USP8 Promotes Smoothened Signaling by Preventing Its Ubiquitination and Changing Its Subcellular Localization

Abstract

Hedgehog regulates the activity of its signal transducer Smoothened by enhancing its interaction with the deubiquitinase USP8, thereby promoting Smoothened translocation to the cell surface and so enhancing Hh signaling.

Abstract

The seven transmembrane protein Smoothened (Smo) is a critical component of the Hedgehog (Hh) signaling pathway and is regulated by phosphorylation, dimerization, and cell-surface accumulation upon Hh stimulation. However, it is not clear how Hh regulates Smo accumulation on the cell surface or how Hh regulates the intracellular trafficking of Smo. In addition, little is known about whether ubiquitination is involved in Smo regulation. In this study, we demonstrate that Smo is multi-monoubiquitinated and that Smo ubiquitination is inhibited by Hh and by phosphorylation. Using an in vivo RNAi screen, we identified ubiquitin-specific protease 8 (USP8) as a deubiquitinase that down-regulates Smo ubiquitination. Inactivation of USP8 increases Smo ubiquitination and attenuates Hh-induced Smo accumulation, leading to decreased Hh signaling activity. Moreover, overexpression of USP8 prevents Smo ubiquitination and elevates Smo accumulation, leading to increased Hh signaling activity. Mechanistically, we show that Hh promotes the interaction of USP8 with Smo aa625–753, which covers the three PKA and CK1 phosphorylation clusters. Finally, USP8 promotes the accumulation of Smo at the cell surface and prevents localization to the early endosomes, presumably by deubiquitinating Smo. Our studies identify USP8 as a positive regulator in Hh signaling by down-regulating Smo ubiquitination and thereby mediating Smo intracellular trafficking.

Author Summary

The Hedgehog (Hh) signaling pathway is well known for its role in directing processes such as cell growth, proliferation, and differentiation during embryogenesis. The signal initiated by Hh binding to its receptor, Patched, is transduced by another protein called Smoothened (Smo), which moves from membranes inside the cell to accumulate on the cell surface when Hh binds. This accumulation of Smo on the cell surface is thought to play a central role in maintaining Hh signaling. In this study, we investigated how Hh controls the stability and movement of Smo inside the cell. We found that Smo is modified by addition of a small protein called ubiquitin (Ub), and that Hh regulates the ubiquitination of Smo. We identified an enzyme called USP8 that can remove the ubiquitin modification from Smo, thereby enhancing its signaling activity. Furthermore we show that Hh can enhance the interaction between Smo and USP8. Finally, we discovered that USP8 promotes the movement of Smo from inside the cell to the cell surface. We conclude that Hh promotes the deubiquitination of Smo by USP8, resulting in the relocation of Smo to the cell surface where it enhances Hh signaling.