BIOLOGICAL OVERVIEW

The Drosophila segment-polarity gene fused (fu) is required for pattern formation within embryonic segments and imaginal discs. Fused has a central role in mediating the Hedgehog signal that activates decapentaplegic (Sánchez-Herrero, 1996). Fused also functions in the maintenance of wingless expression, which in turn is dependent on hedgehog signaling. In order to take a closer look at hedgehog function, and its effects on fused, it becomes necessary to back track for a moment along the developmental chain of events.

From its location in a parasegment adjacent to its target, hedgehog signals from posterior to anterior cells. It is these hedgehog signals that maintain Wingless production. The pathway in the anterior cells is not well understood, but it involves Patched, Fused, and at least two other proteins: Suppressor of fused, and Costal 2. In one model, costal 2 may either be activated by Suppressor of fused, or inactivated by Fused. The function of costal 2 in this model is the activation of an unknown transcription factor, involved in the transcription of wingless (Pham, 1995). An additional target of Fused is cubitus interruptus, another transcription factor implicated in control of wingless (Molzny, 1995). The same pathway functions in the regulation of DPP (Sánchez-Herrero, 1996).

The Suppressor of fused [Su(fu)] gene encodes a protein with a PEST sequence involved in rapid protein turn-over (Pham, 1995). Fused is phosphorylated in response to the Hh signal (Therond, 1996b). A large protein complex that includes Cubitus interruptus, Costal-2 and Fused binds to microtubules and has been implicated in the regulation of Ci cleavage and accumulation, and may be involved in mediating the Hh signal. Although Su(fu) activity is apparently dispensable in a wild-type background, its absence fully suppresses all the fused mutant phenotypes. These data suggest that the activation of Fused in cells receiving the Hh signal relieves the negative effect of Su(fu) on the pathway (Alves, 1998 and references).

The roles of Fused and Su(fu) proteins were examined in the regulation of Hh target gene expression in wing imaginal discs, by using different classes of fu alleles and an amorphic Su(fu) mutation. The fused phenotype consists of a vein 3 thickening and vein 4 disappearance with reduction of the intervein region. At the wing margin, the anterior double row bristles reach the fourth vein. Fused protein is present throughout the entire wing level, but its level is much higher in the anteior compartment. In contrast, fused transcripts are uniformly distributed, suggesting that fused is regulated post-transcriptionally. Observations using fused clones indicate that only fused minus clones located in the region extending between veins 3 and 4 generate a mutant phenotype, consisting of extra-veins, which often bear campaniform sensillae characteristic of vein 3. Thus Fused kinase activity is required at the anterior/posterior (AP) boundary in the anterior compartment. At the AP boundary, Fu kinase activity is involved in the maintenance of high ptc expression and in the induction of late anterior engrailed expression. These combined effects can account for the modulation of Ci accumulation and for the precise localization of the Dpp morphogen stripe. Here, at the AP boundary, Hh signal activates the Fu kinase, leading to a modified active form of Ci required for anterior en expression and high ptc expression. Su(fu) suppresses all fused phenotypes associated with the AP boundary, suggesting that Su(fu) normally functions to antagonize the effects of Fused (Alves, 1998).

Two classes of fused mutants are described with respect to more anterior cells, which are so distant from the AP boundary that they do not receive Hh signal. Class I and class II fused alleles encode structurally different proteins; fused class I alleles encode mutant proteins altered in the catalytic domain but containing at least the 300 C-terminal amino acids, where class II alleles encode proteins truncated in the C-terminal, non-catalytic domain. In class II fused mutant discs, but not in class I mutants, abnormal dpp-lacZ expression is detected at the anterior-dorsal part of the disc in the presumptive hinge region of the wing. This ectopic expression is not correlated with any phenotype, but an interaction of fused with Su(fu) is observed. This interaction consists of an overgrowth of the anterior compartment accompanied by ectopic dpp-lacZ. Taken together, these results demonstrate that whereas at the AP boundary Fu and Su(fu) have opposite effects on the levels of ptc and dpp expression, in the anterior compartment, class II fused mutant products activate dpp expression and this effect is enhanced when Su(fu) is absent. Thus Fu plays a role independent of its kinase function (but dependent on its C-terminal domain) in the regulation of Ci accumulation in the anterior compartment. In these cells, Fu may be involved in the stabilization of a large protein complex that is probably responsible for the regulation of Ci cleavage and/or targeting to nucleus. In the anterior compartment, no Hh signal is received and Ci cleavage gives rise to a short Ci form that represses dpp expression (Alves, 1998).

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

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

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

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

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

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

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

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

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

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

GENE STRUCTURE

cDNA clone length - 3430

Bases in 5' UTR - 903

Exons - three short introns of 72, 60 and 68 bp

Bases in 3' UTR - 749

PROTEIN STRUCTURE AND EVOLUTIONARY HOMOLOGS

Amino Acids - 805

Structural Domains

The segment-polarity gene fused is maternally required for correct patterning in the posterior part of each embryonic metamere. It is also necessary later in development, because fused mutations lead to anomalies of adult cuticular structures and tumorous ovaries. Molecular evidence is provided that this gene encodes a putative serine/threonine protein kinase, a new function for the product of a segmentation gene (Preat, 1990).

The N-terminal part of the fused gene, containing 268 amino acids, is homologous to the catalytic domain of serine/threonine kinases (Therond, 1993). Sequence data suggest that the C-terminal part of Fused corresponds to a putative regulatory domain (Preat, 1993).

The fused homologous gene from Drosophila virilis has been cloned and an interspecific DNA sequence comparison has identified regions that have been conserved during evolution. Comparison of the predicted amino acid (aa) sequences reveal two regions of strong homology, one corresponding to the kinase domain (268 aa), the other located in the third exon of the Dm fu gene, suggesting a functional importance for this region (Blanchet-Tournier, 1995).

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 here. 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, as recombinant Su(fu) enhances the ability of Gli proteins to bind DNA in electrophoretic mobility shift assays (Pearse, 1999).

Drosophila Suppressor of fused [Su(fu)] encodes a novel 468-amino-acid cytoplasmic protein that, by genetic analysis, functions as a negative regulator of the Hedgehog segment polarity pathway. The primary structure, tissue distribution, biochemical and functional analyses of a human Su(fu) [hSu(fu)] is described. Two alternatively spliced isoforms of hSu(fu) were identified, predicting proteins of 433 and 484 amino acids, with a calculated molecular mass of 48 and 54 kDa, respectively. The two proteins differ only by the inclusion or exclusion of a 52 amino-acid extension at the carboxy terminus. Both isoforms are expressed in multiple embryonic and adult tissues, and exhibit a developmental profile consistent with a role in Hedgehog signaling. The hSu(fu) contains a high-scoring PEST-domain, and exhibits an overall 37% sequence identity (63% similarity) with the Drosophila protein and 97% sequence identity with the mouse Su(fu). The hSu(fu) locus maps to chromosome 10q24-q25, a region that is deleted in glioblastomas, prostate cancer, malignant melanoma and endometrial cancer. HSu(fu) represses activity of the zinc-finger transcription factor Gli, which mediates Hedgehog signaling in vertebrates, and physically interacts with Gli, Gli2 and Gli3 as well as with Supernumerary limbs (Slimb), an F-box containing protein that, in the fly, suppresses the Hedgehog response, in part by stimulating the degradation of the fly Gli homolog. Coexpression of Slimb with Su(fu) potentiates the Su(fu)-mediated repression of Gli. Taken together, these data provide biochemical and functional evidence for the hypothesis that Su(fu) is a key negative regulator in the vertebrate Hedgehog signaling pathway. The data further suggest that Su(fu) can act by binding to Gli and inhibiting Gli-mediated transactivation as well as by serving as an adaptor protein, which links Gli to the Slimb-dependent proteasomal degradation pathway (Stone, 1999).

The human Suppressor-of-Fused (SUFUH) complementary DNA has been identified and the gene product has been shown to interact physically with the transcriptional effector GLI-1. SUFUH can sequester GLI-1 in the cytoplasm, but can also interact with GLI-1 on DNA. Functionally, SUFUH inhibits transcriptional activation by GLI-1, as well as osteogenic differentiation in response to signaling from Sonic hedgehog. Localization of GLI-1 is influenced by the presence of a GLI-1 nuclear-export signal, and GLI-1 becomes constitutively nuclear when this signal is mutated or nuclear export is inhibited. These results show that SUFUH is a conserved negative regulator of GLI-1 signaling that may affect nuclear-cytoplasmic shuttling of GLI-1 or the activity of GLI-1 in the nucleus and thereby modulate cellular responses (Kogerman, 1999).

To test whether vertebrate Sufu is expressed in a pattern consistent with a potential role in mediating Shh signaling during embryogenesis, whole-mount in situ hybridization was used to analyse Sufu expression in mouse embryos at days 8.5 to 15.5 of development. Throughout the entire period signals were observed in the neural tube and, at the later stages, in the neural tube derivatives -- the brain and spinal cord. The somites express Sufu at all stages; the vibrissae field stain positively for Sufu from day 12.5 and onwards, with the vibrissae themselves being spared. The Sufu expression pattern during limb-bud development appears to be separated into two distinct phases, with strong homogeneous staining all over the limb buds being observed from their emergence at 9.5 days, whereas at 12.5 days only the interdigital mesenchyme of the limbs stain positively. This expression pattern partially overlaps with the expression of Ptch and the Ci homologs Gli 1-3, and is compatible with a conserved role for Sufu in Shh signaling (Kogerman, 1999).

To substantiate this observation in more detail and in the human system, the expression of SUFUH and PTCH1 was analyzed in the developing limb of a 12-week-old human embryo by radioactive in situ hybridization. The results show marked SUFUH expression in the osteoblasts of the perichondrium, where PTCH1 is also highly expressed. These findings are consistent with earlier observations in the avian and murine systems, in which Ptch1 and Gli1 are highly expressed in the same type of cells in response to Ihh secretion by prehypertrophic chondrocytes. Taken together, these results show that SUFUH is preferentially expressed in cells that receive a Hedgehog signal, and indicate that, during embryogenesis, SUFUH may be co-regulated with PTCH1 and GLI1 (Kogerman, 1999).

The retention of GLI-1 in the cytoplasm by SUFUH when nuclear export is compromised, and the similar SUFUH-mediated retention in the cytoplasm of an otherwise constitutively nuclear GLI-1 variant (truncated so that it lacks the NES) indicates that SUFUH could block nuclear entry of GLI-1, possibly by masking a nuclear-localization signal, and thereby inhibit transcriptional activation of target genes. Consistent with this idea, a truncated SUFUH variant unable to repress GLI1-induced transcriptional activation is also unable to modify the subcellular localization of GLI-1. What remains an interesting question for future studies is whether or not binding of SUFUH to GLI-1 on DNA, or elsewhere in the nuclear compartment, actually acts to repress or block activation of transcription, alone or in combination with cytoplasmic retention of GLI-1. The expression of Sufu in cells next to Shh- or Ihh-producing cells during mouse and human embryogenesis, coupled with the ability of Sufu to inhibit Gli-mediated transcriptional activation, indicates that an important function of Sufu may be to act in an intracellular negative feedback mechanism and to impose thresholds on the responsiveness of cells to Shh and Ihh. A similar role for D-Axin has been proposed as regards Wingless signaling in Drosophila (Kogerman, 1999).

Hedgehog (Hh) proteins are secreted factors that control cell proliferation and cell-fate specification. Hh signaling is mediated in vertebrates by the Gli zinc-finger transcription factors (Gli1, Gli2 and Gli3) and in Drosophila by the Gli homolog Cubitus interruptus (Ci). However, the mechanisms that regulate Gli/Ci activity are not fully understood. Genetic studies in Drosophila have identified a putative serine-threonine kinase, Fused (Fu), and a new protein, Suppressor of Fused [Su(fu)], as modulators of Ci activity. A human homologue of Drosophila Fu, hFu, regulates the activity of Gli1 and Gli2 on several levels. hFu converts Gli2 from a weak to a strong transcriptional activator, antagonizes the repressive effect of the human Su(fu) homolog, [hSu(fu)], on Gli1 and Gli2, and promotes nuclear localization of Gli1 and Gli2 (Murone, 2000).

To identify possible regulators of Gli proteins, complementary DNAs were isolated encoding hFu, which shares a significant level of homology with Drosophila Fu in the kinase domain (55%), but only a limited amount of homology over the remaining 1,052 amino acids. The gene encoding hFu was mapped to chromosome 2q35, close to the PAX3 gene, which is implicated in the Klein-Waardenburg syndrome. PAX3 is a target of Sonic hedgehog (Shh) and it has been suggested that additional loci in the 2q35 region may regulate the PAX3 locus, thereby influencing the Klein-Waardenburg phenotype. Northern-blot analysis has showen that a single 5-kb hFu transcript is expressed at low levels in most fetal tissues and adult ovaries, and at high levels in adult testes, where it is localized in germ cells with other components of the Hh pathway. Examination of a mouse embryo at day 13.5 of development by in situ hybridization shows that mouse Fu (mFu) mRNA is widely distributed in Shh-responsive tissues, including the forebrain, midbrain, hindbrain, spinal cord, somites, developing limb buds and skin (Murone, 2000).

To determine whether hFu can regulate Gli activity, hFu was cotransfected with a Gli-binding-site (Gli-BS) luciferase reporter in the Hh-responsive cell line C3H10T1/2. hFu alone is capable of weakly inducing transcription of the Gli-BS reporter, indicating that it may be a positive regulator of the Hh pathway. Although hFu contains a putative kinase domain, no substantial kinase activity for hFu was detected; a similar lack of kinase activity has been reported for Drosophila Fused (Murone, 2000).

To determine the function of the kinase domain of hFu, a putative catalytically dead version of hFu [hFu(K33R)] was constructed by mutating a conserved lysine residue in the ATP-binding site at position 33. This residue is crucial to the catalytic activity of all kinases, and the corresponding mutation in Drosophila leads to a fu phenotype. hFu(K33R) is able to activate the Gli-BS reporter as efficiently as wild-type hFu, indicating that the putative kinase activity of hFu may not contribute significantly to Gli activation under these conditions. A similar result has been obtained for a hFu construct [hFu(270-1,315)] lacking the entire kinase domain (amino acids 1-269). The activity of hFu was tested in combination with various Gli-family members. Whereas human Gli1 alone strongly induces the luciferase reporter, mouse Gli2 exhibits only weak activity and human Gli3 shows no activity at all. hFu does not affect the activity of Gli1 and Gli3, but strongly synergizes with Gli2. Moreover, activation of Gli2 by hFu is antagonized by hSu(fu). In contrast, Gli1 is constitutively active and its ability to activate the Gli-BS reporter is inhibited by hSu(fu) and restored in the presence of hFu (Murone, 2000).

To investigate further the mechanisms by which hFu regulates Gli activity, whether hFu forms a physical complex with hSu(fu) or the various Gli proteins was determined. Cultured cells were cotransfected with epitope-tagged versions of hFu, hSu(fu), Gli1, Gli2 and Gli3 and the resulting interactions were observed. hFu co-immunoprecipitates with hSu(fu) and with Gli1, Gli2 and Gli3. In vertebrates, Su(fu) represses Gli1 function in part by tethering it in the cytoplasm. In contrast, hFu and hFu(K 33R) promote nuclear localization of Gli1. An assessment was made of whether hFu could influence the subcellular localization of Gli1 when co-expressed with hSu(fu). In the presence of hSu(fu), roughly 3% of cells exhibit nuclear staining of Gli1. In contrast, when both hSu(fu) and hFu are present, 20% of cells possess nuclear Gli1. Identical results are obtained for Gli2. Overall, these results indicate that hFu controls the activity of Gli1 and Gli2 by opposing the effect of hSu(fu). Whereas hSu(fu) constrains Gli1 and Gli2 in the cytoplasm, hFu promotes their nuclear localization. Gli2 also requires an additional function of hFu to become transcriptionally active, as Gli2 transfected in the absence of hSu(fu) is unable to activate transcription unless hFu is present, despite the fact that it enters the nucleus. The mechanisms by which hFu activates Gli2 remain to be elucidated but may include a hFu-mediated modification of Gli2 to mask the inhibitory Gli2 amino-terminal domain (Murone, 2000).

The activity of hFu described here does not seem to require a functional kinase domain, since overexpression of kinase-mutant forms of Fu are as active as wild-type forms. Catalytically dead versions of other serine-threonine kinases, such as the RIPs8 and IRAKs14, show comparable activity to their wild-type counterparts in inducing apoptosis or activating NFkappaB respectively. Although some Drosophila kinase-domain fu mutants suffer a complete lack of induction of Hh target genes in the embryo, they show only a partial fu phenotype in the wing discs, indicating that there may be different requirements for the kinase activity of Fu in different cellular contexts (Murone, 2000).

The Suppressor of fused [Su(fu)] gene of Drosophila encodes a protein containing a PEST sequence [a sequence enriched in proline (P), glutamic acid (E), serine (S) and threonine (T)] that acts as an antagonist to the serine-threonine kinase Fused in Hedgehog (Hh) signal transduction during embryogenesis. The Su(fu) gene isolated from a distantly related Drosophila species, D. virilis, shows significantly high homology throughout its protein sequence with its D. melanogaster counterpart. These two Drosophila homologs of Su(fu) are functionally interchangeable in enhancing the fused phenotype. Mammalian homologs of Su(fu) have been isolated. The absence of the PEST sequence in the mammalian Su(fu) protein suggests a different regulation for this product between fly and vertebrates. Using the yeast two-hybrid method, the murine Su(fu) protein is shown to interact directly with the Fused and Cubitus interruptus proteins, known partners of Su(fu) in Drosophila. Su(fu) could be regulated posttranslationally in the fly and at another level in vertebrates. A similar divergence is observed for the regulation of the ci gene and its homologs, the Gli genes: in Drosophila, there is only one ci gene whose product is regulated posttranslationally; in vertebrates, there are three ci-related genes Gli, Gli2 and Gli3 that are regulated at a transcriptional level (Delattre, 1999).

date revised: 10 September 2000

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