Kinetics of regulated protein-protein interactions revealed with firefly luciferase complementation imaging in cells and living animals

Proc Natl Acad Sci U S A. 2004 Aug 17;101(33):12288-93. doi: 10.1073/pnas.0404041101. Epub 2004 Jul 29.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 14-3-3 Proteins
  • Animals
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism
  • Cell Line
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Dimerization
  • Genetic Complementation Test
  • Humans
  • Kinetics
  • Luciferases / genetics*
  • Luciferases / metabolism*
  • Mice
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Protein Binding
  • Protein Kinases / genetics
  • Protein Kinases / metabolism
  • Proteins / genetics*
  • Proteins / metabolism*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • STAT1 Transcription Factor
  • Sequence Deletion
  • Signal Transduction
  • Sirolimus / pharmacology
  • TOR Serine-Threonine Kinases
  • Tacrolimus Binding Proteins / genetics
  • Tacrolimus Binding Proteins / metabolism
  • Trans-Activators / chemistry
  • Trans-Activators / genetics
  • Trans-Activators / metabolism
  • Tyrosine 3-Monooxygenase / genetics
  • Tyrosine 3-Monooxygenase / metabolism
  • cdc25 Phosphatases / genetics
  • cdc25 Phosphatases / metabolism

Substances

  • 14-3-3 Proteins
  • Cell Cycle Proteins
  • DNA-Binding Proteins
  • Peptide Fragments
  • Proteins
  • Recombinant Proteins
  • STAT1 Transcription Factor
  • STAT1 protein, human
  • Stat1 protein, mouse
  • Trans-Activators
  • Luciferases
  • Tyrosine 3-Monooxygenase
  • Protein Kinases
  • MTOR protein, human
  • mTOR protein, mouse
  • TOR Serine-Threonine Kinases
  • CDC25C protein, human
  • Cdc25c protein, mouse
  • cdc25 Phosphatases
  • Tacrolimus Binding Proteins
  • Sirolimus