Gene name - fused
Cytological map position - 17C4-6
Function - kinase - signaling
Keywords - segment polarity
Symbol - fu
Genetic map position - 1-59.5
Classification - serine-threonine kinase
Cellular location - cytoplasmic
|Recent literature||Sanial, M., Becam, I., Hofmann, L., Behague, J., Arguelles, C., Gourhand, V., Bruzzone, L., Holmgren, R. A. and Plessis, A. (2017). Dose dependent transduction of Hedgehog relies on phosphorylation-based feedback between the GPCR Smoothened and the kinase Fused. Development 144(10):1841-1850. PubMed ID: 28360132
Smoothened (SMO) is a GPCR-related protein required for the transduction of Hedgehog (HH). The HH gradient leads to graded phosphorylation of SMO, mainly by the PKA and CKI kinases. How thresholds in HH morphogen regulate SMO to promote switch-like transcriptional responses is a central unsolved issue. Using the wing imaginal disc model in Drosophila, this study identified novel SMO phosphosites that enhance the effects of the PKA/CKI kinases on SMO accumulation, its localization at the plasma membrane and its activity. Surprisingly, phosphorylation at these sites is induced by the kinase Fused (FU), a known downstream effector of SMO. In turn activation of SMO induces FU to act on its downstream targets. Together these data provide evidence for a SMO/FU positive regulatory loop nested within a multi-kinase phosphorylation cascade. It is proposed that this complex interplay amplifies signaling above a threshold that allows high HH signaling.
|Li, H., Wang, W., Zhang, W. and Wu, G. (2020). Structural insight into the recognition between Sufu and fused in the Hedgehog signal transduction pathway. J Struct Biol 212(2): 107614. PubMed ID: 32911070
Hedgehog signaling plays a crucial role in embryogenesis and adult tissue homeostasis, and mutations of its key components such as Suppressor of fused (Sufu) are closely associated with human diseases. The Ser/Thr kinase Fused (Fu) promotes Hedgehog signaling by phosphorylating the Cubitus interruptus (Ci)/Glioma-associated oncogene homologue (Gli) family of transcription factors. Sufu associates with both Fu and Ci/Gli, but the recognition mechanism between Sufu and Fu remains obscure. The structure of the N-terminal domain (NTD) of Drosophila Sufu (dSufu) in complex with the Sufu-binding site (SBS) of Fu reveals that both main-chain β sheet formation and side-chain hydrophobic interactions contribute to the recognition between Sufu and Fu, and point mutations of highly conserved interface residues eliminated their association. Structural comparison suggests that Fu and Ci/Gli bind on opposite sides of dSufu-NTD, allowing the formation of a Fu-dSufu-Ci ternary complex which facilitates the phosphorylation of Ci/Gli by Fu. Hence, these results provide insights into the Sufu-Fu recognition mechanism.
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).
The Hedgehog (Hh) family of secreted proteins is involved both in developmental and tumorigenic processes. Although many members of this important pathway are known, the mechanism of Hh signal transduction is still poorly understood. In this study, the regulation of the kinesin-like protein Costal2 (Cos2) by Hh was analyzed. A residue on Cos2, serine 572 (Ser572), is necessary for normal transduction of the Hh signal from the transmembrane protein Smoothened (Smo) to the transcriptional mediator Cubitus interruptus (Ci). This residue is located in the serine/threonine kinase Fused (Fu)-binding domain and is phosphorylated as a consequence of Fu activation. Although Ser572 does not overlap with known Smo- or Ci-binding domains, the expression of a Cos2 variant mimicking constitutive phosphorylation and the use of a specific antibody to phosphorylated Ser572 showed a reduction in the association of phosphorylated Cos2 with Smo and Ci, both in vitro and in vivo. Moreover, Cos2 proteins with an Ala or Asp substitution of Ser572 were impaired in their regulation of Ci activity. It is proposed that, after activation of Smo, the Fu kinase induces a conformational change in Cos2 that allows the disassembly of the Smo-Fu-Cos2-Ci complex and consequent activation of Hh target genes. This study provides new insight into the mechanistic regulation of the protein complex that mediates Hh signalling and a unique antibody tool for directly monitoring Hh receptor activity in all activated cells (Reul, 2007).
These data show that phosphorylation of Cos2 residue Ser572 is necessary for the full activation of Hh signalling, and that this phosphorylation is dependent on the kinase Fu. It is likely that Fu directly phosphorylates Cos2 on Ser572, but it was not possible to purify an activated Fu kinase to confirm this. The phosphorylation of this residue strongly decreased the association of Cos2 with both Ci and Smo, an important step in the regulation of the cytoplasmic anchoring of Ci. By contrast, Cos2-572A, a Cos2 mutant that cannot be phosphorylated at Ser 572, remained associated with Smo and Ci but was much less sensitive to Hh regulation; this is because both its restraining activity on Ci and its association with Ci were only minimally sensitive to the presence of Fu and to the activation of Hh signalling. Phosphorylation of Ser572 of Cos2 induces the partial disassembly of the protein complex (Reul, 2007).
The data show that Cos2 phosphorylated on Ser572 does not bind Smo. However, previous studies have shown that Cos2 is phosphorylated and is pulled down by Smo in response to Hh stimulation. How can these data be reconciled? First, it is possible that not all Cos2 proteins that bind to Smo are phosphorylated. Indeed, only a limited fraction of Cos2 and Fu are sensitive to Hh activation. This is clearly observed with Fu (only 50% of the protein undergoes an electromobility shift upon Hh activation), but is more difficult to quantify with Cos2 because of its very small and diffused electromobility shift. Nevertheless, if Cos2 behaves similarly to Fu, it would mean that 50% of the total Cos2 (corresponding to the non-modified protein in Hh-treated cells) should be able to bring enough Smo down to be detectable in immunoprecipitates. Second, it is possible that Smo still binds to phosphorylated Cos2 on Ser572, but with much less affinity. Third, phosphorylation on Ser572 is not responsible for all Cos2 mobility shift, because Cos2-572A still shifts upon OA treatment, suggesting that other phosphorylated sites are present. Therefore, some phosphorylated isoforms that are not phosphorylated on Ser572 might also be associated with Smo. It is thus possible that this study has revealed only one of a series of sequential phosphorylation events on Cos2 that ultimately lead to the complete dissociation of Cos2 from Smo. Finally, it is worth mentioning that more Smo is present in the Cos2 IP from Hh-treated cells than in non-Hh treated cells. This is thought to simply reflect an increased level of Smo resulting from Hh signalling activation, and not the Hh-dependent regulation of the efficiency of the interaction of Smo with Cos2 (Reul, 2007).
The role of the Cos2 protein in the complex is to serve as a platform to allow both positive and negative regulators to be brought into close proximity with Smo and Ci. Thus, the role of Cos2 in transmitting a response can be masked by the role of Cos2 in limiting pathway activity in the absence of Hh. At low concentrations, it is able to stimulate Hh reporter activity in vitro and engrailed expression in vivo. But in Cos2-572A-expressing cells, engrailed expression was lower than in wild-type discs, and the in vitro stimulation of Hh signalling could not be potentiated by Fu activity. Moreover, the restraining activity of Cos2-572A on Ci could not be counteracted by Hh or Fu in vitro. Therefore, it is proposed that the Ser572 to Ala substitution on Cos2 rendered Cos2 less sensitive to Hh and Fu regulation. Because Cos2-572A still binds to its partners, it could bring Fu into proximity with its other targets. Indeed, it is likely that Fu activation leads not only to the direct phosphorylation of Cos2 but also to direct changes in Ci and/or other partners, such as Sufu. This explains why Cos2-572A is still able to stimulate Hh signalling, albeit not to its highest level (Reul, 2007).
From the Cos2-572A results, one could wonder why Cos2-572D did not constitutively activate the pathway. Because the Cos2-572D form is in a 'frozen' state compared with the wild-type form, cycles of phosphorylation/dephosphorylation are blocked and thus Cos-572D cannot participate in the Hh complex signalling anymore. The data show that constitutively phosphorylated Cos2 and endogenous phospho-Cos2 are bound to Fu but are dissociated from Smo and Ci. Therefore, Fu bound to phosphorylated Cos2 would be absent from the complex, preventing the release of all the cytoplasmic anchors from Ci (Reul, 2007).
Because the Cos2 Ser572 residue is not part of the Ci- or Smo-binding domains, but phosphorylation of this site nevertheless leads to the dissociation of these two proteins from Cos2, it is proposed that the Fu-mediated modification of Cos2 induces the protein to undergo a conformational change that leads to the disassembly of the complex. The disassembly is partial because phosphorylated Cos2 and Fu are still associated. Interestingly, it has been proposed that the binding of Cos2, Sufu and Fu to Ci masks a nuclear localisation site on Ci (Ci-NLS). A conformational change that supports this idea: that disassembly of the complex is necessary to expose the Ci-NLS and for consequent nuclear translocation (Reul, 2007).
cDNA clone length - 3430
Bases in 5' UTR - 903
Exons - three short introns of 72, 60 and 68 bp
Bases in 3' UTR - 749
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).
In a forward genetic screen for chemotaxis mutants in Dictyostelium discoideum, a loss-of-function mutation, designated tsunami, was identified encoding a homolog of the Fused kinase. Cells lacking tsuA function could not effectively perform chemotaxis and were unable to become polarized or correctly orient pseudopods in chemotactic gradients. While tsuA- cells were able to couple receptor occupancy to phosphatidylinositol (3,4,5) trisphosphate (PIP3) production and actin polymerization, the PIP3 response was prolonged and basal F-actin levels were increased. Interestingly, TsuA localizes to the microtubule network and puncta mainly found at the cell periphery. Analysis of the gene uncovered a novel C-terminal domain that was designated the Tsunami Homology (TH) domain. Both the kinase domain and the TH domain are required to rescue the phenotypic defects of tsuA- cells. While kinase activity is not required for localization to microtubules, the TH domain is essential. Thus, localization of kinase activity to microtubules is critical for TsuA function. It is proposed that functions in association with the microtubule network may underlie the divergent roles of Fused kinase proteins in different organisms (Tang, 2008).
date revised: 10 September 2000
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