ENGINEERING UNNATURAL NUCLEOTIDE SPECIFICITY FOR ROUS-SARCOMA VIRUS TYROSINE KINASE TO UNIQUELY LABEL ITS DIRECT SUBSTRATES

Protein phosphorylation plays a central role in controlling many diver se signal transduction pathways in all cells. Novel protein kinases ar e identified at a rapid rate using homology cloning methods and geneti c screens or selections; however identification of the direct substrat es of kinases has proven elusive to genetic methods because of the tre mendous redundancy and overlapping of substrate specificities among pr otein kinases. We describe the development of a protein engineering-ba sed method to identify the direct substrates of the prototypical prote in tyrosine kinase v-Src, which controls fibroblast transformation by the Rous sarcoma virus. To differentiate the substrates of v-Src from all other kinase substrates, we mutated the ATP binding site of v-Src such that the engineered v-Src uniquely accepted an ATP analog. We sho w that the engineered v-Src kinase displayed catalytic efficiency with the ATP analog, N-6-(cyclopentyl) ATP, which is similar to the wild-t ype kinase catalytic efficiency with ATP itself. However, the N-6-(cyc lopentyl) ATP analog was not accepted by the wild-type kinase. Further more, the engineered v-Src exhibited the same protein target specifici ty as wild-type v-Src despite the proximity of the reengineered nucleo tide binding site to the phosphoacceptor binding site. The successful engineering of v-Src's active site to accept a unique nucleotide analo g provides a unique handle by which the direct substrates of one kinas e (v-Src) can be traced in the presence of any number of cellular kina ses.