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Chemical Rescue of a Mutant Enzyme in Living Cells
Yingfeng Qiao,1
Henrik Molina,2
Akhilesh Pandey,2
Jin Zhang,1
Philip A. Cole1*
Abstract:
The restoration of catalytic activity to mutant enzymes by smallmolecules is well established for in vitro systems. Here, weshow that the protein tyrosine kinase Src arginine-388alanine(R388A) mutant can be rescued in live cells with the use ofthe small molecule imidazole. Cellular rescue of a viral Srchomolog was rapid and reversible and conferred predicted oncogenicproperties. Using chemical rescue in combination with mass spectrometry,we confirmed six known Src kinase substrates and identifiedseveral new protein targets. Chemical rescue data suggest thatcellular Src is active under basal conditions. Rescue of R388Acellular Src provided insights into the mitogen-activated proteinkinase pathway. This chemical rescue approach will likely havemany applications in cell signaling.
1 Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. 2 Department of Biological Chemistry and the Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
* To whom correspondence should be addressed. E-mail: pcole{at}jhmi.edu
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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. 
