Engineering Src family protein kinases with unnatural nucleotide specificity

Background: Protein kinases play a central role in controlling diverse signal transduction pathways in all cells. The identification of the direct cellular substrates of individual protein kinases remains the key challenge in the field. Results: We describe the protein engineering of v-Src to produce a kinase which preferentially uses an ATP analog, N 6 -(benzyl) ATP, as a substrate, rather than the natural v-Src substrate, ATP. The sidechain of a single residue (Ile338) controls specificity for N 6 -substituted ATP analogs in the binding pocket of v-Src. Elimination of this sidechain by mutation to glycine produces a v-Src kinase which preferentially utilizes N 6 -(benzyl) ATP as a phosphodonor substrate. Our engineering strategy is generally applicable to the Src family kinases: mutation of the corresponding residue (Thr339 to glycine) in the Fyn kinase confers specificity for N 6 -(benzyl) ATP on Fyn. Condusions: The v-Src tyrosine kinase has been engineered to exhibit specificity for an unnatural ATP analog, N6-(benzyl) ATP, even in a cellular context where high concentrations of natural ATP are present (1–5 mM), where preferential use of the ATP analog by the mutant kinase is essential. The mutant v-Src transfers phosphate more efficiently with the designed unnatural analog than with ATP. As the identical mutation in the Src-family kinase Fyn confers on Fyn the ability to recognize the same unnatural ATP analog, our strategy is likely to be generally applicable to other protein kinases and may help to identify the direct targets of specific kinases.