Genetically Engineered Kinases

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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

Products Available for this Protocol
Protocol Material Description	Product #	Product Name	Add to Cart
Buffers, Solutions, and Reagents
Acetic acid	A9967	Acetic acid
Tris-buffered saline (TBS)	T6664	Tris Buffered Saline
NaCl	S3014	Sodium chloride
Water	W4502	Water
KCl	P9541	Potassium chloride
Tris-base	T6066	Trizma® base
Phosphate-buffered saline (PBS)	P5368	Phosphate buffered saline
KH2PO4	60353	Potassium phosphate dibasic
Sodium dodecyl sulfate (SDS)	L3771	Sodium dodecyl sulfate
Ponceau S solution	P7170	Ponceau S solution
Glycerol	G5516	Glycerol for molecular biology
Magnesium acetate	63052	Magnesium acetate solution, for molecular biology
Dithiothreitol (DTT)	D9779	DL-Dithiothreitol for molecular biology
β-Mercaptoethanol	M3148	2-Mercaptoethanol for molecular biology
Na2HPO4•2H2O	S9763	Sodium phosphate dibasic
2x Laemmli sample buffer	S3401	Sample Buffer, Laemmli 2× Concentrate
DMEM	M3942	MegaCell™ Dulbecco's Modified Eagle's Medium, liquid, sterile-filtered, cell culture tested
Fetal Bovine Serum	F2442	Fetal Bovine Serum sterile-filtered, cell culture tested, hybridoma tested
NaF	S7920	Sodium fluoride
EGTA	E3889	Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid
EDTA	E7889	Ethylenediaminetetraacetic acid disodium salt solution
Triton X-100	T8787	Triton® X-100, for molecular biology
Tetrasodium pyrophosphate	S6422	Sodium pyrophosphate tetrabasic decahydrate
Sodium vanadate	590088	Sodium metavanadate
Aprotinin	A3428	Aprotinin from bovine lung, lyophilized powder, 3-7 TIU/mg solid, cell culture tested
Leupeptin	L9783	Leupeptin hydrochloride
Phenylmethylsulfonyl fluoride (PMSF), fresh	P7626	Phenylmethanesulfonyl fluoride
HEPES (pH 7.4)	70216	HEPES Sodium salt buffer solution
ATP	A7699	Adenosine 5'-triphosphate disodium salt
BCA protein assay kit	BCA1	Bicinchoninic Acid Kit for Protein Determination for 200-1000 µg/ml protein
Plasmids
Expression plasmid of choice	Cloning & Expression	Expression plasmids
Cells and Antibodies
COS-1 African green monkey kidney epithelial cells	88031701	COS-1 Cell Line from African green monkey kidney, SV40 transformed
Antibodies to the epitope tag on the protein kinase of interest	Antibody Explorer	Antibodies
A phospho-specific antibody to a known substrate of your kinase	Antibody Explorer	Antibodies

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