Journal Article

Kinetics of Regulated Protein-Protein Interactions Revealed with Firefly Luciferase Complementation Imaging in Cells and Living Animals

Luker, Kathryn E., Matthew C. P. Smith, Gammon, Seth T., Piwnica-Worms, David, Luker, Gary D., Piwnica-Worms, Helen, Cantley, Lewis C.

2004

Overview

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 secreening 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.

• Publisher: National Academy of Sciences,National Acad Sciences
• Subject: 14-3-3 Proteins / Animals / Biological Sciences / Bioluminescence / cdc25 Phosphatases - genetics / cdc25 Phosphatases - metabolism / Cell Cycle Proteins - genetics / Cell Cycle Proteins - metabolism / Cell Line / Cell lines / Cells / Dimerization / DNA-Binding Proteins - chemistry / DNA-Binding Proteins - genetics / DNA-Binding Proteins - metabolism / Drug interactions / Enzymes / Genetic Complementation Test / Genetics / HEK293 cells / Humans / Imaging / Insects / Kinetics / Luciferases - genetics / Luciferases - metabolism / Mice / Peptide Fragments - genetics / Peptide Fragments - metabolism / Phosphorylation / Photons / Plasmids / Protein Binding / Protein Kinases - genetics / Protein Kinases - metabolism / Proteins / Proteins - genetics / Proteins - metabolism / Recombinant Proteins - genetics / Recombinant Proteins - metabolism / Scientific imaging / Sequence Deletion / Signal Transduction / Sirolimus - pharmacology / STAT1 Transcription Factor / Tacrolimus Binding Proteins - genetics / Tacrolimus Binding Proteins - metabolism / TOR Serine-Threonine Kinases / Trans-Activators - chemistry / Trans-Activators - genetics / Trans-Activators - metabolism / Tyrosine 3-Monooxygenase - genetics / Tyrosine 3-Monooxygenase - metabolism