CSH Protocols’ April Issue features a set of protocols from Michael Weber and colleagues at the University of Virginia detailing methods for using genetically engineered kinases to screen for novel protein kinase substrates. Determining the direct substrate of a protein kinase is important for understanding its function in vivo.

The methods described here (also here, here and here) take advantage of conserved residues in the ATP-binding domain of protein kinases. These conserved residues contain large side chains, and mutating them to alanine or glycine creates a “pocket” in the ATP-binding site. This allows the newly generated mutant to utilize ATP analogs with bulky substituents added on. Using a mutated kinase and radiolabeled ATP, one can run a kinase reaction which will result in specific labeling of direct substrates of the mutant kinase. The substrates can then be identified by mass spec.