title: Circular Permutation Probes for Illuminating Phosphorylation of Estrogen Receptor.
abstract: The present protocol demonstrates a new strategy for imaging ligand-triggered protein phosphorylation using circularly permutated luciferases (cpLuc): (1) a luciferase is first fragmented into two segments for creating new N- and C-terminal ends in the hydrophilic region, (2) the original N- and C-terminal ends are circularly permutated and linked via a GS linker, whereas the new ends made by fragmentation are correspondingly linked with two proteins of interest. When the new ends of the cpLuc are linked with the ligand-binding domain of estrogen receptor (ER LBD) and Src homology two domain of Src (SH2), the estrogen can trigger phosphorylation of the ER LBD and consequent intramolecular ER LBD-SH2 binding. This interaction triggers an approximation of the adjacent fragments of split-cpLuc recovering the enzyme activity. This probe design greatly improves signal-to-noise (S/N) ratios upon tracing weak protein-protein interactions (PPIs) in mammalian cells.
authors.affiliation: Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba, 305-8569, Japan. kimu-sb@aist.go.jp.
authors.forename: Sung-Bae
authors.initials: SB
authors.lastname: Kim
authors.affiliation: AIST Shikoku center, National Institute of Advanced Industrial Science and Technology (AIST), 2217-14 Hayashi-cho, Takamatsu, Kagawa, 761-0395, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Tao
mesh_terms: D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D007700:Kinetics; D008024:Ligands; D008163:Luminescent Measurements / Q000379:methods*; D015335:Molecular Probes / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism*
chemical_list: D008024:Ligands; D015335:Molecular Probes; D011960:Receptors, Estrogen
keywords: Bioluminescence; Circular permutation; Estrogen receptor; Luciferase; Phosphorylation