Using genetically engineered kinases to screen for novel protein kinase substrates: phosphorylation of kinase-associated substrates

Abstract

This protocol describes a method for detection of direct substrates of a protein kinase in cell lysates or fractions. The approach involves identification of kinase-associated substrates by immunoprecipitating a tagged form of the mutant kinase from transfected COS-1 cells and performing a kinase reaction by the addition of [γ-³²P]ATP analog. This technique has been used for the phosphorylation of Extracellular signal-Regulated Kinase 2 (ERK2) substrates; however, the methodology can be applied to other protein kinases as well.