The Drosophila protein Shaggy (Sgg, also known as Zeste-white3, Zw3) and its vertebrate orthologue glycogen synthase kinase 3 (GSK3) are inhibitory components of the Wingless (Wg) and Wnt pathways. Here we show that Sgg is also a negative regulator in the Hedgehog (Hh) pathway. In Drosophila, Hh acts both by blocking the proteolytic processing of full-length Cubitus interruptus, Ci (Ci155), to generate a truncated repressor form (Ci75), and by stimulating the activity of accumulated Ci155 (refs 2-6). Loss of sgg gene function results in a cell-autonomous accumulation of high levels of Ci155 and the ectopic expression of Hh-responsive genes including decapentaplegic (dpp) and wg. Simultaneous removal of sgg and Suppressor of fused, Su(fu), results in wing duplications similar to those caused by ectopic Hh signalling. Ci is phosphorylated by GSK3 after a primed phosphorylation by protein kinase A (PKA), and mutating GSK3-phosphorylation sites in Ci blocks its processing and prevents the production of the repressor form. We propose that Sgg/GSK3 acts in conjunction with PKA to cause hyperphosphorylation of Ci, which targets it for proteolytic processing, and that Hh opposes Ci proteolysis by promoting its dephosphorylation.
The Hedgehog (Hh) family of secreted proteins controls many aspects of growth and patterning in animal development. In Drosophila, Hh acts by preventing the formation of a truncated repressor form of Cubitus interruptus (Ci) and stimulating the transcriptional activity of full-length Ci. Here, we provide evidence that Costal2 (Cos2) and Suppressor of Fused [Su(fu)] inhibit Ci by tethering it in the cytoplasm, whereas Hh induces nuclear translocaltion of Ci through Fused (Fu). We have identified a 125 amino acid domain in the C-terminal part of Ci that mediates response to Cos2 inhibition. We show that Cos2 binds Ci, prevents its nuclear import, and inhibits its activity via this domain. We also provide evidence that Su(fu) regulates Ci through two distinct mechanisms: (1) Su(fu) blocks Ci nuclear import through the N-terminal region of Ci , and (2) it inhibits the activity of Ci through a mechanism independent of Ci nuclear translocation. Finally, we show that Cos2 is required for transducing high levels of Hh signaling activity, and it does so by alleviating the blockage of Ci activity imposed by Su(fu).
Hedgehog (Hh) proteins govern animal development by regulating the Gli/Ci family of transcription factors. In Drosophila, Hh signaling blocks proteolytic processing of full-length Ci to generate a truncated repressor form. Ci processing requires sequential phosphorylation by PKA, GSK3, and a casein kinase I (CKI) family member(s). Here we show that Double-time (DBT)/CKIepsilon and CKIalpha act in conjunction to promote Ci processing. CKI phosphorylates Ci at three clusters of serine residues primed by PKA and GSK3 phosphorylation. CKI phosphorylation of Ci confers binding to the F-box protein Slimb/beta-TRCP, the substrate recognition component of the SCF(Slimb/beta-TRCP) ubiquitin ligase required for Ci processing. CKI phosphorylation sites act cooperatively to promote Ci processing in vivo. Substitution of Ci phosphorylation clusters with a canonical Slimb/beta-TRCP recognition motif in beta-catenin renders Slimb/beta-TRCP binding and Ci processing independent of CKI. We propose that phosphorylation of Ci by CKI creates multiple Slimb/beta-TRCP binding sites that act cooperatively to recruit SCF(Slimb/beta-TRCP).
Ubiquitin is thought to be a stress protein that plays an important role in protecting cells under stress conditions; however, its precise role is unclear. Ubiquitin expression level is controlled by the balance of ubiquitinating and deubiquitinating enzymes. To investigate the function of deubiquitinating enzymes on ischemia-induced neural cell apoptosis in vivo, we analyzed gracile axonal dystrophy (gad) mice with an exon deletion for ubiquitin carboxy terminal hydrolase-L1 (UCH-L1), a neuron-specific deubiquitinating enzyme. In wild-type mouse retina, light stimuli and ischemic retinal injury induced strong ubiquitin expression in the inner retina, and its expression pattern was similar to that of UCH-L1. On the other hand, gad mice showed reduced ubiquitin induction after light stimuli and ischemia, whereas expression levels of antiapoptotic (Bcl-2 and XIAP) and prosurvival (brain-derived neurotrophic factor) proteins that are normally degraded by an ubiquitin-proteasome pathway were significantly higher. Consistently, ischemia-induced caspase activity and neural cell apoptosis were suppressed ϳ70% in gad mice. These results demonstrate that UCH-L1 is involved in ubiquitin expression after stress stimuli, but excessive ubiquitin induction following ischemic injury may rather lead to neural cell apoptosis in vivo. The small 76 amino acid protein ubiquitin plays a critical role in many cellular processes, including cell cycle control, transcriptional regulation, and synaptic development.
Photoreceptor differentiation in the Drosophila eye disc progresses from posterior to anterior in a wave driven by the Hedgehog and Decapentaplegic signals. Cells mutant for the hyperplastic discs gene misexpress both of these signaling molecules in anterior regions of the disc, leading to premature photoreceptor differentiation and overgrowth of surrounding tissue. The two genes are independently regulated by hyperplastic discs; decapentaplegic can still be misexpressed in cells mutant for both hyperplastic discs and hedgehog, and a repressor form of the transcription factor Cubitus interruptus can block decapentaplegic misexpression but not hedgehog misexpression. Loss of hyperplastic discs causes the accumulation of full-length Cubitus interruptus protein, but not of Smoothened, in both the eye and wing discs. hyperplastic discs encodes a HECT domain E3 ubiquitin ligase that is likely to act by targeting Cubitus interruptus and an unknown activator of hedgehog expression for proteolysis.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.