title: Chemical rescue of a mutant enzyme in living cells.
abstract: The restoration of catalytic activity to mutant enzymes by small molecules is well established for in vitro systems. Here, we show that the protein tyrosine kinase Src arginine-388-->alanine (R388A) mutant can be rescued in live cells with the use of the small molecule imidazole. Cellular rescue of a viral Src homolog was rapid and reversible and conferred predicted oncogenic properties. Using chemical rescue in combination with mass spectrometry, we confirmed six known Src kinase substrates and identified several new protein targets. Chemical rescue data suggest that cellular Src is active under basal conditions. Rescue of R388A cellular Src provided insights into the mitogen-activated protein kinase pathway. This chemical rescue approach will likely have many applications in cell signaling.
authors.affiliation: Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: Yingfeng
authors.initials: Y
authors.lastname: Qiao
authors.forename: Henrik
authors.initials: H
authors.lastname: Molina
authors.forename: Akhilesh
authors.initials: A
authors.lastname: Pandey
authors.forename: Jin
authors.initials: J
authors.lastname: Zhang
authors.forename: Philip A
authors.initials: PA
authors.lastname: Cole
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D031541:Fluorescence Resonance Energy Transfer; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006133:Growth Substances / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007093:Imidazoles / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D010641:Phenotype; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D018909:src Homology Domains
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C082869:CRKL protein; D006133:Growth Substances; D007093:Imidazoles; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D019000:Phosphotyrosine; C029899:imidazole; D016392:Proto-Oncogene Proteins pp60(c-src); D020928:Mitogen-Activated Protein Kinases