Detecting protein–protein interactions based on kinase-mediated growth induction of mammalian cells

• DOI: 10.1038/srep06127
• Publication Date: 2014-08-19T09:04:48Z
• Authors: Satoru Mabe; Teruyuki Nagamune; Masahiro Kawahara
• Keywords: 0303 health sciences; Proto-Oncogene Proteins c-mdm2; Article; Tacrolimus; Cell Line; Tacrolimus Binding Proteins; Mice; Proto-Oncogene Proteins c-kit; 03 medical and health sciences; Amino Acid Substitution; Protein Interaction Mapping; Animals; Protein Interaction Domains and Motifs; Tumor Suppressor Protein p53; Peptides; Dimerization; Cell Proliferation

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.