Product List for "Using Genetically Engineered Kinases to Screen for Novel Protein Kinase Substrates" Protocol

The following Cold Spring Harbor Laboratory Press protocols are brought to you by BioSupplyNet in a partnership with Sigma-Aldrich. A list of products is available to use in each protocol.

Selected Protocol from "Protein-Protein Interactions", 2nd Edition, E. Golemis, P. Adam.

Using Genetically Engineered Kinases to Screen for Novel Protein Kinase Substrates

Scott T. Eblen, N. Vinay Kumar, and Michael J. Weber
Department of Microbiology and Cancer Center, University of Virginia Health System, Charlottesville, VA 22908

ABSTRACT
Protein kinase cascades regulate many aspects of cellular biochemistry and physiology. Determining the direct substrates of protein kinases is important in understanding how these signaling enzymes exert their effects on cellular processes. A recent development in this area takes advantage of the similarity in the ATP-binding domains of protein kinases. Conserved residues in the ATP-binding site contain large side chains that come into close contact with the N6 position of bound ATP. Mutation of one or more of these large residues to alanine or glycine generates a "pocket" in the ATP-binding site that allows the mutant kinase, but not the wild-type kinase or other cellular kinases, to utilize analogs of ATP with bulky substituents synthesized onto the N6 position. Kinase reactions performed with a mutated kinase and radiolabeled ATP analogs in a cellular lysate allow specific labeling of direct substrates of the mutant kinase, which can later be identified by mass spectrometry or other means. We have recently reported the generation of "pocket" mutants of extracellular regulated kinase 2 (ERK2) and the use of one such mutant to identify two novel substrates of ERK2 (Eblen et al. 2003). In this chapter, we discuss the generation and characterization of kinase mutants that utilize analogs of ATP, and describe the methods used to identify direct kinase substrates. These methods can be adapted for use with other protein kinases to increase the understanding of intracellular signal transduction.

Buy this book, Z719145