Cell-Based Assays for Smoothened Ubiquitination and Sumoylation

Yuhong Han, Jin Jiang

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

The Hedgehog (Hh) family of secreted proteins governs embryonic development and adult tissue homeostasis by regulating the abundance, localization, and activity of the GPCR family protein Smoothened (Smo). Smo trafficking and subcellular accumulation are controlled by multiple posttranslational modifications (PTMs) including phosphorylation, ubiquitination, and sumoylation, which appears to be conserved from Drosophila to mammals. Smo ubiquitination is dynamically regulated by E3 ubiquitin ligases and deubiquitinases (dubs) and is opposed by Hh signaling. By contrast, Smo sumoylation is stimulated by Hh, which counteracts Smo ubiquitination by recruiting the dub USP8. We describe cell-base assays for Smo ubiquitination and its regulation by Hh and the E3 ligases in Drosophila. We also describe assays for Smo sumoylation in both Drosophila and mammalian cultured cells.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages139-147
Number of pages9
DOIs
StatePublished - 2022

Publication series

NameMethods in Molecular Biology
Volume2374
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • Cul4
  • DDB1
  • Hedgehog
  • Nedd4
  • Smo
  • Smurf
  • Sumoylation
  • USP8
  • Ubiquitination
  • Ulp1

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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