Kinetics of regulated protein-protein interactions revealed with firefly luciferase complementation imaging in cells and living animals.
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-14-3-3epsilon 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-gamma-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.
Luker
Kathryn E
KE
Molecular Imaging Center, Mallinckrodt Institute of Radiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
Smith
Matthew C P
MC
Luker
Gary D
GD
Gammon
Seth T
ST
Piwnica-Worms
Helen
H
Piwnica-Worms
David
D
D048948:14-3-3 Proteins; D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D005816:Genetic Complementation Test; D006801:Humans; D007700:Kinetics; D008156:Luciferases / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D050794:STAT1 Transcription Factor; D017384:Sequence Deletion; D015398:Signal Transduction; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases; D022021:Tacrolimus Binding Proteins / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics / Q000378:metabolism; D020687:cdc25 Phosphatases / Q000235:genetics / Q000378:metabolism
D048948:14-3-3 Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D011506:Proteins; D011994:Recombinant Proteins; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; C494080:Stat1 protein, mouse; D015534:Trans-Activators; D008156:Luciferases; D014446:Tyrosine 3-Monooxygenase; D011494:Protein Kinases; C546842:MTOR protein, human; C546843:mTOR protein, mouse; D058570:TOR Serine-Threonine Kinases; C517009:CDC25C protein, human; C517010:Cdc25c protein, mouse; D020687:cdc25 Phosphatases; D022021:Tacrolimus Binding Proteins; D020123:Sirolimus