The Hedgehog (Hh) family of signaling proteins mediate 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. 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 that are bound to microtubules in a 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 have been 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).
Su(fu) is a novel protein containing a PEST sequence, a sequence that has been associated with rapidly degraded proteins. Su(fu) has been shown by genetic means to antagonize fu in a dose-dependent manner. To establish whether Fu could interact with Su(fu) at the molecular level, a two-hybrid assay was used. Two fusion proteins were co-expressed in yeast: Su(fu) fused to the DNA-binding domain of LexA [LexA-Su(fu)], and the carboxy-terminal putative regulatory domain of Fu (Fu-reg; amino acids 306-805) fused to a transactivation domain B42 (B42-Fu-reg). The two reporter genes (lacZ and LEU2) were specifically activated in the presence of the two-hybrid proteins, as attested by the appearance of a Leu+ phenotype and the production of beta-galactosidase. In the controls, no such activation was observed. The beta-galactosidase activity induced by interaction of LexA-Su(fu) and B42-Fu-reg was about 70-fold higher than the sum of the activities of the corresponding control strains. Using deletion analysis, it was established that the region of Fu-reg that spans amino-acid residues 306-436 is sufficient for its interaction with Su(fu). Three sequence elements that are highly conserved between D. virilis and D. melanogaster are present within this domain (Monnier, 1998).
Although no direct target for Fu has been identified so far, it has been shown to act upstream of Ci. Ci plays a dual role in Hh signaling. In the absence of the Hh signal, cleavage of Ci turns it into a transcriptional repressor, whereas Hh signaling prevents Ci truncation, leading to the accumulation of a full-length form of Ci which is thought to be transcriptionally active. To test whether Fu affects Ci directly, or through Su(fu), interactions between Ci and Fu or Su(fu) were tested. Using the two-hybrid assay, no interaction between Ci and Fu-reg could be detected. In contrast, co-expression of B42-Ci and LexA-Su(fu) specifically activates both reporter genes, leading to a more than 30-fold increase in beta-galactosidase activity. The first 346 amino-terminal residues of Ci (Ci-Delta346) are sufficient to mediate the latter's interaction with Su(fu). This domain contains several sequences that are conserved among Gli protein. [35S]methionine-labelled Ci synthesized in vitro is specifically retained by the GST-Su(fu) fusion protein, but not by GST alone, thus confirming the existence of a direct physical interaction between Su(fu) and Ci proteins (Monnier, 1998).
The biological relevance of these interactions is supported by previously reported genetic data and by the fact that Ci and Fu co-immunoprecipitate (Robbins, 1997). Furthermore, using an anti-Su(fu) immunoserum, full-length Ci could be co-immunoprecipitated from wild-type embryo extracts but not from mutant embryos deficient for Su(fu). These results shed new light on the recent observation that large complexes that contain Fu and Ci as well as Cos-2 are present in embryonic extracts. The Fu-Ci interaction is not direct but is mediated by Su(fu). It is proposed that Su(fu) prevents Ci transcriptional activity and that, upon Hh reception, this inhibition is opposed by the negative effect of Fu on Su(fu). This model is in agreement with the embryonic expression pattern of one Hh target gene, wingless (wg), in fu- and Su(fu)- mutants (Preat, 1993). In fact, it is possible that the absence of wg expression in fu- mutants results from a constitutive inhibition of Ci by Su(fu). In contrast, in Su(fu)-, as in fu- Su(fu)- double mutants, Ci escapes from the control of Su(fu) and as a consequence no longer requires the action of Fu to transactivate wg. The control of Ci activity by Su(fu) might rely on the cytoplasmic retention of Ci, like the Rel transcription factor NF-kappaB/Dorsal which is regulated by its cytoplasmic retention by I-kappaB/Cactus. Here, Fu activation possibly triggers the degradation of Su(fu) through the phosphorylation of its PEST sequence. However, no significant change in the subcellular localization of full-length Ci could be detected in wing imaginal discs of Su(fu)- mutants. One interpretation of this observation is that the control exerted by Su(fu) on Ci occurs at another level, for instance by controlling Ci processing (Monnier, 1998).
Su(fu) function is probably redundant, since it is dispensable in an otherwise wild-type context. Ci transactivating activity is probably also under the negative control of another Hh antagonist the kinesin-related protein Cos-2. Several lines of evidence uphold this hypothesis. (1) In cos-2 mutants, the processing of Ci into a repressor form is reduced; (2) Cos-2, like Su(fu), acts as an antagonist of Fu: the loss of Fu kinase activity is suppressed by the loss of function of either Cos-2 or Su(fu), whereas the loss of Su(fu) function enhances the Cos-2 phenotype; (3) Cos-2 co-immunoprecipitates with both Fu and Ci in embryo extracts. Thus it is likely that, in the absence of Su(fu), Fu and Ci would remain associated through Cos-2. An attractive hypothesis is that Cos-2 retains Ci within the cytoplasm by anchoring it to microtubules. In the future, the exploration of the interactions between other known components of the Hh signaling pathway and a search for new components will be important for the elucidation of the molecular mechanisms involved. Given the high level of evolutionary conservation of the Hh signaling pathway and the fact that the domains mediating the molecular interactions described here have also been conserved throughout evolution, it is highly probable that comparable molecular interactions also exist in vertebrates (Monnier, 1998 and references therein).
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).
The Hedgehog (Hh) signaling pathway is crucial for pattern formation during metazoan development. Although originially characterized in Drosophila, vertebrate homologs have been identified for several, but not all, genes in the pathway. Analysis of mutants in Drosophila demonstrates that Suppressor of fused [Su(fu)] interacts genetically with genes encoding proteins in the Hh signal transduction pathway, and its protein product physically interacts with two of the proteins in the Hh pathway. The molecular cloning and characterization of chicken and mouse homologs of Su(fu) is reported. The chick and mouse proteins are 27% identical and 53% similar at the amino acid level to the Drosophila melanogaster and Drosophila virilis proteins. Vertebrate Su(fu) is widely expressed in the developing embryo with higher levels in tissues that are known to be patterned by Hh signaling. The chick Su(fu) protein can physically interact with factors known to function in Hh signal transduction including the Drosophila serine/threonine kinase, Fused, and the vertebrate transcriptional regulators Gli1 and Gli3. This interaction may be significant for transcriptional regulation, since recombinant Su(fu) enhances the ability of Gli proteins to bind DNA in electrophoretic mobility shift assays (Pearse, 1999).
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).
Costal2 (Cos2) and Suppressor of Fused [Su(fu)] inhibit Ci by tethering it in the cytoplasm, whereas Hh induces nuclear translocaltion of Ci through Fused (Fu). A 125 amino acid domain in the C-terminal part of Ci has been identiifed that mediates response to Cos2 inhibition. Cos2 binds Ci, prevents its nuclear import, and inhibits its activity via this domain. Su(fu) regulates Ci through two distinct mechanisms: (1) Su(fu) blocks Ci nuclear import through the N-terminal region of Ci, and (2) it inhibits the activity of Ci through a mechanism independent of Ci nuclear translocation. Cos2 is required for transducing high levels of Hh signaling activity, and it does so by alleviating the blockage of Ci activity imposed by Su(fu) (Wang, 2000).
Wild-type wing discs accumulate Ci in the nucleus in Hh receiving cells after treatment with Leptomycin B (LMB), a drug that blocks CRM1 dependent nuclear export. Ectopic hh expression in anterior (A) compartment cells away from the compartment boundary induces LMB-dependent nuclear translocation of Ci in these cells. The stimulation of LMB-dependent nuclear import by Hh appears to be much more efficient in the developing imaginal discs than in cultured cl-8 cells. One possible explanation is that cl-8 cells might not fully recapitulate all the Hh signaling properties. The ability of LMB to block nuclear export of Ci in cultured imaginal discs provides an opportunity to address the roles of Cos2 and other Hh signaling components in regulating Ci nuclear import. cos2 mutation results in constitutive nuclear translocation of Ci independent of Hh signaling. In contrast, fu mutation attenuates Ci nuclear translocation induced by Hh. Taken together, these experiments show that Cos2 and Hh have opposing influences on Ci nuclear import: Cos2 exerts a block on Ci nuclear translocation, whereas Hh stimulates Ci nuclear translocation through Fu (Wang, 2000).
Using deletion analysis coupled with in vivo coexpression assays, a 125 amino acid domain has been identified in the C-terminal part of Ci (aa 961-1065) that mediates transcriptional repression and cytoplasmic retention by Cos2. This domain has been named CORD for Cos2 responsive domain. Ci deletion mutants that lack CORD are insensitive to Cos2 repression and are no longer sequestered in the cytoplasm by Cos2 in these assays. Moreover, CORD is sufficient to mediate Cos2-dependent cytoplasmic retention when fused to a heterologous protein. In yeast two hybrid assay, CORD is found to be the only region of Ci that binds Cos2. Taken together, these data provide strong evidence that Cos2 inhibits Ci activity by tethering it in the cytoplasm via directly binding to CORD (Wang, 2000).
A Ci region from aa 703 to aa 850 can act to sequester heterologous proteins in the cytoplasm. However, this region does not mediate cytoplasmic retention by Cos2 because Ci deletion mutants that retain it fail to be sequestered by Cos2 and are resistant to Cos2 inhibition in the in vivo assay. Moreover, Ci fragments containing this region fail to bind Cos2 in yeast. Rather, this Ci domain appears to mediate Ci nuclear export as its effect on nuclear localization is abolished by LMB treatment (Wang, 2000).
The mechanism by which Su(fu) inhibits Hh signaling has remained controversial. Overexpression studies using mammalian cultured cells have shown that Su(fu) can sequester Gli1 in the cytoplasm. However, a different result was obtained from overexpression study using Drosophila cultured cells. For example, overexpressing Su(fu) in Drosophila cl-8 cells fails to block LMB-induced nuclear accumulation of Ci. In addition, several studies have revealed that Su(fu) can interact with Gli1 on DNA, raising the possibility that Su(fu) might affect Gli activity in the nucleus. In this study, genetic evidence is provided that Su(fu) regulates Ci/Gli by both blocking its nuclear import and affecting its activity after nuclear translocation. To overcome the problem of Ci instability in Su(fu) mutant cells, the effect of Su(fu) mutation was examined on nuclear translocation of overexpressed Ci that appears to saturate the mechanism responsible for degrading Ci in the absence of Su(fu). Overexpressed Ci is significantly retained in the cytoplasm in A compartment cells of wild-type wing discs but is largely accumulated in the nucleus in Su(fu) mutant wing discs. Removal of Su(fu) binding domain has a similar effect on Ci nuclear translocation to Su(fu) mutation, suggesting that Su(fu) sequesters Ci in the cytoplasm by directly binding to the N-terminal region of Ci. The ability of Su(fu) to sequester Ci in the cytoplasm appears to depend on Cos2, as Su(fu) does not prevent LMB-dependent Ci nuclear import in cos2 mutant cells (Wang, 2000).
Evidence arguing that Su(fu) affects Ci transcriptional activity in the nucleus comes from analysis of cos2 mutant phenotypes. In wild-type wing discs, A compartment cells abutting the A/P compartment boundary transduce high levels of Hh signaling activity; these high levels convert Ci into a labile transcription activator by antagonizing the inhibitory role of Su(fu). As a consequence, these cells activate en and show low levels of Ci staining. cos2 mutant cells abutting the compartment boundary accumulate high levels of Ci and show low levels of Hh signaling activity as they fail to activate en. Thus, it appears that the majority of Ci in cos2 mutant cells remains in a latent stable form, likely in a complex with Su(fu). In support of this view, it has been shown that removal of Su(fu) from cos2 mutant cells restores high levels of Hh signaling activity and simultaneously decreases the concentration of Ci in these cells. Because Hh induction of Ci nuclear import is not affected by Su(fu) in cos2 mutant cells near the A/P compartment boundary, it is concluded that Su(fu) inhibits Ci activity at a step after it translocates into the nucleus. A possible mechanism by which Su(fu) inhibits Ci activity in the nucleus is to prevent it from forming an active transcriptional complex, since it has been shown that Su(fu) can interact with Gli on DNA (Wang, 2000).
Cos2 was identified as a negative component in the hh pathway by previous genetic studies. A novel, positive role for Cos2 in the hh pathway has now been uncovered. In addition to blocking Hh signal transduction in A compartment cells away from the compartment boundary, Cos2 is required for transducing high levels of Hh signaling activity by antagonizing Su(fu) in A compartment cells near the A/P compartment boundary. In addition, this requirement is a general property of Cos2 that applies to all A compartment cells. Thus, these results underscore an unusual relationship between Cos2 and Su(fu): in the absence of Hh signaling, Cos2 acts cooperatively with Su(fu) to block Ci nuclear import by forming a complex with Ci; in cells receiving high dose of Hh signal, Cos2 is required to alleviate the block on Ci transcriptional activity imposed by Su(fu) (Wang, 2000).
Based on the evidence presented here and elsewhere, a working model for how Cos2, Fu and Su(fu) regulate the nuclear translocation and activity of Ci is proposed. Su(fu) and Cos2 bind Ci via the N- and C-terminal domains, respectively, and the complex binds microtubules through Cos2 and retains Ci in the cytoplasm. In addition, Cos2 promotes the proteolysis of Ci to generate a truncated repressor form (Ci75), a process that also requires the activities of PKA, Slimb, and proteasome. Hh stimulates Ci nuclear translocation through Fu kinase and inhibits Ci processing possibly through dephosphorylating Ci. The transcriptional activity of full-length Ci is attenuated in the nucleus by Su(fu), which also stabilizes the latent form of Ci. High levels of Hh signaling activity convert Ci into a labile and active form, possibly by dissociating it from Su(fu), and this process requires the activities of Fu and Cos2 (Wang, 2000).
Several important issues regarding this model need to be addressed. For example, how Hh antagonizes Cos2 and Su(fu) to promote Ci nuclear translocation remains an important unsolved problem. It is likely that Hh stimulates Ci nuclear import by dissociating Ci complex from microtubules and, further, by releasing Ci from the complex. In support of this view, it has been shown that Hh can induce dissociation of Cos2 from microtubules. Moreover, it has also been implicated that dissociation of Ci tetrameric complex might proceed the nuclear translocation of Ci. However, no biochemical evidence has been obtained indicating that Hh induces dissociation of Ci from Cos2 and Su(fu) (Wang, 2000).
Fu kinase appears to be required for Hh to stimulate Ci nuclear translocation, because Ci is retained significantly in the cytoplasm in fu mutant cells that receive Hh signal. The substrate for Fu kinase still remains a mystery. One attractive candidate is Su(fu), which binds Fu and whose function is antagonized by Fu. Since Su(fu) is a PEST domain protein, phosphorylation of Su(fu) might cause its degradation and subsequent disassembly of Ci complex. Another good candidate for a Fu substrate is Cos2, which also interacts with Fu. It has been shown that Hh induces phosphorylation of Cos2; however, the kinase responsible for Hh-dependent phosphorylation of Cos2 has not been identified. It remains to be determined if Fu contributes to Cos2 phosphorylation. Although the biological significance of Cos2 phosphorylation has not been shown yet, it is conceivable that such phosphorylation could cause dissociation of Cos2 from microtubules or from Ci, leading to Ci nuclear translocation. In support of this view, it has been found that the effect of fu mutation on Ci nuclear translocation can be suppressed by removal of Cos2, arguing that Fu promotes Ci nuclear import by antagonizing Cos2 (Wang, 2000).
Finally, how Cos2 positively regulates Hh signaling activity remains to be determined. The finding that Cos2 is required for Hh to antagonize Su(fu) could be explained by the observation that Cos2 forms a complex with Fu and Su(fu). One scenario is that Cos2 might simply play a structural role in which it antagonizes Su(fu) by recruiting Fu. Alternatively, Cos2 might play a more active role in which it recruits other positive components in close proximity to Fu while also regulating Fused activity in response to Hh signaling. Structure and functional analysis of Cos2 and identifying other Cos2 interacting proteins may help to resolve this important issue (Wang, 2000).
Much 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).
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).
Hedgehog signaling is required for the development of many organisms, including Drosophila. In flies, Hh patterns the embryonic epidermis and larval imaginal discs by regulating the transcription factor, Cubitus interruptus (Ci). To date, three levels of regulation have been identified: proteolytic processing into a repressor, nuclear import, and activation. In this report, the function of two Ci domains that are conserved in the vertebrate homologues, GLI1, GLI2, and GLI3 has been tested. One domain includes the first two of five C2-H2 zinc-fingers. While conserved in all members of the GLI/Ci family, the first two fingers do not appear to make significant contacts with the DNA target sequence. Ci protein lacking this region is still able to interact with the cytoplasmic complex and activate transcription in embryos and wing imaginal discs, but it is no longer processed into the repressor form. The second domain, termed NR for 'N-terminal Regulatory', binds Suppressor of Fused. Deletion of this region has little effect on embryonic patterning, but compromises cytoplasmic retention of Ci. Analysis of the amino acid sequence of this domain identifies 11 perfectly conserved serines and one tyrosine. It is proposed that this region may be modified, possibly by phosphorylation, to regulate Ci nuclear import (Croker, 2006).
Despite 500 million years of divergence between higher vertebrates and Drosophila, many proteins have homologues with extensive sequence conservation. This is well exemplified by the GLI family of transcription factors and their fly counterpart, Ci. Over their entirety, Ci and the human GLIs share over 22% amino acid identity. Ci and human GLI3 are even more similar, at greater than 27% identity. Though the proteins' sequences have diverged, the two domains studied here remain strikingly similar. This information was exploited to further the understanding of the complex regulation of Ci in Drosophila (Croker, 2006).
Amino acid comparisons of the mouse and human GLI1, GLI2, and GLI3 proteins with Drosophila Ci show an expected high degree of conservation in the DNA-binding domain, which consists of five tandem C2–H2 zinc-fingers. This domain in all of the GLI homologs appears to recognize the same DNA target sequence. Furthermore, the Gli proteins have been shown to recapitulate the multifaceted functions of Ci in flies. This study focused attention on the first two of five zinc-fingers, which have not been implicated in significant DNA contacts. Sequence analysis shows 58% identity over the 60 amino acids that constitute the region. While this is substantially less than the 91% identity of the three DNA binding zinc-fingers, it is nonetheless impressive (Croker, 2006).
A second domain N-terminal to the DNA-binding domain also shares considerable sequence identity with the vertebrate homologues. In this case, the region, which has been termed “NR”, appears to be present in Ci, GLI1, GLI2, and GLI3. There exists no obvious NR motif in the nematode homologue Tra-1. This region retains greater than 57% identity over 49 amino acids examined in the mouse, human, and fly proteins (72% identity between Ci and its closest homolog GLI3). Over one quarter of this domain consists of perfectly conserved serines, threonines, and tyrosines. It is speculated that this module contains phosphorylation sites that contribute to the proper regulation of these transcription factors, perhaps by modulating interaction with Su(fu) (Croker, 2006).
Analysis of en and 4bsLacZ (a wing-specific decapentaplegic enhancer) gene expression in wing disc clones expressing UAS-CiΔZ2myc induced by the Actin5Cp-“flip-out”-GAL4 driver shows that a Ci molecule lacking the first two zinc-fingers retains the ability to bind DNA and activate transcription. This is consistent with the crystal structure of the DNA–Gli1 complex. Gene expression in this context is Hh-independent, but it is a possible consequence of overexpression, since a wild type UAS-Ci-cDNA can activate these target genes if it is expressed at very high levels (Croker, 2006).
To better gauge the regulation of CiΔZ2myc, it was also analyzed in ci94 null mutant embryos using the ci-GAL4 driver. Embryonic cuticles expressing UAS-CiΔZ2myc in the anterior compartment of every segment show a gain-of-function phenotype reminiscent of cos2 and ptc mutants. This suggests that the first two zinc-fingers provide a negative regulatory or repressive function. Supporting this hypothesis is the observation that CiΔZ2myc fails to repress hh-lacZ expression in posterior smo clones. The lost repressive activity could be accounted for in a number of different ways. It is possible that the first two zinc fingers recruit a co-repressor protein or that they are required for proper modification of Ci. Alternately, it is possible that this construct is not processed into the repressor form in the absence of Hh signaling. The latter hypothesis is favored since Western blot analysis of the CiΔZ2myc protein fails to show a truncated repressor form. Further, there is a broadening of the domain of ectopic CiΔZ2myc in embryos compared to overexpression of both wild type Ci and CiΔNRmyc, which is consistent with a processing defect of this molecule. These results extend previous work showing that the zinc finger domain as a whole is required for processing into the Ci-75 repressor. In addition to the apparent defect in proteolytic processing, the nuclear import of CiΔZ2myc appears to be Hh-independent. These phenotypes could be caused by a failure of the CiΔZ2myc protein to efficiently complex with Fu, Su(fu), and Cos2, but the known binding sites for these proteins are all retained in CiΔZ2myc. Rather, this is likely an effect of overexpression as co-expression with Su(fu) leads to cytoplasmic retention of CiΔZ2myc (Croker, 2006).
While the CiΔZ2myc construct shows some Hh-independent function, the protein does not appear to be fully active in the absence of Hh signaling. In ptc mutant embryos, where the pathway is fully activated, the stripe of en expression is duplicated anterior to each expanded Wg stripe. This does not occur with CiΔZ2myc (Croker, 2006).
Studies on the function of a Ci construct in which sequences amino-terminal to the zinc-finger DNA binding domain were deleted have been reported previously. Expression of the CiZnC protein in the posterior compartment of embryos leads to ectopic ptc activation. These findings extend the former observations by using ci-GAL4 to drive continuous expression of transgenes in the anterior compartment. In the case of embryonic cuticle patterning, it is shown that the CiZnC protein induced by ci-GAL4 actually has a gain-of-function phenotype when expressed in embryos lacking endogenous Ci. The gain-of-function phenotype suggests the existence of domains in the amino-terminus of the Ci protein that regulate the function of the full-length molecule or contribute to the repressor function of Ci-75. Two obvious candidates are amino acid regions 1-346 and 346-430 which bind Su(fu) and Cos2, respectively. The NR region (AA 208-260) is likely to be important for Ci binding to Su(fu); amino acids 116-125 of the human Gli1 protein are critical for Su(fu) binding, especially the SYGHLS amino acid motif at the end of the domain. This interaction domain sequence corresponds to amino acids 245-254 of the Ci NR domain, which retains perfect conservation of the SYGHXS motif (Croker, 2006).
When the amino-terminal deletion is restricted specifically to the NR region (208-260), embryonic development is essentially normal. The CiΔNRmyc protein retains the ability to repress hh-lacZ in posterior smo clones and thus is presumably processed into the repressor form. In contrast, when CiΔNRmyc is expressed in wing imaginal disc clones using 'flip-out' Gal4, it is not properly sequestered in the cytoplasm. This phenotype may be in part a consequence of protein overexpression, but wild type Ci expressed at similar levels is properly sequestered in the cytoplasm in the absence of Hh signaling. These results might be expected if the role of the NR domain is to regulate the interaction of Ci with Su(fu). Su(fu) mutants are viable and only have subtle patterning defects, and thus, a mutation in Ci that disrupted Su(fu) interaction should be relatively normal. However, in conditions of Ci overexpression, it has been shown that Su(fu) is required for Ci cytoplasmic retention. Indeed, overexpression of Su(fu) can restore proper cytoplasmic localization of a highly expressed wild type Ci molecule, but cannot rescue the CiΔNRmyc nuclear localization phenotype suggesting that the NR domain does modulate Ci/Su(fu) interaction. Yeast two-hybrid results show that this conserved interaction between Ci and Su(fu) is direct. It is postulated that modification of the NR domain at conserved putative phosphorylation sites modulates the molecule's interaction with Su(fu). Su(fu) appears able to act as a Ci sponge and to sequester excess Ci in the cytoplasm. This may explain in part why Su(fu) mutants have such a subtle phenotype. Under normal circumstances, Cos2 and the other components of the cytoplasmic complex are able to retain Ci in the cytoplasm. Su(fu) provides a redundant mechanism for sequestering any Ci that escapes (Croker, 2006).
Given that deletion of the NR region fails to generate a strong gain-of-function phenotype, an additional negative regulatory element must exist in the amino-terminus outside of the NR region. Two candidates are the Cos2 binding region (346–430) and a pair alanine-rich clusters in the Ci N-terminus that may function as a repressor domain (Croker, 2006).
This analysis of the NR region and the first two zinc-fingers demonstrates that both are required for negative regulation of the Ci protein. The NR domain functions to regulate interaction with the Su(fu) protein which in turn modulates the subcellular localization of the transcription factor. In the case of the first two zinc-fingers, the interacting proteins are unknown, but are likely to play important roles in Ci regulation (Croker, 2006).
The Hedgehog (Hh) signal transduction pathway plays a central role in the development of invertebrates and vertebrates. While much is known about the pathway, the role of Suppressor of fused [Su(fu)], a component of the pathway's signaling complex has remained enigmatic. Previous studies have linked Su(fu) to the cytoplasmic sequestration of the zinc finger transcription factor, Cubitus interruptus (Ci), while other studies suggest a role in modulating target gene expression. In examining the cell biology of the pathway, it was found that like its vertebrate homologue, Drosophila Su(fu) enters the nucleus. Furthermore, the nuclear import of Su(fu) occurs in concert with that of Ci in response to Hh signaling. This study examines the mechanism by which Su(fu) regulates Ci import by investigating the importance of the Ci nuclear localization signal (NLS) and the effect of adding an additional NLS. Finally, it is demonstrated that Ci can bring Su(fu) with it to a multimerized Ci DNA binding site. These results provide a basis for understanding the dual roles played by Su(fu) in the regulation of Ci (Sisson, 2006).
This study establishes the Hh dependent translocation of the Su(fu)-Ci complex into the nucleus, illustrating further conservation between the mammalian and Drosophila Hedgehog signaling pathways. This result is somewhat of a paradox since Su(fu) has been shown to assist in the sequestration of Ci in the cytoplasm. If Su(fu) contributes to the cytoplasmic sequestration of Ci, what is the mechanism that allows its release in response to Hh signaling? The likely event is phosphorylation by the Fu kinase. In the absence of Fu kinase function, Ci is not released in response to Hh signaling. However, in double mutants lacking both Su(fu) and Fu kinase activity, Ci is now able to enter the nucleus, suggesting that Fu kinase activity is required to regulate the cytoplasmic retention of the Su(fu)-Ci complex. It is not known whether Su(fu) or Ci are direct targets of the Fu kinase, and this need not be the case, since modification of other components of the pathway such as Cos2 could allow Su(fu)-Ci complex release (Sisson, 2006).
While previous studies demonstrated the functionality of the Ci NLS at AA R596-K600 and K611-K614, the data presented here indicate that Ci nuclear import in salivary glands and in wing discs does not absolutely require the presence of this NLS. Consistent with some decrease in NLS function, Ci-mutNLS gives substantial, but not complete, rescue of a ci null mutation. This suggests either the existence of an additional NLS within Ci or the presence of an additional protein that brings Ci into the nucleus (Sisson, 2006).
The results from salivary glands may favor the hypothesis that an additional NLS is present in Ci. Ci-mutNLS nuclear import is impeded but not prevented, implying that if an additional protein were necessary to bring Ci into the nucleus, it too would have to be present in salivary glands and would not be Ci specific. Another consideration is that perhaps the Ci-mutNLS mutation does not entirely destroy NLS function. This mutation only disrupts the second basic cluster within a bipartite NLS, because altering the first cluster would disrupt the last zinc finger and DNA binding (Sisson, 2006).
Addition of an exogenous SV40NLS to Ci leads to more rapid nuclear import in salivary glands and a variable gain of function phenotype in embryos. The increased rate of nuclear import appears to compromise the ability of Su(fu) to sequester Ci-SV40NLS in the cytoplasm of anterior wing imaginal disc clones that are away from Hh signaling. One could interpret this result to suggest that Su(fu) masks the endogenous Ci NLS but not the added SV40NLS. This seems unlikely since wild-type Su(fu)-Ci complexes readily enter the nuclei of salivary glands. An alternative explanation is that away from Hh signaling the Su(fu)-Ci complex has some affinity for a cytoplasmic tether and some low probability of being imported into the nucleus; increasing the rate of nuclear import shifts this equilibrium resulting in nuclear accumulation of Su(fu)-Ci (Sisson, 2006).
Potentially consistent with the role of Su(fu) in sequestering Ci in the cytoplasm is the observation that N-terminally myc-tagged Su(fu) appears to be tightly tethered in the cytoplasm resulting in the cytoplasmic retention of both it and Ci. It is possible that the addition of the myc tag causes a spurious interaction between Su(fu) and an unidentified cytoplasmic component, or it may be the case that the interaction is normal, but the addition of the myc tag prevents the release of Su(fu) from this component. Since these experiments were carried out in salivary glands, where there is little if any Fu, Smo or Cos2, the component tethering myc-Su(fu) is distinct from the known proteins in the Hh signal transduction cascade (Sisson, 2006).
Ci-SV40NLS is not sequestered in the cytoplasm of salivary glands by myc-Su(fu). Instead much of it escapes into the nucleus, but it does not bring myc-Su(fu) with it. Again, this result is likely a consequence of the increased rate of Ci-SV40NLS nuclear import. Myc-Su(fu) remains tightly tethered in the cytoplasm, and the distribution of Ci-SV40NLS will be determined by the relationship between the rate of nuclear import and the binding affinity to myc-Su(fu) (Sisson, 2006).
Mammalian studies have found that the addition of mouse Su(fu) can increase the binding affinity of the Glis to target DNA sequences. The salivary gland model system demonstrates that Su(fu), along with Ci, clearly bind an introduced target within the polytene chromosomes. The presence of Su(fu) at Hh target gene enhancers provides the opportunity for another level of Ci regulation. This regulatory role is likely to be restricted to the full-length form of Ci. The Ci repressor form is missing the C-terminal Su(fu) binding site and Ci-N[HA]Zn, which closely resembles the Ci repressor, does not bring Su(fu) with it to the DNA (Sisson, 2006).
Given the high degree of conservation between the mammalian and Drosophila Hh signaling pathways, one might expect Su(fu) to play homologous roles in the negative regulation of the two pathways. Su(fu) has been shown to act in the cytoplasm as a negative regulator of the pathway by contributing to the sequestration of Ci. In the absence of Hh, Fu and Ci are tethered to microtubules via their interaction with Cos2. In the presence of Hh, Smo is phosphorylated, the complex is released from the microtubules, and a tetrameric cytoplasmic complex is formed with the addition of Su(fu). Su(fu) may contribute to Ci sequestration in two ways, as part of the tetrameric cytoplasmic complex and as a heterodimer with Ci where it could act as a sink to sequester any excess Ci that is not bound to the Cos2-Fu complex (Sisson, 2006).
The presence of Su(fu) in the nucleus suggests a dual role for Su(fu) in the regulation of Ci. A direct nuclear role for Su(fu) negative regulation has been inferred by vertebrate studies documenting an interaction of Su(fu) with SAP18. While mammalian Su(fu) has been show to interact with SAP18 through GST pull-downs and yeast two-hybrid analysis, initial yeast two-hybrid studies did not reveal an interaction between Drosophila SAP18 and Drosophila Su(fu). Therefore, further studies are needed to delineate the function of Drosophila Su(fu) in the nucleus and to determine if SAP18 is indeed involved in the negative regulation of Ci (Sisson, 2006).
The question still remains of how differential responses to Hh signaling are generated. In the presence of Hh, Su(fu) is phosphorylated and it has been suggested that this modification at the A/P boundary may reduce Su(fu) repressive activity, thus allowing Ci to activate target genes requiring the highest levels of Hh. The modification of Su(fu) or Ci could change the nature of cofactors recruited to target enhancers and account for differential gene regulation (Sisson, 2006).
This model is consistent with observations on the phenotypes of cos2 and cos2; Su(fu) double mutant clones. In cos2 mutant clones, targets that require modest levels of Hh are activated while those that require high-level Hh are not. This suggests that the Cos2 protein is required for some modification of the Ci-Su(fu) heterodimer that is essential for 'activation'. When the Su(fu) gene is also eliminated, now target genes requiring high level Hh are activated. Thus, Su(fu) must contribute to the attenuation of Ci activity in response to modest levels of Hh. However, Su(fu) cannot be the entire story as animals mutant for Su(fu) are essentially normal. There must be a second factor that is partially redundant with Su(fu) in attenuating Ci activity. Since cos2; Su(fu) mutant clones have 'activated' Ci, it would seem that Cos2 is required for the function of this second factor (Sisson, 2006).
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).
In Drosophila and vertebrates, Suppressor of fused [Su(fu)] proteins act as negative regulators of the Gli/Ci transcription factors, which mediate the transcriptional effects of Hh signalling. This study sought novel partners of Su(fu) in fly using the two-hybrid method. Most of the Su(fu) interactors thus identified are (or are likely to be) able to enter the nucleus. This study focused on one of these putative partners, dMLF (Myelodysplasia/myeloid leukemia factor), which resembles vertebrate myelodysplasia/myeloid leukaemia factors 1 and 2. dMLF binds specifically to Su(fu) in vitro and in vivo. Using a novel anti-dMLF antibody, it was shown, that dMLF is a nuclear, chromosome-associated protein. A dMLF transgene was overexpressed in the fly using an inducible expression system. dMLF over-expression disrupts normal development, leading to either a lethal phenotype or adult structural defects associated with apoptosis and increased DNA synthesis. Furthermore, the dMLF-induced eye phenotype is enhanced by the loss of Su(fu) function, suggesting a genetic interaction between Su(fu) and dMLF. It is proposed that Su(fu) and dMLF act together at the transcriptional level to coordinate patterning and proliferation during development (Fouix, 2003).
In human, the myeloid leukemia factor 1 (hMLF1) has been shown to be involved in acute leukemia, and mlf related genes are present in many animals. Despite their extensive representation and their good conservation, very little is understood about their function. In Drosophila, Myelodysplasia/myeloid leukemia factor (dMLF) physically interacts with both the transcription regulatory factor DREF and an antagonist of the Hedgehog pathway, Suppressor of Fused, whose over-expression in the fly suppresses the toxicity induced by polyglutamine. No connection between these data has, however, been established. This study shows that dmlf is widely and dynamically expressed during fly development. The first dmlf mutants were isolated and analyzed: embryos lacking maternal dmlf product have a low viability with no specific defect, and dmlf mutant adults display weak phenotypes. dMLF subcellular localization in the fly and cultured cells was monitored. Although generally nuclear, dMLF can also be cytoplasmic, depending on the developmental context. Furthermore, two differently spliced variants of dMLF display differential subcellular localization, allowing the identification of regions of dMLF potentially important for its localization. Finally, it was demonstrated that dMLF can act developmentally and postdevelopmentally to suppress neurodegeneration and premature aging in a cerebellar ataxia model (Martin-Lanneree, 2006).
Hedgehog (Hh) proteins signal by inhibiting the proteolytic processing of Ci/Gli family transcription factors and by increasing Ci/Gli-specific activity. When Hh is absent, phosphorylation of Ci/Gli triggers binding to SCF ubiquitin ligase complexes and consequent proteolysis. This study shows that multiple successively phosphorylated CK1 sites on Ci create an atypical extended binding site for the SCF substrate recognition component Slimb. GSK3 enhances binding primarily through a nearby region of Ci, which might contact an SCF component other than Slimb. Studies of Ci variants with altered CK1 and GSK3 sites suggest that the large number of phosphorylation sites that direct SCFSlimb binding confers a sensitive and graded proteolytic response to Hh, which collaborates with changes in Ci-specific activity to elicit a morphogenetic response. When Ci proteolysis is compromised, its specific activity is limited principally by Su(fu), and not by Cos2 cytoplasmic tethering or PKA phosphorylation (Smelkinson, 2007).
The central task of Hh signal transduction in Drosophila is to regulate the activity of the transcription factor Ci. This is accomplished by regulating the levels of Ci-155 activator and Ci-75 repressor and the specific activity of Ci-155. To examine the regulation of Ci-155-specific activity in isolation, a Ci variant (Ci-S849A) was developed that escapes PKA-dependent proteolysis but has a minimally altered pattern of PKA-initiated phosphorylation. At 29°C, Ci-S849A was expressed in wing discs (via C765-GAL4) at roughly the same level as endogenous Ci, but at 20°C, the levels of Ci-S849A were distinctly lower. At 29°C, Ci-S849A induced the Hh target gene reporter ptc-lacZ in A cells that are not stimulated by Hh. Since ptc induction requires Ci-155 activator and is not accomplished simply by loss of Ci-75 repressor, it is concluded that elevated levels of Ci-155 can suffice to confer some activator function. At 20°C, Ci-S849A induced ptc-lacZ in A cells only when Su(fu) was removed. By contrast, Ci-S849A activity was not detectably increased by loss of either PKA activity or Cos2 activity in P smo mutant clones, where Hh signaling is blocked. Thus, Su(fu) is the principal component that limits Ci-155-specific activity when Ci-155 is protected from PKA-dependent proteolysis, whereas PKA and Cos2 normally limit Ci-155 activity simply by promoting its proteolysis (Smelkinson, 2007).
The prior assertion that PKA limits Ci-155-specific activity by direct phosphorylation was based on the use of an inappropriate reagent (Ci-U), which was mistakenly thought to be inert to PKA-dependent proteolysis. A similar role for Cos2 was previously inferred principally from the observation that Ci-155 accumulated more rapidly in the nuclei of anterior Leptomycin B-treated wing disc cells when those cells lack . It appears that the inferred cytoplasmic retention of Ci-155 by Cos2 contributes very little quantitatively to limiting the activity of stabilized Ci-155. That conclusion is supported by the observation that loss of Cos2 function did not enhance the weak induction of En seen in pka mutant clones of otherwise WT wing discs. The remaining, long-standing observations suggesting roles for PKA and Cos2 in limiting Ci-155-specific activity are the different degrees of Hh target gene induction in pka (strongest), cos2 (intermediate), and slimb (weakest) mutant wing disc clones. It is suggested that this might result from different degrees of disruption of PKA-dependent proteolysis in these clones. This suggestion is consistent with the proposed role of Cos2 in facilitating Ci-155 phosphorylation and with the observation that ptc-lacZ can be induced in slimb mutant clones when PKA activity is halved (Smelkinson, 2007).
Both GST-Ci association with SCFSlimb and Ci-155 proteolysis depend on two phosphorylated regions of Ci. The first region provides an essential Slimb binding site that can be created by five successive CK1 phosphorylations primed initially by PKA site 1. Of the four phosphorylated residues within the motif (844SpTpYYGSpMQSp852) that interacts directly with Slimb, at least one (S849) is essential for binding, and two others (S844 and S852) enhance binding (T845 is essential, but priming and binding functions have not been separated). The second critical phosphorylated region of Ci includes two GSK3 sites (S884 and S888) that are primed by PKA site 3. This region enhances, but is not sufficient for, binding to SCFSlimb (Smelkinson, 2007).
The requirement for multiple successive phosphorylations by PKA, CK1, and GSK3 to create a high-affinity SCFSlimb binding domain on Ci-155 has two important consequences. First, it demands a special mechanism for facilitating Ci phosphorylation that is met by Cos2. Second, it provides a mechanism through which a small change in Ci-155 phosphorylation, induced for example by limited dissociation of protein kinases from Cos2, can be translated into a substantial inhibition of Ci-155 proteolysis. The sharp increase in Ci-155 levels at the anterior limit of Hh signaling territory shows that a low dose of Hh does indeed severely curtail PKA-dependent Ci-155 proteolysis. Hh could inhibit proteolytic processing of Ci variants driven by either PKA-primed GSK3 sites (Ci-SL) or PKA-primed CK1 sites (Ci-G2,3E and Ci-Y846G), but complete inhibition was observed only for the latter pair. This suggests that the sensitive response of Ci proteolysis to Hh depends principally on CK1 (Smelkinson, 2007).
How does inhibition of Ci-155 proteolysis affect Ci-155 activity? Previously, the properties of Ci-U and slimb mutant clones were taken as evidence that inhibition of proteolysis does not suffice for Ci activation. Since Ci-U is subject to PKA-dependent proteolysis and PKA does not affect the specific activity of Ci-155, instead the properties of Ci-S849A and pka mutant clones were relied upon to conclude that complete inhibition of PKA-dependent proteolysis does suffice to induce the Hh target gene ptc. This, in turn, suggests that the high Ci-155 levels anterior to the stripe of elevated ptc expression at the AP border of wing discs result from substantial, but incomplete, inhibition of Ci-155 proteolysis (Smelkinson, 2007).
It has generally been assumed that inhibition of Ci-155 proteolysis is uniformly strong throughout the AP border and that the activation of ptc and en in nested domains is due solely to changes in Ci-155-specific activity elicited by increasing levels of Hh. However, several factors suggest that there may also be a significant gradient of residual PKA-dependent proteolysis at the AP border that contributes to morphogen action (Smelkinson, 2007).
First, the precise degree of substantially inhibited Ci-155 proteolysis can determine whether Hh target genes are induced or not. This is evident from differences in ptc-lacZ induction among proteolytically impaired Ci variants and between pka and slimb mutant clones (Smelkinson, 2007).
Second, Su(fu) is the principal regulator of Ci-155 activity when Ci-155 levels are elevated, yet Hh instructs an almost unchanged morphogenetic response in the absence of Su(fu). It is likely that a proteolytic gradient is critical under these conditions, although it is also possible that Cos2 assumes a more significant role in regulating Ci-155-specific activity when Su(fu) is absent (Smelkinson, 2007).
Third, it was found that the loss of any one of four phosphoserines that contribute to Ci-Slimb binding (S844, S852, S884, and S888) diminishes, but does not abolish, Slimb binding. For S888A (G3A) this results in elevated Ci-155 levels and an increased activity, but residual proteolysis is clearly evident from the generation of sufficient Ci-75 to repress hh-lacZ. Thus, dispersion of direct Slimb binding determinants among several phosphorylatable residues provides a mechanism for Hh to elicit graded inhibition of Ci-155 proteolysis. It is speculated that in response to high levels of Hh, most Ci-155 molecules will not bind to SCFSlimb at all because they lack at least two of the six key phosphorylated residues, whereas a large proportion of Ci-155 molecules may bind SCFSlimb with intermediate affinity in response to low or intermediate Hh levels because they lack only one critical phosphoserine (Smelkinson, 2007).
Fourth, regulation of Ci-155-specific activity depends on Ci-155 levels. Thus, Ci-155 is only activated by loss of Su(fu) when Ci-155 levels are elevated by Hh or appropriate mutations, presumably because other stoichiometric binding partners such as Cos2 act redundantly with Su(fu) when their Ci-155 binding capacity is not saturated. It would therefore be expected that the release of Ci-155 from repressive partners is progressively facilitated as the relative levels of Ci-155 increase. This would allow increasing Hh levels to enhance Ci-155 activity through synergistic effects on Ci-155 levels and Ci-155-specific activity (Smelkinson, 2007).
The archetypal β-TRCP/Slimb substrates, β-catenin and IKB, contain a single, dually phosphorylated, high-affinity binding site (DSpGxxSp) that triggers rapid substrate proteolysis. The primary direct Slimb binding motif that has been defined (SpTpYYGSpMQSp) in Ci differs notably by the presence of Tyr instead of Gly at the third position, by the inclusion of a fourth electronegative residue at its C terminus, and by binding with lower affinity, permitting additional influences on Ci-SCFSlimb association. The fourth electronegative residue (pS852) likely interacts with at least one of two positively charged Slimb surface residues (R333 and R353) based on their potential proximity and reduced GST-Ci binding to the R333A/R353A Slimb variant (Smelkinson, 2007).
Most known β-TRCP substrates include phosphorylated or acidic residues that are two to four residues C-terminal to the standard six amino acid binding motif (DSpGXXSp), but their contribution to binding has not generally been assessed. Even β-catenin includes such phosphorylated residues that are known to have an essential priming role but have not been tested rigorously for direct interactions with β-TRCP. The variant β-TRCP binding motif (EEGFGSpSSp) of mammalian Wee1A presents a notable exception, in which a β-TRCP Arg residue equivalent to R353 of Slimb interacts with the phosphoserines at position 6 and 8 of this motif. This suggests that positive surface residues of β-TRCP/Slimb may commonly stabilize association with extended binding motifs. It is speculated that extended β-TRCP/Slimb binding motifs are likely to be especially important and prevalent in substrates lacking Gly at the third position because it was found that R333 or R353 (or both) of Slimb promotes binding to Ci-WT, but not to Ci-SL, and pS852 contributes significantly to Slimb binding in Ci-WT, but not in Ci-Y846G (Smelkinson, 2007).
Vertebrate Gli homologs of Ci also have a residue other than Gly at the third position (generally Ala) and a potentially phosphorylated Ser at the C terminus of a putative extended β-TRCP binding motif of 9 to 11 residues (SSAYx(x)SRRSS). Both a second Wee1A binding motif (DSAFQEPDS) and a β-TRCP binding motif of the p100 precursor of NFKB p52 (DSAYGSQSVE) also lack a Gly residue at the third position and include residues beyond the six amino acid core motif that might, by analogy to Ci, potentiate binding (Smelkinson, 2007).
Studies of Ci provide a clear precedent for the use of an extended β-TRCP/Slimb binding motif to translate regulated substrate phosphorylation into regulated proteolysis. However, it was also found that Slimb binding cannot be predicted by focusing on only the interactions of charged residues. Thus, fully phosphorylated Ci includes two sequences with a distribution of charged residues similar to that of the primary SCFSlimb binding site (837DSpQNSpTpASpTp and 858SpSpQVSpSpIPTp compared with 844SpTpYYGSpMQSp), but those sites neither suffice for Slimb binding (in Ci-S849A) nor enhance binding significantly in vitro (as revealed by D837A, T842A, S858A, S859A, and Ds2 variants). This probably reflects significant binding contributions of nonpolar residues in positions 3-5 of an extended Slimb/β-TRCP binding motif, as suggested by the presence of Tyr or Phe at position 4 of the functional motifs of Ci, Gli, p100, and Wee1A (Smelkinson, 2007).
Several F-box proteins that use WD40 repeats to bind substrate (FBW proteins) also include a dimerization domain that directs assembly of higher-order SCF complexes. Some substrates of these SCF complexes (for example, Cyclin E for Fbw7 and Wee1A for β-TRCP) contain more than one phosphorylated region capable of interacting with the same WD40 binding surface of the FBW protein. This raises the question of whether the cooperative function of two or more such regions depends on SCF dimerization and simultaneous interaction with two FBW subunits of a dimeric complex (Smelkinson, 2007).
This study found that Ci also contains two phosphorylated regions that contribute to SCF association, and Slimb molecules can bind to each other within higher-order functional SCF complexes. It was also found that Slimb self-association enhanced binding to GST-Ci relative to GST-Ci-SL, which contains a single DSGxxS motif; however, it was not required for both phosphorylated regions of GST-Ci to stimulate binding. Hence, simultaneous binding to separate Slimb monomers within a larger complex can be excluded as a requisite mechanism for cooperativity between the two phosphorylated regions of Ci. This is consistent with recent structural studies that predict a wider separation of WD40 binding surfaces within an SCF dimer than can be spanned readily by the two critical phosphorylated regions of a single Ci molecule (Smelkinson, 2007).
Does the region preceding PKA site 3 of Ci bind directly to the FBW component (Slimb) of an SCF complex as for Cyclin E and Wee1A? That model was proposed for Gli-2/3 proteins, which include a recognizable, potentially extended, variant Slimb-binding motif (DSYDPISTDAS). The analogously positioned sequence in Drosophila (SFYDPISPGCS) retains the YDPIS sequence and the two GSK3 sites at position 7 and 11 (underlined) but lacks a Ser at position 2 (italics), which is required in Gli-2/3 for normal β-TRCP association and proteolysis. Also, Ala substitution of the first Ser in the Drosophila motif (together with three other Ser residues) had only a minor effect on Slimb binding in vitro and Ci-155 proteolysis in vivo. Thus, this region of Ci does not have a clearly recognizable and demonstrably functional, conventional β-TRCP/Slimb binding site. It is, nevertheless, conceivable that the conserved elements of the putative β-TRCP binding motif of Gli-2/3 might provide a very weak direct interaction with the WD40 domain of Slimb that is sufficient to enhance SCFSlimb association (Smelkinson, 2007).
However, this study also found that the GSK3 enhancement of Ci-Slimb binding conferred by the GSK3 sites preceding PKA site 3 was lost if Slimb lacked an F-box domain and consequent direct association with SkpA and its SCF complex partners. This result is interpreted with some caution because Slimb-ΔF also bound less well than wild-type Slimb to a canonical β-catenin substrate and to GST-Ci that was phosphorylated only at its primary Slimb binding site. Nevertheless, the result suggests that the region of Ci immediately preceding PKA site 3 might augment SCF association by binding directly to an SCF component other than Slimb (Smelkinson, 2007).
Whether GSK3 stimulates Ci binding to SCFSlimb via a direct interaction with Slimb, an unprecedented interaction with another SCF component, or a conformational effect on the primary Slimb binding site of Ci, the Ci-G3A transgene reveals that the stimulation conferred by GSK3 phosphorylation is critical for efficient Ci-155 proteolysis and for Hh pathway silencing. Since Slimb self-association enhanced GST-Ci, but not GST-Ci-SL, binding in vitro, it is suspected that this may also be important for Ci-155 proteolysis in vivo. It is not known if SCFSlimb dimerization (or oligomerization) is regulated, but the different modes of association of SCFSlimb with Ci and β-catenin certainly provide several opportunities for SCF regulatory mechanisms or mutations to affect the Hh pathway without altering the Wnt/β-catenin pathway (Smelkinson, 2007).
The Hedgehog (Hh) pathway, an evolutionarily conserved key regulator of embryonic patterning and tissue homeostasis, controls its target genes by managing the processing and activities of the Gli/Ci transcription factors. Little is known about the nuclear co-factors the Gli/Ci proteins recruit, and how they mechanistically control Hh target genes. This study provides evidence for the involvement of Parafibromin/Hyx as a positive component in Hh signaling. hyx RNAi impaired Hh pathway activity in Drosophila cell culture. Consistent with an evolutionarily conserved function in Hh signaling, RNAi-mediated knockdown of Parafibromin in mammalian cell culture experiments diminished the transcriptional activity of Gli1 and Gli2. In vivo, in Drosophila, genetic impairment of hyx decreased the expression of the high-threshold Hh target gene knot/collier. Conversely, hyx overexpression ameliorated the inhibitory activity of Ptc and Ci(75) misexpression during Drosophila wing development. It was subsequently found that Parafibromin can form a complex with all three Glis, and evidence is provided that Parafibromin/Hyx directly binds Region 1, the Su(fu) interaction domain, in the N-terminus of all Glis and Ci. Taken together, these results suggest a target gene-selective involvement of the PAF1 complex (see Drosophila Paf1) in Hh signaling via the Parafibromin/Hyx-mediated recruitment to Gli/Ci (Mosimann, 2009).
Region 1 of Ci/Gli has never revealed any autonomous transactivation potential when tethered to DNA, in contrast to C-terminal Gli fragments. Parafibromin/Hyx binding to Region 1 would not necessarily stimulate transcription on its own, as DNA-tethered Hyx shows no detectable transactivation effect, suggesting that it is not sufficient for triggering RNAPII-mediated transcription. Instead, in agreement with these results, the recruitment of Hyx to Hh target genes by binding to Region 1 probably helps to ensure efficient reoccurring transcription. This function might be particularly important for certain genes induced at high Hh levels and might involve particular chromatin modifications dependent on the PAF1 complex (Mosimann, 2009).
Region 1 is also the minimal interaction site for Su(fu). While competitive Su(fu) binding is an intriguing possibility, the idea of consecutive binding is favored since Parafibromin/Hyx appears to be principally required for high signal output -- conditions under which, due to Fu action, Su(fu) binding is believed not to occur. Su(fu) plays a critical negative regulatory role in the Hh pathway, especially in mammals. How this factor functions is unclear, but it may regulate Gli processing, act as a co-repressor, and/or regulate Gli/Ci localization. The finding that positive and negative regulators bind to Region 1 may explain why its deletion in Ci only had a minor effect (Mosimann, 2009).
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 (Hh) family of secreted proteins governs a myriad of key developmental processes by regulating Ci/Gli transcription factors at multiple levels including nuclear/cytoplasmic shuttling. This study investigated the mechanism underlying the regulation of Ci/Gli subcellular localization by identifying and characterizing a novel nuclear localization sequence (NLS) in the N-terminal conserved domain of Ci/Gli that matches the PY-NLS consensus. This study demonstrates that the PY-NLS functions in parallel with a previously identified bipartite NLS to promote nuclear localization and activity of full-length Ci. Transportin (Trn), a Drosophila homolog of Kapbeta2, is responsible for PY-NLS-mediated nuclear localization of Ci. Furthermore, it was shown that the tumor suppressor and conserved Hh pathway component Suppressor of fused (Sufu) opposes Trn-mediated Ci nuclear import by masking its PY-NLS. Finally, evidence is provided that Gli proteins also contain a functional PY-NLS and that mammal Sufu employs a similar mechanism to regulate Gli nuclear translocation. This study not only provides a mechanistic insight into how Sufu regulates Ci/Gli subcellular localization and Hh signaling but also reveals a role of Trn/Kapbeta2 in developmental regulation (Shi, 2014).
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