title: A Chemically Inducible Helper Module for Detecting Protein-Protein Interactions with Tunable Sensitivity Based on KIPPIS.
abstract: As protein-protein interactions (PPIs) play essential roles in regulating their functional consequences in cells, methods to detect PPIs in living cells are desired for correct understanding of intracellular PPIs and pharmaceutical development therefrom. Here we demonstrate a c-kit-based PPI screening (KIPPIS) system in combination with a chemically inducible helper module for detecting PPIs in living mammalian cells. In this system, a mutant of FK506-binding protein 12 (FKBPF36 V) is fused with a protein of interest and the intracellular domain of a receptor tyrosine kinase c-kit. Constitutive expression of two fusion proteins with interacting proteins of interest in interleukin-3 (IL-3)-dependent cells results in dimerization and subsequent activation of the c-kit intracellular domains, which allows cell proliferation in a culture medium devoid of IL-3. A helper ligand, a small synthetic chemical that homodimerizes FKBPF36 V, assists the formation of stable complexes of the fusion proteins and serves as a tuner for sensitivity of the system. Using this system, two model PPIs were successfully detected on the basis of cell proliferation, which was featured by the helper-ligand- and PPI-dependent phosphorylation of the Src family kinases, a hallmark of the c-kit signaling. Notably, the inclusion of the helper module enabled PPI detection with tunable sensitivity. The helper-assisted KIPPIS allows us to configure various affinity thresholds by changing the concentration of the helper ligand, which may be applied to select affinity-matured variants using the advantage of cell proliferation.
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: Daiki
authors.initials: D
authors.lastname: Kashima
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: Raiji
authors.initials: R
authors.lastname: Kawade
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.identifier: 0000-0003-2301-6171
authors.initials: M
authors.lastname: Kawahara
mesh_terms: D000818:Animals; D001483:Base Sequence; D049109:Cell Proliferation / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D051379:Mice; D055503:Protein Multimerization / Q000187:drug effects; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics / Q000378:metabolism; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D020123:Sirolimus / Q000031:analogs & derivatives / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D016559:Tacrolimus / Q000031:analogs & derivatives / Q000494:pharmacology; D022061:Tacrolimus Binding Protein 1A / Q000235:genetics / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
chemical_list: C539629:AP 21967; C463061:AP20187; D008024:Ligands; D011993:Recombinant Fusion Proteins; C495901:TP53 protein, human; C000628667:Trp53 protein, mouse; D016159:Tumor Suppressor Protein p53; C497206:MDM2 protein, human; C497207:Mdm2 protein, mouse; D051736:Proto-Oncogene Proteins c-mdm2; D019009:Proto-Oncogene Proteins c-kit; D058570:TOR Serine-Threonine Kinases; D022061:Tacrolimus Binding Protein 1A; D020123:Sirolimus; D016559:Tacrolimus