title: Analysis of Smoothened Phosphorylation and Activation in Cultured Cells and Wing Discs of Drosophila.
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.
authors.affiliation: Department of Molecular and Cellular Biochemistry, Markey Cancer Center, University of Kentucky, 741 S. Limestone, BBSRB 373, Lexington, KY, 40536-0509, USA.
authors.forename: Kai
authors.initials: K
authors.lastname: Jiang
authors.forename: Jianhang
authors.initials: J
authors.lastname: Jia
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004330:Drosophila; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D018390:Gene Targeting; D017930:Genes, Reporter; D009154:Mutation; D010766:Phosphorylation; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D000072086:Smoothened Receptor; D014921:Wings, Animal / Q000254:growth & development / Q000378:metabolism*
chemical_list: D029721:Drosophila Proteins; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D000072086:Smoothened Receptor; C100664:smo protein, Drosophila