively acts on CiPKA4 by direct protein-protein interaction, a mutant form of Ci with impaired Su(fu) binding was created. Su(fu) interacts with Ci within a region that encompasses amino acids 244-346 (Monnier, 1998). Indeed, an N-terminal fragment of Ci (amino acids 5-440) interacts with GST-Su(fu). A deletion removing amino acids 212-268 of Ci almost abrogates Su(fu) binding to an N-terminal in vitro translated product of Ci. Removal of amino acids 268 to 346 also reduces Su(fu) binding, but to a lesser extent. The Delta212-268 deletion was introduced into CiPKA4, to create CiDeltaNPKA4. This mutant is constitutively active, with P cells expressing higher ptc-lacZ levels than A cells. The activity of CiDeltaNPKA4 is slightly reduced when introduced into a strong fu background, but the reduction is much less pronounced compared to that observed for CiPKA4. It is concluded that inhibition of Su(fu) activity by Fu kinase is an important step toward stimulating the basal activity of CiPKA4 (and by analogy Ci[act]) (Methot, 2000).

A possible mechanism by which Fu stimulates and Su(fu) counteracts Ci[act] could be the promotion or impediment of nuclear Ci[act] accumulation, respectively. The subcellular distribution of CiPKA in cells expressing or lacking Su(fu) was examined. Strikingly while wild-type cells show a cytoplasmic distribution of CiPKA4, this protein is mostly nuclear in Su(fu) mutant salivary gland cells. Identical results were obtained with wild-type Ci fused N terminally to GFP. This suggests that Su(fu) influences the nuclear localization of Ci. The effect of Su(fu) on Ci localization was further tested by overexpressing Su(fu) with a GFP-tagged form of Ci75, which has been shown to be mainly nuclear. Expression of mycSu(fu) reduces the amount of GFPCi75 that accumulates in the nucleus. These experiments were also performed with full-length Ci (CiGFP). Co-expression of Su(fu) in discs reduces the amount of nuclear CiGFP, both in A and P cells. It is concluded that Su(fu) downregulates the Hh pathway by preventing nuclear accumulation of Ci[act] (Methot, 2000).

It is suggested that Fu, rather than being involved in Ci[act] formation per se, stimulates the Hh pathway by permitting nuclear accumulation of Ci[act]. Su(fu) is not involved in the formation of Ci[rep] or Ci[act]. Rather, Su(fu) appears to restrict the activities of Ci[act]. This is evident from the observation that Su(fu) overexpression substantially curbs the transcriptional activity of constitutively active CiPKA, and is suggestive of Su(fu) acting after Ci[act] formation. There are several ways by which Su(fu) could fulfill such a role. One possibility is that it impedes entry of Ci[act] into the nucleus. Alternatively, Su(fu) might promote nuclear export of Ci[act]. It is difficult to distinguish between these two possibilities. The observation that little Su(fu) accumulates in the nuclei suggests that Su(fu) functions primarily in the cytoplasm and hence might exert a negative effect on Ci[act] by preventing its nuclear entry. It cannot be excluded, however, that a minor fraction of Su(fu) negatively affects the activity, stability or localization of Ci[act] in the nucleus (Methot, 2000).

Fu, as the main regulator of Su(fu) activity, is also controlled by Hh. In fu1 discs, CiPKA expression leads to similar levels of ptc transcription in A and P cells but, in fu+ discs, CiPKA expression causes higher ptc levels in P cells. In other words, Fu enhances CiPKA activity only in Hh-exposed cells. From this, it can be concluded that Fu activity is subject to Hh control (Methot, 2000).

One puzzling aspect regarding Su(fu) is that it is dispensable for viability. Animals that lack Su(fu) protein do not exhibit Hh-independent Ci[act] activity. This paradox can be partly explained by viewing Su(fu) only as a partial inhibitor of Ci[act] activity, which exerts its function subsequent to Ci[act] formation. Other elements ensure tight control over the generation of Ci[act]. The problem of how full-length Ci protein is converted into Ci[act] is more challenging. Fu has been implicated in this process, but as in the case of Su(fu), Fu kinase activity is partially dispensable in wild-type discs and entirely dispensable in animals lacking Su(fu). This suggests that the Fu kinase functions only to prevent Su(fu) from negatively acting on the Hh pathway. If we accept that Su(fu) acts subsequent to Ci[act] formation, it must be concluded that the same is true for the Fu kinase. In short, it is proposed that the activity of the Fu kinase is only required to maximize the output of an already activated form of Ci, for example by opposing cytoplasmic tethering of Ci[act] by Su(fu). The precise mechanism of how these components act is not understood. No substrate for the Fu kinase has been identified and the significance of nuclear Fu protein is unclear (Methot, 2000 and references therein).

PKA and Cos2 prevent Ci[act] formation and the same components are required for Ci[rep] formation (Methot, 2000). This observation closely links the two events. Cos2, Fu and Ci are found in a large cytoplasmic complex that is associated with microtubules. Fu derived from type II alleles, lacking the C-terminal portion, fails to locate to this complex. Indeed, Ci[rep] is not generated in cells expressing only type II-mutant Fu protein. In addition, exposure to Hh releases Cos2 from microtubules. This links Ci[rep] formation to complex formation. Together, these findings lead to the idea that complex formation fulfills two roles: one is to tether Ci to microtubules, thereby preventing nuclear entry. The other is to localize Ci to the site of proteolytic processing for the formation of Ci[rep]. Hh signaling would promote the formation of Ci[act] by releasing this complex (or Ci) from microtubules, and as a consequence would prevent the cleavage of Ci. Upon release, Ci[act] would be subjected to Su(fu) action, possibly by cytoplasmic tethering. Stimulation of Fu kinase activity by Hh inhibits Su(fu) and enables nuclear accumulation of Ci[act]. A challenging question to be answered is whether the Hh-dependent events are all catalyzed by a single biochemical step (Methot, 2000 and references therein).

Hedgehog (Hh) signal transduction requires a large cytoplasmic multi-protein complex that binds microtubules in an Hh-dependent manner. Three members of this complex, Costal2 (Cos2), Fused (Fu), and Cubitus interruptus (Ci), bind each other directly to form a trimeric complex. This trimeric signaling complex exists in Drosophila lacking Suppressor of Fused [Su(fu)], an extragenic suppressor of fu, indicating that Su(fu) is not required for the formation, or apparently function, of the Hh signaling complex. However, Su(fu), although not a requisite component of this complex, does form a tetrameric complex with Fu, Cos2, and Ci. This additional Su(fu)-containing Hh signaling complex does not appear to be enriched on microtubules. Additionally, it has been demonstrated that, in response to Hh, Ci accumulates in the nucleus without its various cytoplasmic binding partners, including Su(fu). A model is discussed in which Su(fu) and Cos2 each bind to Fu and Ci to exert some redundant effect on Ci such as cytoplasmic retention. This model is consistent with genetic data demonstrating that Su(fu) is not required for Hh signal transduction proper and with the elaborate genetic interactions observed among Su(fu), fu, cos2, and ci (Stegman, 2000).

Hedgehog signal transduction in the posterior compartment of the Drosophila wing imaginal disc

Drosophila Hedgehog (Hh) is secreted by Posterior (P) compartment cells and induces Anterior (A) cells to create a developmental organizer at the AP compartment border. Hh signaling converts Fused (Fu) to a hyperphosphorylated form, Fu*. Anterior border cells of wing imaginal discs contain Fu*. Unexpectedly, P cells also produce Fu*, in a Hh-dependent and Ptc-independent manner. Increasing Ptc, the putative Hh receptor expressed specifically by A cells, reduces Fu*. These results are consistent with proposals that Ptc downregulates Hh signaling and suggest that a receptor other than Ptc mediates Hh signaling in P cells of imaginal discs. It is concluded that Hh signals in these P cells and that the outputs of the pathway are blocked by transcriptional repression (Ramírez-Weber, 2000).

Consistent with expectations, Fu* is absent from hh and smo mutant embryos in which Hh signal transduction is blocked, and it accumulates in mutant embryos lacking Ptc, a negative regulator of Hh signaling. These studies confirm Fu* as an indicator of Hh signaling. In addition, ectopic expression of ptc in discs results in a fu phenocopy and abolishes Fu* from the disc. This indicates that Fu* embodies the active form of Fu. However, identification of the cells in normal wing discs that make Fu* did not conform to expectations (Ramírez-Weber, 2000).

Both Fu and Fu* are present in the A cells that express high levels of ptc at the A/P compartment border. In contrast, only Fu is detected in A cells located away from the compartment border near the disc flank, and only Fu* is detected in P cells. The quantitative conversion of Fu to Fu* in P cells shows that all of the Fu protein is responsive to Hh and indicates that P cells transduce the Hh signal. This latter conclusion contradicts a fundamental tenet of Hh signaling -- that the cells that produce Hh do not transduce the Hh signal. P cells do not express Hh target genes such as ptc and dpp, so it had been assumed that they are refractory to Hh. If the Hh signal transduction pathway is indeed active in P cells, as the presence of Fu* suggests, then the output of the pathway must be blocked at some downstream step. This is an unorthodox means of regulating a signal transduction pathway (Ramirez-Weber, 2000).

Although the Hh pathway is active in P cells, Fu function is not required for normal development of the P compartment, and Hh signaling has no apparent role. It is proposed that the Hh signaling pathway does not reach transcriptional fruition in P cells due to the activity of Engrailed (En). En is expressed in P cells and induces these cells to express hh. P cells, as well as their neighbors in the A compartment, respond to Hh, initiate the Hh signal transduction cascade, and generate Fu*. In A border cells, Hh signal transduction modulates Ci to upregulate dpp and ptc expression. In contrast, En represses ci expression in P cells, thereby preventing a transcriptional response (Ramirez-Weber, 2000).

Several related observations support this model of Hh signaling in P cells. (1) When En is absent from P cells, ci, dpp, and ptc are activated. Presumably, En directly represses ci in normal P cells, and the expression of ci in the mutant cells mediates the induction of dpp and ptc as an indirect consequence of Hh signaling. It is also possible that En plays a direct role in repressing dpp and ptc, but the patterns in which dpp and ptc are induced at the periphery of en mutant clones suggests that their expression is dependent upon Hh. (2) Hh seems to influence the activity of Ci when ci is expressed ectopically in P cells. Hh regulates Ci activity in part by converting Ci to an activator form (CiAct) and by inhibiting its conversion to a repressor form (CiRep). When the full-length Ci protein is made ectopically in P cells, dpp and ptc are activated in a smo-dependent manner, and hh, a target of CiRep, is not repressed. These observations indicate that CiAct is functional in these cells and that CiRep is not. Both are hallmarks of Hh signaling. Using a temperature-sensitive allele of hh, the data with Fu* show that the state of the Hh signaling pathway is not constitutively activated in P cells, but that it reflects the activity of Hh (Ramirez-Weber, 2000).

Ptc protein and ptc RNA have been detected only in A cells, so a role for Ptc in suppressing activation of the Hh pathway in the P cells of imaginal discs seems unlikely. For technical reasons, this could not be tested directly by examining Fu* in ptc- P disc cells, so the possibility cannot be ruled out that P cells express ptc RNA and protein at levels that can not be detected. However, since the level of Ptc in P cells is much less than Smo, any model in which Ptc suppresses Smo signaling in the absence of Hh would require that Ptc act catalytically to silence Smo. If Ptc does act catalytically, it is not obvious why the much higher levels of Ptc in the A cells at the border fail to prevent Hh signaling. Moreover, the fact that overexpression of ptc depresses Hh signaling suggests that the relative levels of Hh and Ptc are important and directly influence Hh signaling. It therefore seems more likely that Hh signaling in discs is mediated by a Hh binding protein other than Ptc (Ramirez-Weber, 2000).

Previous work has shown that in embryos the Hh signal transduction pathway becomes Hh independent in the absence of Ptc. Several different ptc;hh allele combinations were examined, RNAi phenocopies of hh and ci were made in ptc mutants, and Fu* was independently monitored. In each assay, the results are consistent with the proposal that Hh signal transduction pathway is activated independently of Hh in ptc mutant embryos. This behavior contrasts with P disc cells, which are Hh dependent and Ptc independent (Ramirez-Weber, 2000).

Two issues that may be relevant to this apparent contradiction are the role of Ptc and the mechanisms involved in transporting Hh from producing to receiving cells. Hh is presumed to bind Ptc, although no binding studies with the Drosophila proteins have been described. In the work reported here, indirect evidence for a Hh-Ptc interaction is provided. Hh adopts a diffuse distribution in P cells and a particulate appearance in A cells. Ptc and Hh colocalize to these particles and ectopic expression of ptc in P cells blocks signaling, suppresses the production of Fu*, and redistributes Hh into Ptc-containing particles. It is not know whether the Hh protein in these punctate structures signals or has been sequestered for lysosomal degradation or whether these particles are heterogeneous and have different functions. The finding that P cells with a diffuse distribution of Hh produce Fu* while P cells with a particulate distribution of Hh do not shows that these particles do not correlate with signaling (Ramirez-Weber, 2000).

Perhaps the role of Ptc is in part to titrate Hh activity by targeting Hh to an endocytic pathway. This proposal places Ptc in a class of proteins that downregulates the signal that induces its own expression. Others in this class include Dad, an antagonist of Drosophila Dpp; Sprouty, an antagonist of Drosophila FGF; Argos, an antagonist of Drosophila EGF, and Naked, an antagonist of Wg. This model also suggests the presence of a Hh receptor other than Ptc that mediates signal transduction. The contrasting behavior of embryos and discs may reflect the use of different receptors, different regulatory components in the pathway, or the existence of compensating signaling systems in embryos that are not present in discs. Given the multiplicity of Hh binding proteins and the large and diverse group of organs in which Hh plays an instructive role, there may be significant heterogeneity in its downstream effectors (Ramirez-Weber, 2000).

Hh signaling in embryos and discs may also differ in the way they transport Hh to the target cells. The distances between Hh-producing cells and Hh-receiving cells does not exceed 2–3 cells in embryos, but may be significantly greater in discs. Different mechanisms may be used to move Hh over long distances or short, requiring distinct ways to engage the receptor. Further studies on the mechanisms that transport and bind Hh should resolve these issues (Ramirez-Weber, 2000).

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).

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).

Smoothened transduces Hedgehog signal by physically interacting with Costal2/Fused complex through its C-terminal tail

The Hedgehog (Hh) family of secreted proteins controls many aspects of growth and patterning in animal development. The seven-transmembrane protein Smoothened (Smo) transduces the Hh signal in both vertebrates and invertebrates; however, the mechanism of its action remains unknown. Smo lacking its C-terminal tail (C-tail) is inactive, whereas membrane-tethered Smo C-tail has constitutive albeit low levels of Hh signaling activity. Smo is shown to physically interact with Costal2 (Cos2) and Fused (Fu) through its C-tail. Deletion of the Cos2/Fu-binding domain from Smo abolishes its signaling activity. Moreover, overexpressing Cos2 mutants that fail to bind Fu and Ci but retain Smo-binding activity blocks Hh signaling. Taken together, these results suggest that Smo transduces the Hh signal by physically interacting with the Cos2/Fu protein complex (Jia, 2003).

The most surprising finding of this study is that the Smo C-tail suffices to induce Hh pathway activation. Overexpressing the membrane-tethered Smo C-tail (Myr-SmoCT, Sev-SmoCT) blocks Ci processing, induces dpp-lacZ expression, and stimulates nuclear translocation of Ci155. Myr-SmoCT is refractory to Ptc inhibition and activates Hh-pathway independent of endogenous Smo. Membrane tethering appears to be crucial for the Smo C-tail to activate the Hh pathway; untethered SmoCT has no signaling activity. This is consistent with observations that cell surface accumulation of Smo correlates with its activity (Jia, 2003).

Although the Smo C-tail has constitutive Hh signaling activity, it does not possess all the activities associated with full-length Smo. For example, overexpressing Myr-SmoCT in A-compartment cells away from the A/P compartment boundary does not significantly activate ptc and en, which are normally induced by high levels of Hh. In addition, Myr-SmoCT cannot substitute endogenous Smo at the A/P compartment boundary to transduce high levels of Hh signaling activity, since boundary smo mutant cells expressing Myr-SmoCT fail to express ptc in response to Hh (Jia, 2003).

The failure of the Smo C-tail to transduce high Hh signaling activity is due to its inability to antagonize Su(fu). Although Myr-SmoCT blocks Ci processing to generate Ci75, the activity of Ci155 accumulated in Myr-SmoCT-expressing cells is still blocked by Su(fu); removal of Su(fu) function from Myr-SmoCT-expressing cells allows Ci155 to activate ptc to high levels. Because Myr-SmoCT stimulates nuclear translocation of Ci155, the inhibition of Ci155 by Su(fu) in Myr-SmoCT-expressing cells must rely on a mechanism that is independent of impeding Ci nuclear translocation (Jia, 2003).

Several observations prompted a determination of whether Smo can transduce the Hh signal by physically interacting with the Cos2/Fu complex: (1) although Smo is related to G-protein-coupled receptors, no genetic or pharmacological evidence has been obtained to support the involvement of a G-protein in a physiological Hh signaling process; (2) Myr-SmoCT can interfere with the ability of endogenous Smo to transduce high levels of Hh signaling activity, which can be offset by increasing the amount of full-length Smo. This implies that Myr-SmoCT may compete with full-length Smo for binding to limiting amounts of downstream signaling components. (3) Extensive genetic screens failed to identify Hh signaling components that may link Smo to the Cos2/Fu complex (Jia, 2003).

Using a coimmunoprecipitation assay, it was demonstrated that Smo interacts with the Cos2/Fu complex both in S2 cells and in wing imaginal discs, and the Smo C-tail appears to be both necessary and sufficient to mediate this interaction. The Cos2/Fu-binding domain was narrowed down to the C-terminal half of the Smo C-tail (between amino acids 818 and 1035). Furthermore, both the microtubule-binding domain (amino acids 1-389) and the C-terminal tail (amino acids 990-1201) of Cos2 interact with Smo. Since none of these Cos2 domains binds Fu, this implies that the Cos2/Smo interaction is not mediated through Fu. Ci is also dispensable for Smo/Cos2/Fu interaction; Smo binds Cos2/Fu in S2 cells in which Ci is not expressed. However, the results did not rule out the possibility that Smo could interact with the Cos2/Fu complex through multiple contacts. For example, Smo could simultaneously contact Cos2 and Fu. Nor was it demonstrated that binding of Cos2 to Smo is direct. Indeed, no protein-protein interaction between Smo and Cos2 was detected in yeast. It is possible that a bridging molecule(s) is required to link Smo to the Cos2/Fu complex. Alternatively, Smo needs to be modified in vivo in order to bind Cos2. It has been shown that Hh stimulates phosphorylation of Smo; hence, it is possible that phosphorylation of Smo might be essential for recruiting the Cos2/Fu complex (Jia, 2003).

Several lines of evidence suggest that Smo/Cos2/Fu interaction is important for Hh signal transduction. (1) Deletion of the Cos2-binding domain from Smo, either in the context of full-length Smo or the Smo C-tail, abolishes Smo signaling activity. (2) Overexpressing Cos2 deletion mutants that no longer bind Fu and Ci but retain a Smo-binding domain intercept Hh signal transduction. Genetic evidence has been provided that Cos2 has a positive role in transducing Hh signal in addition to its negative influence on the Hh pathway, since Ci155 is no longer stimulated into labile and hyperactivity forms by high levels of Hh in cos2 mutant cells. In light of the finding that Smo interacts with Cos2/Fu, the simplest interpretation for a positive role of Cos2 is that it recruits Fu to Smo and allows Fu to be activated by Smo in response to Hh (Jia, 2003).

Of note, interaction between SmoCT and Cos2/Fu per se is not sufficient for triggering Hh pathway activation. For example, Myr-SmoCTDelta625-818, which binds Cos2/Fu to the same extent as Myr-SmoCT, does not possess Hh signaling activity. The fact that Myr-SmoDeltaCT625-730 and Myr-Smo730-1035 can activate the Hh pathway suggests that Smo sequence between amino acids 730 and 818 is essential. This domain may recruit factors other than Cos2/Fu to achieve Hh pathway activation. Alternatively, it might target SmoCT to an appropriate signaling environment (Jia, 2003).

An important property of Hh family members in development is that they can elicit distinct biological responses via different concentrations. How different thresholds of Hh signal are transduced by Smo to generate distinct transcriptional outputs is not understood. The results suggest that Smo can function as a molecular sensor that converts quantitatively different Hh signals into qualitatively distinct outputs. In the absence of Hh, the cell surface levels of Smo are low. In addition, the Smo C-tail may adopt a 'closed' conformation that prevents it from binding to Cos2/Fu. Low levels of Hh partially inhibit Ptc, leading to an increase of Smo on the cell surface. In addition, the Smo C-tail may adopt an 'open' conformation, which allows Smo to bind the Cos2/Fu complex and inhibit its Ci-processing activity. Low levels of Hh signaling activity can be mimicked by overexpression of either full-length Smo or membrane-tethered forms of the Smo C-tail. High levels of Hh completely inhibit Ptc, resulting in a further increase in Smo signaling activity. Hyperactive Smo stimulates the phosphorylation and activity of bound Fu, which in turn antagonizes Su(fu) to activate Ci155. Consistent with this, Fu bound to Myc-Smo was found to became phosphorylated in response to ectopic Hh (Jia, 2003).

The Smo sequence N terminus to SmoCT (SmoN) appears to be essential for conferring high Smo activities. It is not clear how SmoN modulates the activity of SmoCT. SmoN might recruit additional effector(s) or target SmoCT to a microdomain with a more favorable signaling environment. Alternatively, SmoN might function as a dimerization domain that facilitates interaction between two SmoCTs, as in the case of receptor tyrosine kinases. It is also not clear how Smo/Cos2/Fu interaction inhibits Ci processing. One possibility is that Smo/Cos2 interaction may cause disassembly of the Cos2/Ci complex, which could prevent Ci from being hyperphosphorylated; Cos2/Ci complex formation might be essential for targeting Ci to its kinases. Consistent with this view, Ci is barely detectable in the Cos2/Fu complex bound to Smo (Jia, 2003).

Physical association of the receptor complex with a downstream signaling component has also been demonstrated for the canonic Wnt pathway whereby the Wnt coreceptor LRP-5 interacts with Axin, a molecular scaffold in the Wnt pathway. Hence, Hh and Wnt/Wg pathways appear to use a similar mechanism to transmit signal downstream of their receptor complexes (Jia, 2003).

Hedgehog signal transduction via Smoothened association with a cytoplasmic complex scaffolded by the atypical kinesin, Costal-2

The seven-transmembrane protein Smoothened (Smo) transduces extracellular activation of the Hedgehog (Hh) pathway by an unknown mechanism to increase transcriptional activity of the latent cytoplasmic transcription factor Ci (Cubitus interruptus). Evidence is presented that Smo associates directly with a Ci-containing complex that is scaffolded and stabilized by the atypical kinesin, Costal-2 (Cos2). This complex constitutively suppresses pathway activity, but Hh signaling reverses its regulatory effect to promote Ci-mediated transcription. In response to Hh activation of Smo, Cos2 mediates accumulation and phosphorylation of Smo at the membrane as well as phosphorylation of the cytoplasmic components Fu and Su(fu). Positive response of Cos2 to Hh stimulation requires a portion of the Smo cytoplasmic tail and the Cos2 cargo domain, which interacts directly with Smo (Lum, 2003).

Early studies of Cos2 suggested primarily a negative role for Cos2 in pathway regulation, as manifested by phenotypic analysis of cos2 mutations and by a requirement for Cos2 in cytoplasmic retention of Ci and in its proteolytic processing to produce CiR. More recent studies have suggested a potential positive role based on a requirement for Cos2 in transcriptional activation of gene targets associated with highest levels of pathway activity. This study extends the evidence for such a positive role by demonstrating: (1) a requirement for Cos2 in mediating a series of Hh-induced biochemical changes in pathway components; (2) an association between the Cos2/Fu/Ci complex and Smo; (3) a direct interaction between Smo and Cos2, and (4) a requirement for Cos2 in highest level Hh pathway response in cultured cell reporter assays and Hh-induced morphogenesis in the dorsal cuticle (Lum, 2003).

Based on the sequence relationship between Smo and GPCRs, previous speculation and experimental work has focused on the possibility that Smo may interact with heterotrimeric G proteins. G protein components have been systematically targeted using RNAi in a cultured cell signaling assay, and no significant role has been found for G proteins in transcriptional regulation via Ci. A potential role cannot be ruled out for G proteins or other mediators in cellular responses to Hh signaling that do not involve transcriptional regulation via Ci/Gli. For example, a recently described chemoattractant activity for Shh in axon guidance appears to be mediated by Smo, yet proceeds in a short time scale and with a local cell polarity that suggests a possible nonnuclear mechanism of response (Lum, 2003).

In searching for other mediators of information transfer from membrane to cytoplasm, it was surprising to find cytoplasmic components copurifying with Smo. Since these were the only Drosophila proteins identified in the bands excised, it is concluded that these complexes were highly pure, and that Smo associates stably with components of the cytoplasmic complex in vivo. It is further demonstrated that Smo interacts directly with Cos2, which scaffolds this complex. Consistent with these findings, articles presenting genetic evidence for a role of the Smo cytoplasmic tail in Hh signaling and evidence suggesting a physical interaction between Smo and Cos2 were published during final review of this work. Additional direct association of Smo with other complex components have not been ruled out (Lum, 2003 and references therein).

The identification of a complex that includes both Ci and Smo immediately suggested that recruitment of the cytoplasmic complex to Smo upon Hh stimulation might be critical for pathway activity. Cos2 plays a central role in mediating this association, functioning both as a scaffold that brings together cytoplasmic components (the Cos2 complex) and as a sensor that monitors the state of pathway activation by interacting with Smo at the membrane (Lum, 2003).

In the unstimulated state, Smo levels are low, and most of the Cos2 complex therefore is not associated with or influenced by Smo; even the small fraction of Cos2 complex associated with Smo may be negative, since Smo may not be in an active state. The negative form of the Cos2 complex presumably mediates production of CiR and prevents nuclear accumulation of Ci, resulting in a net suppression of transcriptional targets. In the intermediate state, present after a few minutes of stimulation, Smo protein has become activated by Hh stimulation but has not yet accumulated. Therefore, despite a positive state for the specific Cos2 complexes affected by Smo, the low level of activated Smo protein is insufficient for interaction with most of the Cos2 complexes. The net outcome with regard to transcriptional targets thus remains negative (Lum, 2003).

In stimulated cells, activated Smo exerts a pervasive influence on the Cos2 complex, either through a stable association or through a transient association with enduring effects. This association presumably involves a direct binding interaction between a portion of the Smo cytotail and the Cos2 cargo and stalk domains. The evidence suggests that both activation and accumulation of Smo are critical, as evidenced by the observation that moderate Smo overexpression alone is unable to fully activate the pathway in the absence of Hh stimulation. Similarly, a cycloheximide block of Smo accumulation dramatically limits the biochemical changes normally induced by Hh stimulation, and cotransfection experiments demonstrate that transition of Cos2 from a pathway suppressor to activator requires adequate levels of activated Smo. The activation of transcriptional targets resulting from pathway stimulation presumably result from the positive action of Cos2 on Fu and from loss of the ability to produce CiR (Lum, 2003).

It is the dual action of Cos2 in promoting formation of CiR and in stabilization and possible activation of Fu that leads to the apparent dual negative and positive roles of Cos2 in pathway regulation. Pathway activation induced by loss of Cos2 thus results from loss of CiR, but this activation is only partial because Fu is also destabilized, and the pathway-suppressing action of Su(fu) is unchecked. Consistent with this interpretation, a combined loss of Cos2 and Su(fu) results in maximal pathway activation irrespective of the presence of Hh (Lum, 2003).

How does Cos2 function? Motor proteins recently have been found to play a role in regulation of transmembrane receptors, as in the case of rhodopsin and mannose-6-phosphate receptor. These motor proteins apparently regulate receptor localization but do not play a direct role in receptor function. A recent report suggests that Cos2 overexpression indeed may influence Smo localization. The evidence, however, suggests that Cos2 also functions as a primary sensor of the state of pathway activation by interacting with Smo at the membrane and by scaffolding and stabilizing downstream pathway components (Lum, 2003).

Kinesins likely share an evolutionary origin with G proteins and myosins, and all three types of proteins use a conserved mechanism to couple nucleotide hydrolysis to dramatic conformational changes in protein structure. Kinesins utilize this mechanism to generate force that allows movement on microtubules. Sequences essential for microtubule binding, for nucleotide binding, and for motor function in other kinesins, however, are not conserved among Cos2 sequences in the Diptera and are not required for Cos2 function in cultured cells. It is still possible, however, that Cos2 retains the capacity for a conformational shift that may be triggered by Smo activation. If so, then this conformational change may involve the Cos2 cargo domain, which binds to Smo at the cytoplasmic tail and is likely required for Smo responsiveness (Lum, 2003).

Whatever its mechanism of activation, evidence points to a role for the atypical kinesin Cos2 as a scaffold and sensor that functions as the pivotal component in transduction of pathway activation from the seven transmembrane receptor Smo to the latent cytoplasmic transcription factor Ci. In addition to stabilizing Fu and mediating forward signaling events that affect other cytoplasmic components and Ci, Cos2 is also required for the accumulation of activated Smo, a critical aspect of producing a full response to Hh signal. Cos2 thus functions not just as a passive sensor for the state of pathway activation at the membrane, but is also an active participant in the cellular dynamics of transition from the unstimulated to the stimulated state. These activities are all the more remarkable in view of the well-recognized role played by Cos2 in maintaining the unstimulated state of the Hh pathway, and together these findings suggest that Cos2 dynamics are a critical determinant of intracellular Hh pathway response and regulation (Lum, 2003).

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).

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

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; Fused kinase activity increases the specific activity of Ci-155

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Evidence for a novel feedback loop in the Hedgehog pathway involving Smoothened and Fused

Hedgehog (HH) is a major secreted morphogen involved in development, stem cell maintenance and oncogenesis. In Drosophila wing imaginal discs, Hh produced in the posterior compartment diffuses into the anterior compartment to control target gene transcription via the transcription factor Cubitus interruptus (Ci). The first steps in the reception and transduction of the Hh signal are mediated by its receptor Patched (Ptc) and the seven-transmembrane-domain protein Smoothened (Smo). Ptc and Hh control Smo by regulating its stability, trafficking, and phosphorylation. Smo interacts directly with the Ser-Thr protein kinase Fused (Fu) and the kinesin-related protein Costal2 (Cos2), which interact with each other and with Ci in an intracellular Hedgehog transducing complex. Hh induces Fu targeting to the plasma membrane in a Smo-dependent fashion and, reciprocally, Fu controls Smo stability and phosphorylation. Fu anchorage to the membrane is sufficient to make it a potent Smo-dependent, Ptc-resistant activator of the pathway. These findings reveal a novel positive-feedback loop in Hh transduction and are consistent with a model in which Fu and Smo, by mutually enhancing each other's activities, sustain high levels of signaling and render the pathway robust to Ptc level fluctuations (Claret, 2007).

This work provides new information about (1) the mechanisms by which the activation of Smo is transduced to its cytoplasmic effector Fu, (2) the mechanisms of Fu activation, and (3) a novel positive-feedback loop between Fu and Smo. Evidence is provided that Smo controls the subcellular distribution of two of its physical partners, Fu and Cos2, recruiting them to the plasma membrane in response to Hh. The data also suggest that Fu might link Cos2 to Smo in a vesicle-associated complex in the absence of Hh, whereas Fu and Cos2 might independently bind Smo at the plasma membrane in the presence of Hh. Thus, Hh might not only promote, via Smo, the recruitment of Fu and Cos2 to the plasma membrane; it might also modulate the nature of interactions between these three proteins (Claret, 2007).

Several nonexclusive mechanisms seem to be involved in controlling Fu activity. (1) The forced localization of Fu at the membrane induces strong Smo-dependent activation of the pathway in the wing. This study is the first to report a dominant active form of this type of kinase. (2) This study shows that the presence of a conserved Thr in the activating loop is important for the promotion of full Smo phosphorylation and for the activating effects of GAP-Fu. Thus, because Fu is known to be phosphorylated in response to Hh, the phosphorylation of this loop (by autophosphorylation or by other kinases) might be a key element in Fu regulation. (3) Hh might regulate Fu by controlling its dimerization or its interaction with potential regulatory proteins. Possible Fu dimerization is consistent with (1) the reported interaction between the regulatory domain of Fu and its kinase domain, (2) the recruitment to the plasma membrane of wild-type Fu by the wild-type and mutant forms of GAP-Fu, and (3) the dominant negative effects of Fu mutants with modified kinase domains (Claret, 2007).

Evidence is presented for of a novel, positive-feedback loop in which Smo and Fu enhance each other's activities. Indeed, Smo promotes the relocalization of Fu to the plasma membrane and is required for the activating effects of GAP-Fu, whereas both GAP-Fu and Fu control Smo stability and phosphorylation. Fu kinase activity is required for Smo phosphorylation and for the activating effects of GAP-Fu, but not for its association with Smo. Fu might phosphorylate Smo directly or might act on other substrates, indirectly facilitating Smo phosphorylation, inhibiting phosphatases, or stabilizing phosphorylated Smo. Both Fu activity and its interaction with Smo seem to be required for full hyperphosphorylation of Smo in response to Hh (Claret, 2007).

In the wing imaginal disc, Fu is required principally for responses to the highest levels of Hh present at the anteroposterior border, where Smo is active despite the strong upregulation of ptc. The effects of GAP-Fu and Fu on Smo provide the first clues to a putative mechanism (Fu-dependent phosphorylation and stabilization of Smo), potentially accounting for the resistance of Smo to the high level of Ptc induced by Hh in responding cells (Claret, 2007).

The following model is proposed: (1) The Hh-induced relocalization of Smo to the plasma membrane leads to the recruitment of Fu and Cos2 at this membrane. (2) Fu, in turn, acts on Smo, probably by further enhancing its phosphorylation, to stabilize it further and prevent its inhibition by Ptc. It is not yet possible to determine whether Fu regulates Smo directly or indirectly. The kinesin Cos2 may be also part of this regulatory loop. (3) The stabilized, activated Smo/Fu/Cos2 complex at the plasma membrane then promotes the accumulation and activation of Ci-FL, leading to the activation of Hh target genes, including ptc (Claret, 2007).

SmoδFu, which does not bind Fu, is constitutively active, suggesting that Fu might also act as a negative regulator of Smo in the absence of the Hh signal. Thus, Fu might act as a switch, sensing the level of Hh, inhibiting Smo in the absence of Hh or activating the pathway in response to high levels of Hh. Interestingly, the existence of such regulatory loops might account for the bistability properties of signaling pathways and explain how graded levels of signal might act as morphogens, leading to differential cell responses (Claret, 2007).

In conclusion, Fu was found to be recruited by Smo at the plasma membrane in response to Hh and this recruitment was found to be directly dependent on the physical interaction of Fu with Smo. The expression of a membrane-anchored form of Fu (GAP-Fu) constitutively activates the Hh pathway, indicating that Fu activity might be regulated by its subcellular location. Surprisingly, the activating effects of GAP-Fu require a wild-type dose of endogenous Smo. Evidence is reported that (1) Fu and GAP-Fu induce the phosphorylation of Smo, (2) GAP-Fu recruits Smo to the plasma membrane, (3) GAP-Fu renders Smo resistant to the destabilizing effects of Ptc, and (4) Fu controls the level of accumulation of Smo in the wing imaginal disc. Thus, these data demonstrate that Fu, which is generally considered to be an effector of Smo, can also act on Smo (Claret, 2007).

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

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

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

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

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

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

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

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

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

The Hedgehog-induced Smoothened conformational switch assembles a signaling complex that activates Fused by promoting its dimerization and phosphorylation

Hedgehog (Hh) transduces signal by regulating the subcellular localization and conformational state of the GPCR-like protein Smoothened (Smo) but how Smo relays the signal to cytoplasmic signaling components remains poorly understood. This study shows that Hh-induced Smo conformational change recruits Costal2 (Cos2)/Fused (Fu) and promotes Fu kinase domain dimerization. Induced dimerization through the Fu kinase domain activates Fu by inducing multi-site phosphorylation of its activation loop (AL) and phospho-mimetic mutations of AL activate the Hh pathway. Interestingly, it was observed that graded Hh signals progressively increase Fu kinase domain dimerization and AL phosphorylation, suggesting that Hh activates Fu in a dose-dependent manner. Moreover, it was found that activated Fu regulates Cubitus interruptus (Ci) by both promoting its transcriptional activator activity and inhibiting its proteolysis into a repressor form. Evidence is provided that activated Fu exerts these regulations by interfering with the formation of Ci-Sufu and Ci-Cos2-kinase complexes that normally inhibit Ci activity and promote its processing. Taken together, these results suggest that Hh-induced Smo conformational change facilitates the assembly of active Smo-Cos2-Fu signaling complexes that promote Fu kinase domain dimerization, phosphorylation and activation, and that Fu regulates both the activator and repressor forms of Ci (Shi, 2011).

How Hh signal is transduced from the GPCR-like receptor Smo to the transcription factor Ci/Gli is still poorly understood. A major unsolved issue is how a change in the Smo activation state is translated into a change in the activity of intracellular signaling complexes, which ultimately changes the balance between CiR/GliR and CiA/GliA. The current study suggests that Hh-induced conformational change of Smo exposes a Cos2 docking site(s) near the Smo C terminus that facilitates the assembly of an active Smo-Cos2-Fu complex, and that Smo activates Fu by promoting its kinase domain dimerization and phosphorylation. Evidence is provided that graded Hh signals progressively increase Fu kinase domain dimerization and phosphorylation, which may generate a Fu activity gradient, and that activated Fu regulates both CiR and CiA by controlling Ci-Sufu and Ci-Cos2-kinase complex formation (Shi, 2011).

Previous immunoprecipitation studies have revealed that Smo pulled down Cos2/Fu in both quiescent cells and Hh-stimulated cells, suggesting that Smo can form a complex with Cos2/Fu even in the absence of Hh. Furthermore, deletion analyses have indicated that both a membrane proximal domain and a C-terminal region of Smo C-tail can mediate the interaction between Smo and Cos2/Fu. Intriguingly, deleting the C-terminal region impaired, whereas deleting the membrane proximal domain potentiated, Smo activity in vivo. Further study suggested that the membrane proximal domain recruits Cos2/PP4 to inhibit Smo phosphorylation and cell-surface accumulation, which is released by Fu-mediated phosphorylation of Cos2 Ser572 in response to Hh. These observations suggest that Smo-Cos2-Fu interaction is likely to be dynamic and that distinct complexes may exist depending on the Hh signaling status. For example, Cos2 may associate with the membrane proximal region of Smo to inhibit Smo phosphorylation in quiescent cells. Upon Hh stimulation, Cos2/Fu may interact with the C-terminal region of Smo to transduce the Hh signal. In support of this model, it was found that Hh stimulated the recruitment of Cos2/Fu to the C-terminal region rather than the membrane proximal region of the Smo C tail. The increased binding depends on phosphorylation-induced conformational change of Smo C-tail that may expose the C-terminal Cos2 binding pocket(s) (Shi, 2011).

Hh signaling induces Fu kinase domain dimerization in a dose-dependent manner, most probably as a consequence of phosphorylation-induced conformational change and dimerization of Smo C tails. In addition, Hh-induced Fu dimerization depends on Cos2. Importantly, dimerization through the Fu kinase domain (CC-Fu) triggers Fu activation both in vitro and in vivo. Furthermore, CC-Fu can activate Ci in smo mutant clones and restore high levels of Hh signaling activity in cos2 mutant discs. Taken together, these results support a model in which Hh-induced Fu dimerization via Smo/Cos2 leads to Fu activation (Shi, 2011).

Both Fu dimerization and Hh stimulation induce phosphorylation of multiple Thr/Ser residues in the Fu activation loop that are important for Fu activation. Fu phosphorylation depends on its kinase activity and Fu can trans-phosphorylate itself, suggesting that Hh and dimerization may induce Fu autophosphorylation, although the results do not exclude the involvement of additional kinase(s). CC-induced dimerization does not fully activate Fu, suggesting that Smo may promote Fu activation through additional mechanisms. Activated Fu can promote phosphorylation of its C-terminal regulatory fragment, raising a possibility that Fu activation may also involve phosphorylation of its regulatory domain. Indeed, while this manuscript was under review, Zhou and Kalderon provided evidence that phosphorylation of several Ser/Thr residues in the Fu regulatory domain, likely by CK1, modulates the activity of an activated form of Fu (Zhou, 2011; Shi, 2011 and references therein).

The involvement of multiple phosphorylation events in Fu activation may provide a mechanism for fine-tuning Fu activity in response to different levels of Hh. Indeed, the efficiency of Fu dimerization and the level of activation loop phosphorylation correlate with the level of Hh signaling. Furthermore, the level of Fu activity correlates with the level of its activation loop phosphorylation. Thus, graded Hh signals may generate a Fu activity gradient by progressively increasing its dimerization and phosphorylation in response to a gradual increase in Smo phosphorylation and C-tail dimerization (Shi, 2011).

The conventional view is that Fu is required for high levels of Hh signaling by converting CiF into CiA. In support of this notion, fu mutations only affect the high, but not low, threshold Hh responsive genes. However, Fu function could have been underestimated because none of the fu mutations examined so far represents a null mutation. In addition, the existence of paralleled mechanisms, such as Gαi activation, could mask the contribution of Fu to low levels of Hh signaling. Nevertheless, a recent study using the phospho-specific antibody against Cos2 Ser572 revealed that Fu kinase activity could be induced by low levels of Hh, raising an interesting possibility that Fu may contribute to all levels of Hh signaling (Raisin, 2010). However, the lack of a fu-null mutation and the involvement of Fu in promoting Ci processing, probably through a structural role, make it difficult to directly demonstrate a role of Fu in blocking Ci processing. Using an in vivo assay for Ci processing, it was demonstrated that activated forms of Fu block Ci processing into CiR. In addition, this study found that activated Fu attenuates the association between Cos2 and Ci, as well as their association with PKA/CK1/GSK3, probably by phosphorylating Cos2, suggesting that activated Fu may block Ci processing by impeding the formation of the kinase complex required for efficient Ci phosphorylation (Shi, 2011).

Evidence is provided that activated Fu attenuates Ci/Sufu interaction. Because Sufu impedes Ci nuclear localization and may recruit a co-repressor(s) to further inhibit Ci activity in the nucleus, dissociation of Ci from Sufu may lead to the conversion of CiF to CiA. Interestingly, recent studies using mammalian cultured cells revealed that Shh signaling induces dissociation of full-length Gli proteins from Sufu (Humke, 2010; Tukachinsky, 2010), suggesting that inhibition of Sufu-Ci/Gli complex formation could be a conserved mechanism for Ci/Gli activation. Although activated forms of Fu promote Sufu phosphorylation, phospho-deficient and phospho-mimetic forms of Sufu behaved in a similar manner to wild-type Sufu in functional assays (Zhou, 2011), implying that phosphorylation of Sufu might not be a major mechanism through which Fu activates Ci. It has been shown that Shh also induces phosphorylation of full-length Gli3 that correlates with its nuclear localization (Humke, 2010). Furthermore, a Fu-related kinase Ulk3 can phosphorylate Gli proteins and promote their transcriptional activities (Maloverjan, 2010). Thus, Fu may activate Ci by promoting its phosphorylation, an interesting possibility that awaits further investigation (Shi, 2011).

The Fused/Smurf complex controls the fate of Drosophila germline stem cells by generating a gradient BMP response

In the Drosophila ovary, germline stem cells (GSCs) are maintained primarily by bone morphogenetic protein (BMP) ligands produced by the stromal cells of the niche. This signaling represses GSC differentiation by blocking the transcription of the differentiation factor Bam. Remarkably, bam transcription begins only one cell diameter away from the GSC in the daughter cystoblasts (CBs). How this steep gradient of response to BMP signaling is formed has been unclear. This study shows that Fused (Fu), a serine/threonine kinase that regulates Hedgehog, functions in concert with the E3 ligase Smurf to regulate ubiquitination and proteolysis of the BMP receptor Thickveins in CBs. This regulation generates a steep gradient of BMP activity between GSCs and CBs, allowing for bam expression on CBs and concomitant differentiation. Similar roles for Fu were observed during embryonic development in zebrafish and in human cell culture, implying broad conservation of this mechanism (Xia, 2010).

Previous studies have demonstrated that BMP/Dpp signals from the niche play primary roles in the self-renewal of GSCs by silencing bam transcription. However, the mechanism by which the differentiating CBs avoid the control of BMP/Dpp and activate bam remains poorly understood. This study has provided direct evidence that the differentiating daughter cells of GSCs, known as CBs, become resistant to BMP signaling through degradation of Tkv in CBs. Fu functions as an antagonistic factor in BMP/Dpp signaling by regulating Tkv degradation during the differentiation of CBs. Moreover, both genetic and biochemical evidence is provided that Fu acts in concert with Smurf, a HECT domain-containing ubiquitin E3 ligase, to regulate the ubiquitination of Tkv in the CB, thereby generating a steep gradient of response to BMP signaling between GSCs and CBs for their fate determination. Finally, a conserved role is shown for fu in antagonizing BMP/ TGFβ signals in zebrafish embryonic development as well as in human cell cultures. These findings not only reveal a conserved function of fu in controlling BMP/TGFβ signal-mediated developmental processes, but also provide a comprehensive view of mechanisms that produce both self-renewal and asymmetry in the division of stem cells (Xia, 2010).

Observations of the existence of a BMP resistance mechanism that controls the proper division of GSCs through the regulation of Tkv prompted an exploration of how Tkv was regulated. Using immunoprecipitation followed by mass spectrometry analysis, it was identified that Fu associates with the Tkv protein. Given that previous studies demonstrated that a loss of fu leads to early germ cell proliferation and a tumorous germarium phenotype and that biochemical evidence showed that Fu forms a complex with Tkv and affects its stability, it was subsequently identified that Fu as a component negatively regulates BMP/Dpp signaling by interacting with the BMP/Dpp type I receptor, Tkv (Xia, 2010).

BMP/TGFβ signals play pivotal roles in controlling diverse normal developmental and cellular processes. In the canonical BMP/TGFβ pathway, the receptors and Smad proteins are the essential components for BMP/TGFβ signal transduction. However, this pathway is known to be modulated by additional factors to reach physiological levels in a cellular context-dependent manner. Smurfs and HECT domain-containing proteins have been shown to antagonize BMP/TGFβ signals through the regulation of the stability of either receptors or Smads in vertebrates. In Drosophila, Smurf has previously been implicated in regulating proteolysis of phosphorylated Smad proteins in somatic cells. In the ovary, Smurf was also proposed to downregulate the level of BMP to promote CB differentiation. The mechanism underlying the action of Smurf in Drosophila early germline cells remains elusive. This study has shown that Fu, Smurf, and Tkv could form a trimeric complex in S2 cells. Importantly, both Fu and Smurf are required for ubiquitination of Tkv in S2 cells and for turnover of Tkv in germ cells. Combined with genetic evidence, it is proposed that Fu and Smurf likely function in a common biochemical process by controlling Tkv degradation. The present study reveals a mechanism by which Fu serves as an essential component in the Smurf-mediated degradation of the BMP/TGFβ receptor, thereby terminating BMP/TGFβ signaling and negatively regulating the downstream target genes of BMP/TGFβ (Xia, 2010).

Because Fu is a putative serine/threonine protein kinase, the question becomes how Fu acts on Tkv regulation in concert with Smurf. Given that knockdown of fu does not significantly change the pattern of autoubiquitination of Smurf itself, it is therefore likely that Tkv is a strong candidate substrate for Fu kinase. Although there is no assay system for analyzing the kinase activity of Fu presently, in this study, mutagenesis assays were perfomred and it was identified that the S238 in Tkv is important for Tkvca to respond to Fu and is critical for Tkvca ubiquitination and degradation. Of note, it was found that the ubiquitin- resistant form of Tkvca [TkvcaS238A] blocks CB differentiation. A previous study has shown that the S189 site in TGF-β type-I receptor, the corresponding site of S238 in Tkv, was phosphorylated in the cell culture system. The current results suggest that Fu likely acts on Tkv through targeting and phosphorylating the S238 site and subsequently leads to Tkv ubiquitination and degradation by Smurf. Nevertheless, it would be advantageous to develop a kinase assay system for Fu to determine whether the S238 site in Tkv is an authentic phosphorylation site for Fu kinase in the future (Xia, 2010).

Previous genetic analyses revealed that Fu plays an evolutionarily conserved role in the proper activation of the Hh pathway and functions downstream of the Hh receptor. Increasing evidence has shown that the kinase Fu regulates the Hh-signaling complex by targeting Cos2. However, the function of Fu as a component in the Hh pathway is not consistent with its spatiotemporal expression pattern during development. For example, Hh signaling only plays a role in zebrafish embryonic development at late stages, but Fu is expressed ubiquitously at both the early and the late stages of zebrafish embryonic development. These findings suggest that Fu may have Hh-independent functions in different physiological conditions. In this study, by using several different systems, including Drosophila germline, zebrafish embryo, and human tissue cultures, it was demonstrated that Fu is indeed required for balancing proper BMP/TGFβ signals in different developmental processes. Given that both Fu and Smurf are evolutionarily conserved proteins, it would be interesting to determine whether the Fu/Smurf complex also plays roles in other signaling pathways (Xia, 2010).

The niche-dependent feedback loop generates a BMP activity gradient to determine the germline stem cell fate.

Stem cells interact with surrounding stromal cells (or niche) via signaling pathways to precisely balance stem cell self-renewal and differentiation. However, little is known about how niche signals are transduced dynamically and differentially to stem cells and their intermediate progeny and how the fate switch of stem cell to differentiating cell is initiated. The Drosophila ovarian germline stem cells (GSCs) have provided a heuristic model for studying the stem cell and niche interaction. Previous studies demonstrated that the niche-dependent BMP signaling is essential for GSC self-renewal via silencing bam transcription in GSCs. The Fused (Fu)/Smurf complex has been shown to degrade the BMP type I receptor Tkv allowing for bam expression in differentiating cystoblasts (CBs). However, how the Fu is differentially regulated in GSCs and CBs remains unclear. This study reports that a niche-dependent feedback loop involving Tkv and Fu produces a steep gradient of BMP activity and determines GSC fate. Importantly, it was shown that Fu and graded BMP activity dynamically develop within an intermediate cell, the precursor of CBs, during GSC-to-CB transition. Mathematic modeling reveals a bistable behavior of the feedback-loop system in controlling the bam transcriptional on/off switch and determining GSC fate (Xia, 2012).

In the feedback loop model to show how the GSC fate is regulated. In the model, the external BMP signal cues stimulate phosphorylation of Tkv protein, the activated Tkv then promotes the synthesis rate of phosphorylated Mad (pMad), and pMad promotes the degradation of Fu protein and represses the transcription of bam. Meanwhile, degradation of the activated Tkv is also controlled by Fu. To assess the dynamic properties of this feedback loop, it was assumed that the transcriptions of genes tkv, mad, and fu are sufficient and that the degradation rate of pMad and the synthesis rate of Fu protein are constants. The network diagram of the feedback loop clearly points out two characteristics of the model: first, the microenvironment-derived BMP ligands serve as a key external signal, the strengths of which are differentially sensed by GSCs, pre-CBs, and CBs, thereby regulating the dynamic expression of the activated Tkv, pMad, and Fu during the asymmetric division of GSCs. Second, although the transcription of the bam gene is regulated negatively by Tkv/pMad, the expressions (and/or regulations) of the activated Tkv, pMad, and Fu are independently of the status of the Bam protein (Xia, 2012).

The dynamic analysis reveals the bistable behavior (i.e., switch behavior) of the system and how the system dynamics respond to the strength of external BMP ligand activity. Specifically, the strong external BMP ligand activity (in GSCs) will lead to a low expression level of Fu as well as high expression levels of the activated Tkv and pMad. Conversely, the weak external BMP ligand activity (in CBs) will lead to a high level of Fu expression (and low levels of the activated Tkv and pMad expression). However, for the transitional stage with intermediate BMP signaling (in pre-CBs), both high and low levels of Fu and pMad expression exist. These theoretical predictions not only exactly match the experimental data, but they also bring an insightful physical interpretation for why the niche dependence of BMP signaling determines the fate of stem cells by precisely balancing of stem cell renewal and differentiation. The current model permits the proposal of a comprehensive description of the action of niche signaling that governs the decision between stem cells and differentiating cells (Xia, 2012).

Cilia-mediated Hedgehog signaling in Drosophila

Cilia mediate Hedgehog (Hh) signaling in vertebrates and Hh deregulation results in several clinical manifestations, such as obesity, cognitive disabilities, developmental malformations, and various cancers. Drosophila cells are nonciliated during development, which has led to the assumption that cilia-mediated Hh signaling is restricted to vertebrates. This study identified and characterized a cilia-mediated Hh pathway in Drosophila olfactory sensory neurons. Several fundamental key aspects of the vertebrate cilia pathway, such as ciliary localization of Smoothened and the requirement of the intraflagellar transport system, are present in Drosophila. Cos2 and Fused are required for the ciliary transport of Smoothened and cilia mediate the expression of the Hh pathway target genes. Taken together, these data demonstrate that Hh signaling in Drosophila can be mediated by two pathways and that the ciliary Hh pathway is conserved from Drosophila to vertebrates (Kuzhandaivel, 2014).

The existence of this second cilia-dependent Hh pathway in Drosophila shows that Hh signaling can be mediated via two pathways within a single organism. The results further demonstrate that the core components are shared between the two Hh pathways in Drosophila. The function of Cos2 as a putative kinesin in the ciliary compartment indicates that the ancestral Hh signaling pathway may have been cilia specific and that invertebrate cells did not maintain this specialization. Interestingly, not all vertebrate cells have primary cilia, and different types of tumors react differently to Shh depending on whether they are ciliated, indicating that there might be a second, overlooked nonciliary pathway in vertebrates (Kuzhandaivel, 2014).

Genetic in vivo analysis of Smo ciliary localization revealed that, as in vertebrates, the ciliary IFT system and a ciliary localization signal are required for localization of Smo to cilia in Drosophila. The results further show that the Hh receptor Ptc regulates Smo stability and that ciliary localization depends on the activation of the kinesin-like protein Cos2. In the Drosophila wing disc, Fu regulates Cos2 function and is required for most aspects of Hh signaling. The current data show that Fu is also required for Cos2 ciliary localization and Smo transport within the cilia. However, Fu is not essential for mammalian Hh signaling, and in zebrafish, loss of Fu results in weak Hh-related morphological phenotypes. These differences from the Drosophila pathway and vertebrate ciliary signaling could be explained by the existence of a second, as yet unidentified kinase with an analogous function. Cell culture and in vivo studies in vertebrates led to the identification of four kinases with phenotypes related to Fu: Ulk3, Kif11, Map3K10, and Dyrk2. Further investigation is required to determine whether these kinases control the ciliary transport of Smo and whether Cos2 Smo transport is conserved in vertebrates. Yet, the current results demonstrate that cilia-mediated Hh signaling does occur in Drosophila and that this pathway is conserved in vertebrates, which makes the Drosophila OSN a powerful in vivo model for studying Hh signaling and its ciliary transport regulation (Kuzhandaivel, 2014).


fused: Biological Overview | Evolutionary Homologs | Developmental Biology | References

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