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.
Subject(s)
Imidazoles/metabolism , Proto-Oncogene Proteins pp60(c-src)/genetics , Proto-Oncogene Proteins pp60(c-src)/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Animals , Cell Line , Cell Transformation, Neoplastic , Fluorescence Resonance Energy Transfer , Gene Expression Profiling , Gene Expression Regulation , Growth Substances/metabolism , Growth Substances/pharmacology , Humans , Imidazoles/pharmacology , Kinetics , Mice , Mitogen-Activated Protein Kinases/metabolism , Mutation , Nuclear Proteins/metabolism , Oligonucleotide Array Sequence Analysis , Phenotype , Phosphorylation , Phosphotyrosine/metabolism , Proto-Oncogene Proteins/metabolism , Recombinant Proteins/metabolism , Transfection , src Homology DomainsABSTRACT
Csk and Src are two protein tyrosine kinases with similar amino acid sequences but very different structures and functions. Csk catalyzes a tail phosphorylation reaction on Src and thereby restrains Src's activity and oncogenic potential. Comparative analysis of the domain interactions in these enzymes provides a lesson in signalling diversity and mechanisms of enzyme regulation. The molecular basis of the specificity of Csk targeting the Src tail appears to involve both local and long-range interactions and illustrates the complexity of selective targeting in post-translational modification.
Subject(s)
Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins pp60(c-src)/metabolism , Animals , CSK Tyrosine-Protein Kinase , Humans , Protein Structure, Tertiary , Protein-Tyrosine Kinases/chemistry , Proto-Oncogene Proteins pp60(c-src)/genetics , Substrate Specificity , src-Family KinasesABSTRACT
Protein phosphorylation catalyzed by protein kinases plays a critical role in cellular signaling. Here we review several chemical approaches to understanding protein kinases and the consequences of protein phosphorylation. We discuss the design of bisubstrate analogue inhibitors based on a dissociative transition state, the development of reagents for cross-linking protein kinases with their substrates, the chemical rescue of mutant protein tyrosine kinases, and the application of expressed protein ligation to understanding protein phosphorylation.
