Engineering unnatural nucleotide specificity for Rous sarcoma virus tyrosine kinase to uniquely label its direct substrates

摘要: Protein phosphorylation plays a central role in controlling many diverse signal transduction pathways all cells. Novel protein kinases are identified at rapid rate using homology cloning methods and genetic screens or selections; however identification of the direct substrates has proven elusive to because tremendous redundancy overlapping substrate specificities among kinases. We describe development engineering-based method identify prototypical tyrosine kinase v-Src, which controls fibroblast transformation by Rous sarcoma virus. To differentiate v-Src from other substrates, we mutated ATP binding site such that engineered uniquely accepted an analog. show displayed catalytic efficiency with analog, N6-(cyclopentyl) ATP, is similar wild-type itself. However, analog was not kinase. Furthermore, exhibited same target specificity as despite proximity reengineered nucleotide phosphoacceptor site. The successful engineering v-Src’s active accept unique provides handle one (v-Src) can be traced presence any number cellular