Hedgehog-regulated ubiquitination controls smoothened trafficking and cell surface expression in Drosophila

PLoS Biol. 2012 Jan;10(1):e1001239. doi: 10.1371/journal.pbio.1001239. Epub 2012 Jan 10.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antigens, Surface / genetics
  • Antigens, Surface / metabolism
  • Cell Membrane / genetics
  • Cell Membrane / metabolism*
  • Cells, Cultured
  • Drosophila Proteins / antagonists & inhibitors
  • Drosophila Proteins / genetics
  • Drosophila Proteins / metabolism*
  • Drosophila Proteins / physiology*
  • Drosophila* / genetics
  • Drosophila* / metabolism
  • Drosophila* / physiology
  • Gene Expression
  • Gene Silencing
  • Hedgehog Proteins / genetics
  • Hedgehog Proteins / metabolism
  • Hedgehog Proteins / physiology*
  • Protein Transport / genetics
  • Protein Transport / physiology
  • Receptors, G-Protein-Coupled / metabolism*
  • Signal Transduction / genetics
  • Signal Transduction / physiology
  • Smoothened Receptor
  • Tissue Distribution
  • Ubiquitin-Activating Enzymes / antagonists & inhibitors
  • Ubiquitin-Activating Enzymes / genetics
  • Ubiquitin-Activating Enzymes / metabolism
  • Ubiquitin-Activating Enzymes / physiology
  • Ubiquitination / physiology*

Substances

  • Antigens, Surface
  • Drosophila Proteins
  • Hedgehog Proteins
  • Receptors, G-Protein-Coupled
  • Smoothened Receptor
  • smo protein, Drosophila
  • hh protein, Drosophila
  • Uba1 protein, Drosophila
  • Ubiquitin-Activating Enzymes