The GPCR-family protein Smoothened (Smo) is an obligatory signal transducer of the Hedgehog (Hh) signaling pathway. Binding of Hh to its receptor Patched (Ptc) alleviates Ptc-mediated inhibition of Smo, allowing Smo to activate the Cubitus interruptus (Ci)/Gli family of zinc finger transcription factors. The activation of Smo is an early and crucial event in Hh signal transduction. 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/GRK2), and atypical PKC (aPKC). Here, we describe the assays used to examine the phosphorylation and activity of Smo, including in vitro kinase assay, phospho-specific antibodies, luciferase reporter assay, cell surface accumulation, and ciliary localization assays. These assays provide powerful tools to study Smo phosphorylation and activation, leading to mechanistic insight into Smo regulation.
|Title of host publication||Methods in Molecular Biology|
|Number of pages||17|
|State||Published - 2022|
|Name||Methods in Molecular Biology|
Bibliographical noteFunding Information:
We thank the members in Dr. Jia’s laboratory for detailed protocols. This work was supported by grants from the National Institutes of Health R35GM131807 to J. Jia and R35GM118063 to J. Jiang, and from Welch Foundation I-1603 to J.Jiang.
© 2022, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
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ASJC Scopus subject areas
- Molecular Biology