Characterization of Smoothened Phosphorylation and Activation

Jianhang Jia; Yuhong Han; Yong Suk Cho; Jin Jiang

doi:10.1007/978-1-0716-1701-4_11

Characterization of Smoothened Phosphorylation and Activation

Jianhang Jia, Yuhong Han, Yong Suk Cho, Jin Jiang

Research output: Chapter in Book/Report/Conference proceeding › Chapter › peer-review

1 Scopus citations

Abstract

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.

Original language	English
Title of host publication	Methods in Molecular Biology
Pages	121-137
Number of pages	17
DOIs	https://doi.org/10.1007/978-1-0716-1701-4_11
State	Published - 2022

Publication series

Name	Methods in Molecular Biology
Volume	2374
ISSN (Print)	1064-3745
ISSN (Electronic)	1940-6029

Bibliographical note

Publisher Copyright:
© 2022, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

Funding

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.

Funders	Funder number
National Institutes of Health (NIH)	R35GM118063
National Institute of General Medical Sciences	R35GM131807
Welch Foundation	I-1603

Keywords

CK1
CK2
GRK2
Hedgehog
PKA
Phosphorylation
Primary cilium
Smoothened
aPKC

ASJC Scopus subject areas

Molecular Biology
Genetics

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10.1007/978-1-0716-1701-4_11

Cite this

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abstract = "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.",

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author = "Jianhang Jia and Yuhong Han and Cho, {Yong Suk} and Jin Jiang",

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AB - 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.

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KW - CK2

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KW - Smoothened

KW - aPKC

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Characterization of Smoothened Phosphorylation and Activation

Abstract

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Bibliographical note

Funding

Keywords

ASJC Scopus subject areas

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