荧光素酶
蛋白质片段互补分析
自噬相关蛋白13
细胞生物学
FKBP公司
磷酸化
蛋白激酶A
化学
分子生物学
生物
蛋白质磷酸化
生物化学
互补
转染
突变体
基因
作者
Gary D. Luker,Madeline Smith,Gary D. Luker,Seth T. Gammon,Helen Piwnica‐Worms,David Piwnica‐Worms
标识
DOI:10.1073/pnas.0404041101
摘要
Signaling pathways regulating proliferation, differentiation, and apoptosis are commonly mediated through protein–protein interactions as well as reversible phosphorylation of proteins. To facilitate the study of regulated protein–protein interactions in cells and living animals, we optimized firefly luciferase protein fragment complementation by screening incremental truncation libraries of N- and C-terminal fragments of luciferase. Fused to the rapamycin-binding domain (FRB) of the kinase mammalian target of rapamycin and FK506-binding protein 12 (FKBP), respectively, the optimized FRB-N-terminal luciferase fragment (NLuc)/C-terminal luciferase fragment (CLuc)-FKBP luciferase complementation imaging (LCI) pair reconstituted luciferase activity in cells upon single-site binding of rapamycin in an FK506-competitive manner. LCI was used in three independent applications. In mice bearing implants of cells expressing the FRB-NLuc/CLuc-FKBP LCI pair, dose- and time-dependent luciferase activity allowed target-specific pharmacodynamic analysis of rapamycin-induced protein–protein interactions in vivo . In cells expressing a Cdc25C-NLuc/CLuc-14-3-3ε LCI pair, drug-mediated disruption of cell cycle regulated protein–protein interactions was demonstrated with the protein kinase inhibitor UCN-01 in a phosphoserine-dependent manner. When applied to IFN-γ-dependent activation of Janus kinase/signal transducer and activator of transcription 1 (STAT1), LCI revealed, in the absence of ligand-induced phosphorylation, STAT1 proteins existing in live cells as preformed dimers. Thus, optimized LCI provides a platform for near real-time detection and characterization of regulated and small molecule-induced protein–protein interactions in intact cells and living animals and should enable a wide range of novel applications in drug discovery, chemical genetics, and proteomics research.
科研通智能强力驱动
Strongly Powered by AbleSci AI