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Cell-Based Assays for Smoothened Ubiquitination and Sumoylation
1Department of Molecular Biology, UT Southwestern Medical Center at Dallas, Dallas, TX, USA.
Methods in Molecular Biology (Clifton, N.J.)
|September 25, 2021
Summary
Hedgehog signaling regulates Smoothened (Smo) protein through ubiquitination and sumoylation. New assays track these posttranslational modifications, revealing how Hedgehog (Hh) signaling impacts Smo protein regulation.
Area of Science:
- Molecular Biology
- Developmental Biology
- Cell Biology
Background:
- The Hedgehog (Hh) signaling pathway is crucial for embryonic development and tissue maintenance.
- The G protein-coupled receptor Smoothened (Smo) is a key mediator of Hh signaling.
- Smo's activity is modulated by posttranslational modifications (PTMs) like ubiquitination and sumoylation, conserved across species.
Purpose of the Study:
- To develop and describe cell-based assays for monitoring Smo ubiquitination and sumoylation.
- To investigate the regulation of Smo ubiquitination and sumoylation by Hh signaling and associated enzymes.
Main Methods:
- Development of cell-based assays in Drosophila to measure Smo ubiquitination.
- Establishment of assays in both Drosophila and mammalian cells to assess Smo sumoylation.
- Analysis of the roles of E3 ubiquitin ligases and deubiquitinases (Dubs) in regulating Smo ubiquitination.
Main Results:
- Smo ubiquitination is dynamically regulated by E3 ligases and Dubs, and is inhibited by Hh signaling.
- Smo sumoylation is promoted by Hh signaling, which involves the deubiquitinase USP8.
- The developed assays allow for the study of Smo PTMs and their regulation.
Conclusions:
- Hedgehog signaling differentially regulates Smo ubiquitination and sumoylation.
- These opposing modifications, controlled by specific enzymes, are critical for Smo protein homeostasis and Hh pathway activity.
- The new assays provide valuable tools for dissecting Smo regulation in both developmental and cellular contexts.
