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: Precomputed BLAST | Entrez Gene

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
Summary:
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
Summary:
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
Summary:
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.
BIOLOGICAL OVERVIEW

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).


GENE STRUCTURE

cDNA clone length - 1863

Bases in 5' UTR - 295

Bases in 3' UTR - 581


PROTEIN STRUCTURE

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: 16 September 2000

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