title: Hedgehog-regulated ubiquitination controls smoothened trafficking and cell surface expression in Drosophila.
abstract: Hedgehog transduces signal by promoting cell surface expression of the seven-transmembrane protein Smoothened (Smo) in Drosophila, but the underlying mechanism remains unknown. Here we demonstrate that Smo is downregulated by ubiquitin-mediated endocytosis and degradation, and that Hh increases Smo cell surface expression by inhibiting its ubiquitination. We find that Smo is ubiquitinated at multiple Lysine residues including those in its autoinhibitory domain (SAID), leading to endocytosis and degradation of Smo by both lysosome- and proteasome-dependent mechanisms. Hh inhibits Smo ubiquitination via PKA/CK1-mediated phosphorylation of SAID, leading to Smo cell surface accumulation. Inactivation of the ubiquitin activating enzyme Uba1 or perturbation of multiple components of the endocytic machinery leads to Smo accumulation and Hh pathway activation. In addition, we find that the non-visual β-arrestin Kurtz (Krz) interacts with Smo and acts in parallel with ubiquitination to downregulate Smo. Finally, we show that Smo ubiquitination is counteracted by the deubiquitinating enzyme UBPY/USP8. Gain and loss of UBPY lead to reciprocal changes in Smo cell surface expression. Taken together, our results suggest that ubiquitination plays a key role in the downregulation of Smo to keep Hh pathway activity off in the absence of the ligand, and that Hh-induced phosphorylation promotes Smo cell surface accumulation by inhibiting its ubiquitination, which contributes to Hh pathway activation.
authors.affiliation: Department of Developmental Biology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, United States of America.
authors.forename: Shuang
authors.initials: S
authors.lastname: Li
authors.forename: Yongbin
authors.initials: Y
authors.lastname: Chen
authors.forename: Qing
authors.initials: Q
authors.lastname: Shi
authors.forename: Tao
authors.initials: T
authors.lastname: Yue
authors.forename: Bing
authors.initials: B
authors.lastname: Wang
authors.forename: Jin
authors.initials: J
authors.lastname: Jiang
mesh_terms: D000818:Animals; D000954:Antigens, Surface / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D004330:Drosophila / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D029721:Drosophila Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D015870:Gene Expression; D020868:Gene Silencing; D053823:Hedgehog Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D021381:Protein Transport / Q000235:genetics / Q000502:physiology; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology; D000072086:Smoothened Receptor; D014018:Tissue Distribution; D044764:Ubiquitin-Activating Enzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D054875:Ubiquitination / Q000502:physiology*
chemical_list: D000954:Antigens, Surface; D029721:Drosophila Proteins; D053823:Hedgehog Proteins; D043562:Receptors, G-Protein-Coupled; D000072086:Smoothened Receptor; C100664:smo protein, Drosophila; C077245:hh protein, Drosophila; C542423:Uba1 protein, Drosophila; D044764:Ubiquitin-Activating Enzymes