Using Genetically Engineered Kinases to Screen for Novel Protein Kinase Substrates: Phosphorylation of Kinase-Associated Substrates
This protocol was adapted from “Using Genetically Engineered Kinases to Screen for Novel Protein Kinase Substrates,” Chapter 24, in Protein-Protein Interactions (ed. Golemis and Adams). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005.INTRODUCTION
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.
