An ATP analog-sensitive version of the tomato cell death suppressor protein kinase Adi3 for use in substrate identification

Adi3 is a protein kinase from tomato that functions as a cell death suppressor and its substrates are not well defined. As a step toward identifying Adi3 substrates we developed an ATP analog-sensitive version of Adi3 in which the ATP-binding pocket is mutated to allow use of bulky ATP analogs. Met385 was identified as the “gatekeeper” residue and the M385G mutation allows for the use of two bulky ATP analogs. Adi3^M385G can also specifically utilize N⁶-benzyl-ATP to phosphorylate a known substrate and provides a tool for identifying Adi3 substrates.