Src-Abl tyrosine kinase chimeras: Replacement of the adenine binding pocket of c-Abl with v-Src to swap nucleotide and inhibitor specificities

Engineered protein kinases with unnatural nucleotide specificity and inhibi tor sensitivity have been developed to trace kinase substrate targets. We f irst engineered unnatural nucleotide specificity into v-Src by mutating one residue, isoleucine 338, to alanine. This position is highly conserved amo ng all kinases in the sense that it is always occupied by either a large hy drophobic residue or threonine. Because of the conservation of this residue and the highly conserved fold of the kinase family, we have attempted to g eneralize the engineering of all kinases on the basis of our success with v -Src. Although many kinases can be similarly engineered using v-Src as a bl ueprint, we encountered one kinase, c-Abl, which when mutated, does not dis play the ability to accept unnatural ATP analogues. To overcome this failur e of the engineered c-Abl (T315A) to accept unnatural nucleotides, we devel oped a new strategy for introducing unnatural nucleotide specificity into k inases, We generated a chimeric kinase in which regions of the kinase domai n of c-Abl were swapped with the corresponding regions of v-Src (I338A). Sp ecifically, we engineered two chimeras in which the N-terminal lobe of the SH1 domain of c-Abl was swapped with that of v-Src. These kinase chimeras w ere found to have the same unnatural nucleotide specificity as that of v-Sr c (I338A), while retaining the peptide specificity of c-Abl. Thus, these ch imeric kinases are suitable for identifying the direct substrates of c-Abl. These engineered chimeric enzymes provide a new strategy for constructing kinases with tailor-made ligand binding properties.