Analysis of smoothened phosphorylation and activation in cultured cells and wing discs of drosophila

Kai Jiang, Jianhang Jia

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

1 Scopus citations


Smoothened (Smo) is essential for transduction of the Hedgehog (Hh) signal in both insects and vertebrates. Binding of Hh to Ptc-Ihog relieves the Patched (Ptc)-mediated inhibition of Smo, which allows Smo to activate the cubitus interruptus (Ci)/Gli family of zinc fi nger transcription factors and thereby induce the expression of Hh target genes, such as decapentaplegic (dpp), ptc, and engrailed (en). The activation of Smo appears to be one of the most important events in Hh signaling. Studies have shown that Hh induces cell surface/ciliary accumulation and phosphorylation of Smo by multiple kinases, including protein kinase A (PKA), casein kinase 1 (CK1), casein kinase 2 (CK2), G protein-coupled receptor kinase 2 (Gprk2), and atypical PKC (aPKC). Here, we describe the assays used to examine the activity of Smo in Hh signaling, including in vitro kinase, ptc -luciferase reporter assay, cell surface accumulation assay, fl uorescence resonance energy transfer (FRET) assay, and wing disc immunostaining. These assays are powerful tools to study Smo phosphorylation and activation, which have provided mechanistic insight into a better understanding the mechanisms of Smo regulation.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
Number of pages16
StatePublished - 2015

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745


  • Anti-SmoP antibody
  • Cell surface accumulation
  • Hedgehog
  • Phosphorylation
  • Smoothened
  • Wing disc immunostaining

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics


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