Suppressor of fused: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - Suppressor of fused

Synonyms -

Cytological map position - 87C7-9

Function - signal transduction

Keywords - Hedgehog pathway, segment polarity

Symbol - Su(fu)

FlyBase ID: FBgn0005355

Genetic map position - 3-50.7-53.1

Classification - novel protein with PEST sequence

Cellular location - cytoplasmic

NCBI links: Entrez Gene
Su(fu) orthologs: Biolitmine

Recent literature
Oh, S., Kato, M., Zhang, C., Guo, Y. and Beachy, P. A. (2015). A comparison of Ci/Gli activity as regulated by Sufu in Drosophila and mammalian hedgehog response. PLoS One 10: e0135804. PubMed ID: 26271100
Suppressor of fused [Su(fu)/Sufu], one of the most conserved components of the Hedgehog (Hh) signaling pathway, binds Ci/Gli transcription factors and impedes activation of target gene expression. In Drosophila, the Su(fu) mutation has a minimal phenotype, and this study shows that Ci transcriptional activity in large part is regulated independently of Su(fu) by other pathway components. Mutant mice lacking Sufu in contrast show excessive pathway activity and die as embryos with patterning defects. In cultured cells Hh stimulation can augment transcriptional activity of a Gli2 variant lacking Sufu interaction and, surprisingly, regulation of Hh pathway targets is nearly normal in the neural tube of Sufu-/- mutant embryos that also lack Gli1 function. Some degree of Hh-induced transcriptional activation of Ci/Gli thus can occur independently of Sufu in both flies and mammals. It is further noted that Sufu loss can also reduce Hh induction of high-threshold neural tube fates, such as floor plate, suggesting a possible positive pathway role for Sufu.

Han, Y., Shi, Q. and Jiang, J. (2015). Multisite interaction with Sufu regulates Ci/Gli activity through distinct mechanisms in Hh signal transduction. Proc Natl Acad Sci U S A 112: 6383-6388. PubMed ID: 25941387
The tumor suppressor protein Suppressor of fused (Sufu) plays a conserved role in the Hedgehog (Hh) signaling pathway by inhibiting Cubitus interruptus (Ci)/Glioma-associated oncogene homolog (Gli) transcription factors, but the molecular mechanism by which Sufu inhibits Ci/Gli activity remains poorly understood. This study shows that Sufu can bind Ci/Gli through a C-terminal Sufu-interacting site (SIC) in addition to a previously identified N-terminal site (SIN), and that both SIC and SIN are required for optimal inhibition of Ci/Gli by Sufu. Sufu can sequester Ci/Gli in the cytoplasm through binding to SIN while inhibiting Ci/Gli activity in the nucleus depending on SIC. It was also found that binding of Sufu to SIC and the middle region of Ci can impede recruitment of the transcriptional coactivator CBP by masking its binding site in the C-terminal region of Ci. Indeed, moving the CBP-binding site to an 'exposed' location can render Ci resistant to Sufu-mediated inhibition in the nucleus. Hence, this study identifies a previously unidentified and conserved Sufu-binding motif in the C-terminal region of Ci/Gli and provides mechanistic insight into how Sufu inhibits Ci/Gli activity in the nucleus.
Jabrani, A., Makamte, S., Moreau, E., Gharbi, Y., Plessis, A., Bruzzone, L., Sanial, M. and Biou, V. (2017). Biophysical characterisation of the novel zinc binding property in Suppressor of Fused. Sci Rep 7(1): 11139. PubMed ID: 28894158
Suppressor of Fused (SUFU) is a highly conserved protein that acts as a negative regulator of the Hedgehog (HH) signalling pathway, a major determinant of cell differentiation and proliferation. Therefore, SUFU deletion in mammals has devastating effects on embryo development. SUFU is part of a multi-protein cytoplasmic signal-transducing complex. Its partners include the Gli family of transcription factors that function either as repressors, or as transcription activators according to the HH activation state. The crystal structure of SUFU revealed a two-domain arrangement, which undergoes a closing movement upon binding a peptide from Gli1. There remains however, much to be discovered about SUFU's behaviour. To this end, recombinant, full-length SUFU from Drosophila, Zebrafish and Human were expressed. Guided by a sequence analysis that revealed a conserved potential metal binding site, it was discovered that SUFU binds zinc. This binding was found to occur with a nanomolar affinity to SUFU from all three species. Mutation of one histidine from the conserved motif induces a moderate decrease in affinity for zinc, while circular dichroism indicates that the mutant remains structured. These results reveal new metal binding affinity characteristics about SUFU that could be of importance for its regulatory function in HH.
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.
Makamte, S., Thureau, A., Jabrani, A., Paquelin, A., Plessis, A., Sanial, M., Rudenko, O., Oteri, F., Baaden, M. and Biou, V. (2022). A large disordered region confers a wide spanning volume to vertebrate Suppressor of Fused as shown in a trans-species solution study J Struct Biol 214(2): 107853. PubMed ID: 35364288
Hedgehog (Hh) pathway inhibition by the conserved protein Suppressor of Fused (SuFu) is crucial to vertebrate development. By constrast, SuFu loss-of-function mutant has little effect in Drosophila. Previous publications showed that the crystal structures of human and Drosophila SuFu consist of two ordered domains that are capable of breathing motions upon ligand binding. However, the crystal structure of human SuFu does not give information about twenty N-terminal residues (IDR1) and an eighty-residue-long region predicted as disordered (IDR2) in the C-terminus, whose function is important for the pathway repression. These two intrinsically disordered regions (IDRs) are species-dependent. To obtain information about the IDR regions, full-length SuFu's structure in solution was studied, both with circular dichroism and small angle X-ray scattering, comparing Drosophila, zebrafish and human species, to better understand this considerable difference. These studies show that, in spite of similar crystal structures restricted to ordered domains, Drosophila and vertebrate SuFu have very different structures in solution. The IDR2 of vertebrates spans a large area, thus enabling it to reach for partners and be accessible for post-translational modifications. Furthermore, this study showed that the IDR2 region is highly conserved within phyla but varies in length and sequence, with insects having a shorter disordered region while that of vertebrates is broad and mobile. This major variation may explain the different phenotypes observed upon SuFu removal.
Zhou, M., Han, Y., Wang, B., Cho, Y. S. and Jiang, J. (2022). Dose-dependent phosphorylation and activation of Hh pathway transcription factors. Life Sci Alliance 5(11). PubMed ID: 36271509
Graded Hedgehog (Hh) signaling is mediated by graded Cubitus interruptus (Ci)/Gli transcriptional activity, but how the Hh gradient is converted into the Ci/Gli activity gradient remains poorly understood. This study shows that graded Hh in Drosophila induces a progressive increase in Ci phosphorylation at multiple Fused (Fu)/CK1 sites including a cluster located in the C-terminal Sufu-binding domain. Fu directly phosphorylated Ci on S1382, priming CK1 phosphorylation on adjacent sites, and that Fu/CK1-mediated phosphorylation of the C-terminal sites interfered with Sufu binding and facilitated Ci activation. Phosphorylation at the N-terminal, middle, and C-terminal Fu/CK1 sites occurred independently of one another and each increased progressively in response to increasing levels of Hh or increasing amounts of Hh exposure time. Increasing the number of phospho-mimetic mutations of Fu/CK1 sites resulted in progressively increased Ci activation by alleviating Sufu-mediated inhibition. C-terminal Fu/CK1 phosphorylation cluster is conserved in Gli2 and contributes to its dose-dependent activation. This study suggests that the Hh signaling gradient is translated into a Ci/Gli phosphorylation gradient that activates Ci/Gli by gradually releasing Sufu-mediated inhibition.
Deng, Y., Peng, D., Xiao, J., Zhao, Y., Ding, W., Yuan, S., Sun, L., Ding, J., Zhou, Z. and Zhan, M. (2022). Inhibition of the transcription factor ZNF281 by SUFU to suppress tumor cell migration. Cell Death Differ. PubMed ID: 36220888
Although the Hedgehog (Hh) pathway plays an evolutionarily conserved role from Drosophila to mammals, some divergences also exist. Loss of Sufu, an important component of the Hh pathway, does not lead to an obvious developmental defect in Drosophila. However, in mammals, loss of SUFU results in serious disorder, even various cancers. This divergence suggests that SUFU plays additional roles in mammalian cells, besides regulating the Hh pathway. This study identified that the transcription factor ZNF281 is a novel binding partner of SUFU. Intriguingly, the Drosophila genome does not encode any homologs of ZNF281. SUFU is able to suppress ZNF281-induced tumor cell migration and DNA damage repair by inhibiting ZNF281 activity. Mechanistically, SUFU binds ZNF281 to mask the nuclear localization signal of ZNF281, culminating in ZNF281 cytoplasmic retention. In addition, SUFU also hampers the interactions between ZNF281 and promoters of target genes. Finally, we show that SUFU is able to inhibit ZNF281-induced tumor cell migration using an in vivo model. Taken together, these results uncover a Hh-independent mechanism of SUFU exerting the anti-tumor role, in which SUFU suppresses tumor cell migration through antagonizing ZNF281. Therefore, this study provides a possible explanation for the functional divergence of SUFU in mammals and Drosophila.

Suppressor of fused [Su(fu)] functions in a complex pathway which may be described, at least in part, in terms of the function of genes that regulate and are regulated by the gene Hedgehog (Hh). Hh function is best understood in the developing Drosophila wing imaginal disc, a tissue that is formed by two cell populations, the anterior (A) and posterior (P) compartments. The selector gene engrailed is expressed in P cells where it permits the synthesis of Hh while at the same time preventing a response to Hh. Hh diffuses into the A compartment and initiates a signaling cascade by binding to its receptor, the multi-transmembrane domain protein Patched (Ptc). Hh neutralizes the inhibitory hold of Ptc on the co-receptor protein Smoothened (Smo), which then signals to downstream effectors. Signaling results in the stabilization of the zinc finger protein Cubitus interruptus (Ci) and the transcriptional activation of target genes. Several proteins, such as Costal2, PKA, Slimb, and the subject of this overview, Su(fu), prevent or downregulate this signaling. Su(fu) is thought to downregulate the Hh pathway by binding to and inhibiting Ci. Upon reception of the Hh signal, Fused is activated and counteracts Su(fu), leading to the activation of Ci and consequently to the transduction of the Hh signal (Monnier, 1998). Some evidence suggests that Cos2 tethers Ci to the cytoplasm, whereas Slimb and PKA are required for the proteolytic processing of Ci. Cleavage of Ci occurs in the absence of Hh and results in the release of a C-terminally truncated Ci, referred to as the repressor form of Ci or Ci[rep]. Ci[rep] translocates to the nucleus and inhibits transcription of target genes such as decapentaplegic and hh itself. Phosphorylation of Ci by Protein kinase A (PKA) is a key event in mediating cleavage. Su(fu) downregulates the Hh pathway by preventing nuclear accumulation of Ci[act], the activated form of Ci (Methot, 2000 and references therein).

Reception of Hh prevents Ci[rep] formation, and activates the transcription of several Hh target genes such as dpp and ptc. A Hh-dependent event other than prevention of cleavage permits full-length Ci to function as a potent transcriptional activator (Ci[act]). The serine-threonine kinase Fused plays a role in the conversion of Ci into a transcriptional activator (Ohlmeyer, 1998). The formation of both Ci[act] and Ci[rep] is tightly controlled by Hh signaling. Formation of Ci[rep] requires PKA, Cos2 and Fused (Fu), while the generation of Ci[act] proceeds through the neutralization of PKA and Cos2 activity. Although Ci can bypass PKA and gain constitutive Ci[act] activity by mutations in its PKA phosphorylation sites, this activity can be further stimulated by Hh signaling. Both Su(fu) and Fu alter the transcriptional output of this mutant form of Ci, by regulating its nuclear-cytoplasmic localization. The accumulation of full-length Ci in the nucleus is Hh-dependent and is blocked by excess Su(fu). It is proposed that Fu kinase stimulates the Hh pathway, not by promoting the formation of Ci[act], but rather by facilitating its entry into the nucleus. Maximal activation of Hh target genes would therefore occur in a two-step process. Complex formation with Fu, Cos2 and microtubules serves to tether Ci to the cytoplasm and to locate Ci to the site of Slimb-dependent proteolytic processing. Hh stimulates the release of this complex from microtubules, leading to the formation of Ci[act]. The latter can still be partially inhibited through the action of Su(fu), either by suppressing nuclear import or by enhancing nuclear export (Methot, 2000 and references therein). It is proposed that, in the absence of Hh signaling, Su(fu) inhibits Ci by binding to it and that, upon reception of the Hh signal, Fu is activated and counteracts Su(fu), leading to the activation of Ci (Monnier, 1998).

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

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

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

Components of the Hh pathway that could stimulate the basal activity of CiPKA were then sought. Fu is one of the few proteins that have a positive input on Hh signaling (Preat, 1990; Ohlmeyer, 1998; Therond, 1999). ptc-lacZ levels were examined in wing imaginal discs that ubiquitously express CiPKA1, in either a wild-type or fu1 background. ptc-lacZ expression is clearly reduced in P cells of fu1 discs. Similar results have also been obtained with CiPKA4. Slightly elevated ptc-lacZ can still be seen near the AP compartment boundary in fu1 discs, and may be the result of cumulative activities of endogenous Ci[act] and CiPKA. It is concluded that Fu kinase enhances the basal activity of CiPKA (Methot, 2000).

Beyond this basal activity, Fu stimulates CiPKA by inhibiting Su(fu) activity. fu is tightly linked to Su(fu), both genetically and biochemically (Preat, 1992; Preat, 1993 and Monnier, 1998). To test whether the modulation of CiPKA activity involves Su(fu), CiPKA4 was ubiquitously expressed together with myc-tagged Su(fu) or GFP as a negative control. CiPKA4 (in the presence of GFP) induces ptc-lacZ expression everywhere in the wing imaginal disc but at higher levels in the P compartment. Co-expression with mycSu(fu) abolishes ptc-lacZ expression in the A compartment and reduces ptc-lacZ levels in P cells. Thus Su(fu) inhibits the activity of CiPKA4. This result is strengthened by the converse experiment, where the absence of Su(fu) [in Su(fu)LP homozygous animals] reduces the difference in ptc-lacZ levels between A and P CiPKA1-expressing cells. To determine whether Su(fu) negatively acts on CiPKA4 by direct protein-protein interaction, a mutant form of Ci with impaired Su(fu) binding was created. Su(fu) interacts with Ci within a region that encompasses amino acids 244-346 (Monnier, 1998). Indeed, an N-terminal fragment of Ci (amino acids 5-440) interacts with GST-Su(fu). A deletion removing amino acids 212-268 of Ci almost abrogates Su(fu) binding to an N-terminal in vitro translated product of Ci. Removal of amino acids 268 to 346 also reduces Su(fu) binding, but to a lesser extent. The Delta212-268 deletion was introduced into CiPKA4, to create CiDeltaNPKA4. This mutant is constitutively active, with P cells expressing higher ptc-lacZ levels than A cells. The activity of CiDeltaNPKA4 is slightly reduced when introduced into a strong fu background, but the reduction is much less pronounced compared to that observed for CiPKA4. It is concluded that inhibition of Su(fu) activity by Fu kinase is an important step toward stimulating the basal activity of CiPKA4 (and by analogy Ci[act]) (Methot, 2000).

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

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

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

One puzzling aspect regarding Su(fu) is that it is dispensable for viability. Animals that lack Su(fu) protein do not exhibit Hh-independent Ci[act] activity. This paradox can be partly explained by viewing Su(fu) only as a partial inhibitor of Ci[act] activity, which exerts its function subsequent to Ci[act] formation. Other elements ensure tight control over the generation of Ci[act]. The problem of how full-length Ci protein is converted into Ci[act] is more challenging. Fu has been implicated in this process (Ohlmeyer, 1998), but as in the case of Su(fu), Fu kinase activity is partially dispensable in wild-type discs and entirely dispensable in animals lacking Su(fu) (Preat, 1992; Preat, 1993). This suggests that the Fu kinase functions only to prevent Su(fu) from negatively acting on the Hh pathway. If it is accepted that Su(fu) acts subsequent to Ci[act] formation, it must be concluded that the same is true for the Fu kinase. In short, it is proposed that the activity of the Fu kinase is only required to maximize the output of an already activated form of Ci, for example by opposing cytoplasmic tethering of Ci[act] by Su(fu). The precise mechanism of how these components act is not understood. No substrate for the Fu kinase has been identified and the significance of nuclear Fu protein is unclear (Methot, 2000).

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

Divergence of hedgehog signal transduction mechanism between Drosophila and mammals

The Hedgehog (Hh) signaling pathway has conserved roles in development of species ranging from Drosophila to humans. Responses to Hh are mediated by the transcription factor Cubitus interruptus (Ci; GLIs 1-3 in mammals), and constitutive activation of Hh target gene expression has been linked to several types of human cancer. In Drosophila, the kinesin-like protein Costal2 (Cos2), which associates directly with the Hh receptor component Smoothened (Smo), is essential for suppression of the transcriptional activity of Ci in the absence of ligand. Another protein, Suppressor of Fused [Su(Fu)], exerts a weak negative influence on Ci activity. Based on analysis of functional and sequence conservation of Cos2 orthologs, Su(Fu), Smo, and Ci/GLI proteins, Drosophila and mammalian Hh signaling mechanisms have been found to diverge; in mouse cells, major Cos2-like activities are absent and the inhibition of the Hh pathway in the absence of ligand critically depends on Su(Fu) (Varjosalo, 2006).

The evidence indicates that a significant divergence in the mechanism of Shh signal transduction has occurred between vertebrates and invertebrates at the level of Smo, Cos2, and Su(Fu). The results indicate that major Cos2-like activities are absent in mouse cells based on four observations: (1) domains in Smo that are required in Drosophila to bind to Cos2 are not required for mSmo function; (2) mouse Shh signaling is insensitive to expression of Drosophila Cos2, but can be rendered Cos2 sensitive by replacing the mSmo C-terminal domain with the dSmo C-terminal domain; (3) expression of the Smo C-terminal domain which, in Drosophila, inactivates Cos2 has no effect in the mouse in vivo or in vitro; (4) overexpression or RNAi-mediated suppression of mouse Cos2 homologs has no effect on Hh signaling, even under sensitized conditions. These results are also consistent with divergence of the sequence of domains involved in Cos2 binding in Ci/GLI proteins and Smo between insects and mammals (Varjosalo, 2006).

Although the RNAi experiments targeting Cos2 orthologs Kif7 and Kif27 were performed under conditions in which negative regulators of GLI2 were limiting, they could be argued to be consistent with a model in which multiple kinesins with Cos2-like activity would act in a redundant fashion in mammals. By loss-of-function studies of individual kinesins in cell culture or in mice it would be difficult to obtain conclusive evidence against such a model due to the potential redundancy of multiple members of the kinesin family. However, several other in vitro and in vivo experiments that were presented directly contradict such a model. These include RNAi analyses targeting multiple Kif proteins, the analysis of loss of function of mSmo domains, and the lack of effect of overexpression of myristoylated-mSmoC and the Cos2 orthologs Kif7 and Kif27. In addition, no kinesin with Cos2-like activity could be found by extending the analyses to several other kinesins, which show homology to Cos2 but have different fly orthologs (Varjosalo, 2006).

In contrast to the case in Drosophila, Su(Fu) has a critical role in suppression of the mammalian Hh pathway in the absence of ligand, and loss of Su(Fu) function results in dramatic induction of GLI transcriptional activity. The results are also consistent with the studies that show that loss of Su(Fu) in mouse embryos results in complete activation of the Hh pathway, in a fashion similar to the loss of Ptc. These results are particularly surprising in light of the central role of Cos2 and a minor role of Su(Fu) in Drosophila. Together, these results also clearly show that mouse cells and embryos lack a Cos2-like activity that, in Drosophila, can completely suppress the Hh pathway in the absence of Su(Fu). However, the results should not be taken as evidence against novel proteins (including kinesins not orthologous to Cos2) acting in mammalian cells between Smo and GLI proteins with mechanisms that are distinct from those used by Drosophila Cos2. Several reports have, in fact, described such vertebrate-specific regulators of Hh signaling, including SIL, Iguana, Rab23, Kif3a, IFT88, IFT172, MIM/BEG4, and β-arrestin2 (Varjosalo, 2006).

The results also shed light on some known differences in the function of the Hh pathway in Drosophila and mammals. Mutations and small molecules affecting conformation of Smo transmembrane domains have a strong effect in mammals, but they have little effect in Drosophila. Interestingly, the Smo transmembrane domain is required for regulation of Su(Fu) activity, whereas the Smo C-terminal domain is critical for inhibition of Cos2 activity. Thus, based on the data, manipulations that affect the Smo transmembrane domain would be predicted to affect Su(Fu) and therefore to have a limited role in Drosophila and a major effect in mammals (Varjosalo, 2006).

Although there are differences in mouse and Drosophila Smo functional domains, and a lack of conservation of Smo phosphorylation sites, conservation of Smo function at a level not involving Cos2 is supported by the observation that mutation of a conserved isoleucine (I573A in mSmo) results in loss of both mouse and Drosophila Smo activity, yet does not result in a loss of Cos2 binding to dSmo. In addition, dSmo proteins that are activated by phosphomimetic mutations are constitutively stabilized; yet, they are partially responsive to Hh, suggesting that, in addition to stabilization and phosphorylation, other, potentially conserved mechanisms could be required to generate fully active Smo in Drosophila as well (Varjosalo, 2006).

In the mSmo C terminus, six residues between amino acids 570 and 580 were identified that resulted in significant loss of mSmo activity. The predicted secondary structure for this region is an α helix, in which these residues would reside on the same side, raising the possibility that, together with the third Smo intracellular loop, this region may form an interaction surface involved in inactivation of Su(Fu) or activation of Ci/GLI (Varjosalo, 2006).

Recent results have indicated that Su(Fu) acts as a tumor suppressor in medulloblastoma, and it has been suggested that medulloblastomas associated with loss of Su(Fu) result, in part, from activation of the Wnt pathway. However, consistent with the lack of a Wnt phenotype of Su(Fu) mutations in Drosophila, in the current experiments, a Wnt pathway-specific reporter is not activated by shRNAs targeting Su(Fu). Given observations that Su(Fu) is critically important in the suppression of the mammalian Hh pathway in the absence of ligand, and the fact that Hh pathway activation is required for growth of a form of medulloblastoma induced by mutations in Patched, it is likely that constitutive activation of the Hh pathway is also essential for growth of medulloblastomas associated with the loss of Su(Fu) (Varjosalo, 2006).

In a wider context, the results demonstrate that signal transduction mechanisms of even the major signaling pathways are not immutable, but that they can be subject to evolutionary change. The divergence may have occurred after the separation of the vertebrate and invertebrate lineages. However, some evidence also suggests that functional divergence may have occurred much later in evolution. Although mutants of Fused or Cos2 orthologs of zebrafish have not been identified, zebrafish homologs of Fused and Cos2 act in the Hh pathway based on morpholino antisense injections. In contrast to these findings, mice deficient in mouse ortholog of Drosophila Fused do not have a Hh-related phenotype, and mouse orthologs of Cos2 do not affect Hh signaling. Hh-related phenotypes can be observed in zebrafish by morpholino-mediated targeting of other genes as well, such as β-arrestin2, whose loss in mice does not result in a Hh-related phenotype. It is widely appreciated that multiple types of embryonic insults result in Hh-like phenotypes, such as holoprosencephaly. Thus, it is possible that the zebrafish phenotypes observed are caused by the morpholino injection process itself. Alternatively, there may also be significant differences between the mechanism of Hh signaling between vertebrate species (Varjosalo, 2006).

Because of the strong conservation of Su(Fu) in both invertebrate and vertebrate phyla, the presence of a Cos2 binding domain only in insect Smo, and the divergence of the Cos2 proteins from the kinesin family, the simplest explanation of the data is that Su(Fu) represents the primordial Ci/GLI repressor, and that the Cos2-like functionality has evolved specifically in the invertebrate lineage. The results, thus, also raise the possibility that multicomponent pathways evolve, in part, by insertion of novel proteins between existing pathway components. This mechanism potentially explains a challenging aspect of evolutionary biology regarding the emergence of signaling pathways with multiple specific components (Varjosalo, 2006).

Engrailed, Suppressor of fused and Roadkill modulate the Drosophila GLI transcription factor Cubitus interruptus at multiple levels

Morphogen gradients need to be robust, but may also need to be tailored for specific tissues. Often this type of regulation is carried out by negative regulators and negative feedback loops. In the Hedgehog (Hh) pathway, activation of patched (ptc) in response to Hh is part of a negative feedback loop limiting the range of the Hh morphogen. This study shows that in the Drosophila wing imaginal disc two other known Hh targets genes feed back to modulate Hh signaling. First, anterior expression of the transcriptional repressor Engrailed modifies the Hh gradient by attenuating the expression of the Hh pathway transcription factor cubitus interruptus (ci), leading to lower levels of ptc expression. Second, the E-3 ligase Roadkill shifts the competition between the full-length activator and truncated repressor forms of Ci by preferentially targeting full-length Ci for degradation. Finally, evidence is provided that Suppressor of fused, a negative regulator of Hh signaling, has an unexpected positive role, specifically protecting full-length Ci but not the Ci repressor from Roadkill (Roberto, 2022).

This study examined the roles of three potential negative regulators of Hh signal transduction, two of which are themselves encoded by Hh target genes. In each case interesting new aspects about the pathway's regulation. Anterior expression of en likely extends the range of the Hh gradient were discovered (Roberto, 2022).

Anterior expression of en in the wing imaginal disc was first observed 30 years ago and en is the Hh target gene requiring the highest level of Hh signaling. Its domain of expression exactly correlates with a region of lower full-length Ci protein levels. It had been proposed that the lower Ci protein levels are a consequence of Ci being particularly active and labile in this region. This study shows that the lower levels of Ci are not primarily due to it being particularly labile, but rather are a consequence of negative transcriptional regulation by En. The role of this negative feedback loop appears to be to modulate the Hh gradient by downregulating the expression of ptc in addition to its effects on dpp. This leads to Hh signaling extending further into the anterior compartment, with a corresponding anterior shift in the location of LV3 and the expression of dpp. A model is prefered in which the attenuation of ptc expression by anterior en is indirect via Ci, but in principle en could also directly negatively regulate ptc. This is thought less likely as, 'flip-out' clones expressing Ci activate high levels of ptc in the posterior compartment in the presence of en. The anterior expression of en occurs late in third instar larvae, which correlates with the downregulation of ci expression as visualized using the UAS-TT transcriptional timer and the refinement of wing vein specification (Roberto, 2022).

Ci function is modulated by two feedback loops acting at different levels. Anterior expression of the En protein attenuates Ci activity directly adjacent to the compartment boundary of the wing disc by downregulating the expression of the ci gene. Rdx and Su(fu) act at the protein level modulating the competition between the full-length (Ci FL) and repressor forms (Ci R) of Ci. Rdx specifically targets full-length Ci, whereas Su(fu) partially protects full-length Ci from Rdx-mediated degradation. Rdx degradation of full-length Ci appears to help downregulate Hh target genes in cells no longer receiving the Hh signal (Roberto, 2022).

Why did this mechanism evolve to modulate the Hh gradient? Morphogen gradients, by virtue of their central roles in the development of multiple tissues, must be robust and resistant to perturbation. Therefore, to specifically expand the range of the Hh gradient in the wing disc a new component was added, anterior expression of the ci repressor en (Roberto, 2022).

The lack of the C-terminal domain in the Ci repressor has multiple consequences. It loses the binding site for the co-activator CBP, and it loses C-terminal binding sites for Su(fu), Cos2 and Rdx. As a consequence, the Ci repressor is not sequestered in the cytoplasm by Cos2 in the absence of Hh signaling and enters the nucleus without Su(fu), whereas full-length Ci enters the nucleus only in the presence of Hh signaling and as a complex with Su(fu) (Roberto, 2022).

In order to better understand the roles of Su(fu) and Rdx, animals heterozygous for the ciCe2 mutation were examined. In this context, overexpression of rdx or loss of Su(fu) function leads to a complete fusion between LV3 and LV4. In addition, clones mutant for Su(fu) show dramatic reduction in the expression of the Hh target genes ptc and dpp. These results show that Su(fu) has a potential novel positive role in Hh signal transduction, improving the ability of full-length Ci to compete with the repressor form. A positive role for Su(fu) has also been found in mammals where Su(fu) appears to function as a chaperone for the full-length Gli proteins, but not the repressor forms, and is required for full activation of Gli target genes. The requirement for Drosophila Su(fu) is obviated in the absence of Rdx, suggesting that Rdx primarily targets full-length Ci and not Ci repressor, even though the repressor is not protected by Su(fu). These results are analogous to what is seen with the mammalian homologue of Rdx, SPOP, indicating that this mechanism has been conserved during evolution. SPOP is opposed by Su(fu) and degrades the full-length forms of the mammalian GLI2 and GLI3 but not the GLI3 repressor form. The competition between Rdx and Su(fu) appears to be rather finely balanced as either increasing the expression of rdx or reducing the expression of Su(fu) enhances the ability of CiCe2 to compete with full-length Ci. This function of protecting full-length Ci from Rdx presumably takes place in the nucleus, as this is where the Rdx protein primarily localizes (Roberto, 2022).

However, the functional relevance of rdx being an Hh target gene has been unclear. Zygotic loss of rdx in the embryo has no visible effect on segmental patterning of the cuticle and, unlike en, knockdown of rdx along the compartment boundary in the wing disc has little effect on wing patterning. Perhaps its role is to clear full-length Ci from cells that were once within the domain of Hh signaling and have moved outside the domain of Hh signaling. Perdurance of Rdx could target full-length Ci in the nucleus allowing the Ci repressor to shut off Hh target genes. This is the situation in the eye disc with the progression of the morphogenetic furrow. Cells that recently received high level Hh signaling and activated Ci must now downregulate Ci to allow proper differentiation of the ommatidia. Rdx appears to be important for this process, as loss of rdx leads to defects in the eye. A similar situation may exist in other tissues. Looking at the temporal regulation of ptc expression with UAS-TT, cells removed from the compartment boundary in the wing disc have lower levels of destabilized GFP relative to RFP and appear to be in the process of shutting off ptc. This distinction is lost following downregulation of rdx by RNAi (Roberto, 2022).

In the domain of modest level Hh signaling (in which dpp is expressed), both full-length Ci and Ci repressor must be present in some form of reciprocal gradients. In this domain, enhancers with perfect Ci consensus binding sites are silent due to binding of Ci repressor. The dpp enhancer with imperfect Ci binding sites is expressed, and for it to be completely active, full-length Ci must be bound. Why is full-length Ci able to better compete with Ci repressor for the imperfect binding sites? Full-length Ci and the Ci repressor share the same DNA binding domain, and it would be expected that the repressor would outcompete full-length Ci for binding to target sites because the repressor is primarily nuclear, whereas full-length Ci is primarily cytoplasmic, even in the presence of Hh signaling, due to a strong nuclear export signal (NES). I suggest that cooperativity between Ci repressor proteins at perfect Ci binding sites can account for this distinction. Another potential mechanism for preferentially recruiting full-length Ci to imperfect binding sites might be suggested by the different protein interactions observed with full-length Ci and CiCe2. Full-length Ci enters the nucleus with Su(fu) while the Ci repressor is not bound to Su(fu). In addition, the Ci repressor is missing the CBP binding site. As a consequence, full-length Ci could engage in protein-protein interactions with other transcription factors that are not available to the Ci repressor. This added affinity to other proteins within the enhanceosome could allow the preferential recruitment of full-length Ci to enhancers with imperfect Ci binding sites. Differential protein-protein interactions may also explain why full-length Ci is still able to activate ptc-lacZ expression along the compartment boundary in ciCe2/+ heterozygotes (Fig. S4) but not the artificial enhancer 4bs-lacZ. The ptc-lacZ enhancer is a bona fide Drosophila enhancer and is likely to recruit a constellation of proteins that could interact with full-length Ci, whereas protein-protein interactions are likely to be much less robust at 4bs (Roberto, 2022).

In conclusion, these results highlight the complexity of Hh signal transduction and its modulation. Expressing en in the anterior compartment of the wing pouch modulates the Hh gradient, whereas Su(fu) has a surprising positive role in the pathway, acting to partially protect full-length Ci from the E-3 ligase Rdx that Ci activates (Roberto, 2022).


cDNA clone length - 1863

Bases in 5' UTR - 295

Bases in 3' UTR - 581


Amino Acids - 468

Structural Domains

Suppressor of fused has been identified in Drosophila as a semi-dominant suppressor of the putative serine/threonine kinase encoded by the segment polarity gene fused . The amorphic Su(fu) mutation is viable, shows a maternal effect and displays no phenotype by itself. Su(fu) mutations are often found associated to karmoisin (kar) mutations but two complementation groups can be clearly identified. By using a differential hybridization screening method, the Su(fu) region has been cloned and chromosomal rearrangements associated with Su(fu) mutations have been identifed. Two classes of cDNAs with similar developmental patterns, including a maternal contribution, are detectable in the region. Transformation experiments clearly assign the Su(fu)+ function to one of these transcription units while the other one can be most likely assigned to the kar+ function. Surprisingly the 5' end of the KAR mRNA maps within the 3' untranslated region of the Su(fu) transcribed sequence. The Su(fu) gene encodes a 53-kD protein, which contains a PEST sequence and shows no significant homologies with known proteins. Genetic analysis shows that proper development requires a fine tuning of the genetic doses of fu and Su(fu) both maternally and zygotically. These results, together with previous genetic and molecular data, suggest that fused and Suppressor of fused could act through a competitive posttransductional modification of a common target in the Hedgehog signaling pathway (Pham, 1995).

Suppressor of fused: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 12 July 2023

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