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Analysis of Smoothened Phosphorylation and Activation in Cultured Cells and Wing Discs of Drosophila

  • Protocol
Hedgehog Signaling Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1322))

Abstract

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 finger 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, fluorescence 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.

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Acknowledgment

We thank the members in Dr. Jia’s Lab for their efforts to this protocol. This work was supported by grant from the National Institutes of Health (GM079684) to J. Jia.

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Authors and Affiliations

  1. Department of Molecular and Cellular Biochemistry, Markey Cancer Center, University of Kentucky, 741 S. Limestone, BBSRB 373, Lexington, KY, 40536-0509, USA

    Kai Jiang & Jianhang Jia

Authors
  1. Kai Jiang
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  2. Jianhang Jia
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Corresponding author

Correspondence to Jianhang Jia .

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Editors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania, USA

    Natalia A. Riobo

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Jiang, K., Jia, J. (2015). Analysis of Smoothened Phosphorylation and Activation in Cultured Cells and Wing Discs of Drosophila . In: Riobo, N. (eds) Hedgehog Signaling Protocols. Methods in Molecular Biology, vol 1322. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2772-2_5

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  • DOI: https://doi.org/10.1007/978-1-4939-2772-2_5

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2771-5

  • Online ISBN: 978-1-4939-2772-2

  • eBook Packages: Springer Protocols

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