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

• DOI: 10.1073/pnas.94.8.3565
• Publication Date: 2002-07-26T14:40:52Z
• Authors: Kavita Shah; Yi Liu; Carl Deirmengian; Kevan M. Shokat
• Keywords: Male; 0301 basic medicine; 0303 health sciences; Nucleotides; Molecular Sequence Data; Fibroblasts; Cell Transformation, Viral; Protein Engineering; Oncogene Protein pp60(v-src); Substrate Specificity; 3. Good health; Mice, Inbred C57BL; Mice; 03 medical and health sciences; Avian Sarcoma Viruses; Mutation; Animals; Female; Amino Acid Sequence; Phosphorylation

Protein phosphorylation plays a central role in controlling many diverse signal transduction pathways in all cells. Novel protein kinases are identified at a rapid rate using homology cloning methods and genetic screens or selections; however identification of the direct substrates of kinases has proven elusive to genetic methods because of the tremendous redundancy and overlapping of substrate specificities among protein kinases. We describe the development of a protein engineering-based method to identify the direct substrates of the prototypical protein 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 show that the engineered v-Src kinase displayed catalytic efficiency with the ATP analog, N 6 -(cyclopentyl) ATP, which is similar to the wild-type kinase catalytic efficiency with ATP itself. However, the N 6 -(cyclopentyl) ATP analog was not accepted by the wild-type kinase. Furthermore, the engineered v-Src exhibited the same protein target specificity as wild-type v-Src despite the proximity of the reengineered nucleotide binding site to the phosphoacceptor binding site. The successful engineering of v-Src’s active site to accept a unique nucleotide analog provides a unique handle by which the direct substrates of one kinase (v-Src) can be traced in the presence of any number of cellular kinases.