title: Detecting protein-protein interactions based on kinase-mediated growth induction of mammalian cells.
abstract: Detection of protein-protein interactions (PPIs) is important for understanding numerous processes in mammalian cells; however, existing PPI detection methods often give significant background signals. Here, we propose a novel PPI-detection method based on kinase-mediated growth induction of mammalian cells. In this method, target proteins are fused to the intracellular domain of c-kit (c-kit ICD) and expressed in interleukin-3-dependent mammalian cells. The PPI induces dimerization and activation of c-kit ICDs, which leads to cell growth in the absence of interleukin-3. Using this system, we successfully detected the ligand-dependent homo-interaction of FKBPF36V and hetero-interaction of FKBP and FRBT2098L, as well as the constitutive interaction between MDM2 and a known peptide inhibitor. Intriguingly, cells expressing high-affinity peptide chimeras are selected from the mixture of the cell populations dominantly expressing low-affinity peptide chimeras. These results indicate that this method can detect PPIs with low background levels and is suitable for peptide inhibitor screening.
authors.affiliation: Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
authors.forename: Satoru
authors.initials: S
authors.lastname: Mabe
authors.affiliation: Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
authors.forename: Teruyuki
authors.initials: T
authors.lastname: Nagamune
authors.affiliation: Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Kawahara
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D019281:Dimerization; D051379:Mice; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping / Q000379:methods*; D019009:Proto-Oncogene Proteins c-kit / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051736:Proto-Oncogene Proteins c-mdm2 / Q000037:antagonists & inhibitors / Q000378:metabolism; D016559:Tacrolimus / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D022021:Tacrolimus Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000378:metabolism
chemical_list: C463061:AP20187; D010455:Peptides; D016159:Tumor Suppressor Protein p53; D051736:Proto-Oncogene Proteins c-mdm2; D019009:Proto-Oncogene Proteins c-kit; D022021:Tacrolimus Binding Proteins; D016559:Tacrolimus