Using genetically engineered kinases to screen for novel protein kinase substrates: identification of a mutant kinase/ATP analog pair

Abstract

This protocol describes a method for studying protein kinases and their substrates by generation of mutant kinase enzymes that can incorporate ATP analogs into the substrate. This method was used to identify substrates of extracellular signal-regulated kinase 2 (ERK2). Once mutations (called “pocket mutations”) have been generated in the kinase, the next step is to screen ATP analogs for their compatibility with the kinase mutant. This screening is best performed in a two-step process. The first step involves assaying the ability of an ATP analog to inhibit the incorporation of radioactive phosphate from normal [γ-³²P]ATP into a known substrate in an in vitro kinase reaction. Because certain analogs might be able to interact with the ATP-binding site of the kinase but be unable to be used by the kinase as an ATP source, it is also necessary to directly test the ability of the mutant kinase to phosphorylate a substrate with analog ATP. Doing this requires either radiolabeled ATP analogs or, preferably, a phospho-specific antibody to the phosphorylation site on the known substrate. (A phospho-specific antibody allows a large number of ATP analogs to be screened without the need for radioactivity.) The kinase/ATP analog pair that provides the best phosphorylation of the substrate is then used for future experiments. Only those analogs that cannot be used by the wild-type kinase and other cellular kinases are chosen.