Identifying specific kinase substrates through engineered kinases and ATP analogs.
Intracellular signaling by protein kinases controls many aspects of cellular biochemistry and physiology. Determining the direct substrates of protein kinases is important in understanding how these signaling enzymes exert their effect on cellular functions. One of the recent developments in this area takes advantage of the similarity in the ATP binding domains of protein kinases, where a few conserved amino acids containing large side chains come in close contact with the N-6 position of bound ATP. Mutation of one or more of these residues generates a "pocket" in the ATP binding site that allows the mutant kinase, but not other cellular kinases, to utilize analogs of ATP with bulky substituents synthesized onto the N-6 position. The use of such a mutated kinase and radiolabeled ATP analogs allows for the specific labeling of direct substrates of the kinase within a mixture of cellular proteins. We have recently reported the generation of "pocket" mutants of extracellular regulated kinase 2 (ERK2) and their use in the identification of two novel substrates of ERK2. In this report, we discuss the generation and characterization of ERK2 mutants that utilize analogs of ATP and describe the methodology used to identify ERK2-associated substrates. We also describe the direct labeling of ERK2 substrates in cell lysates. These methodologies can be adapted for use with other protein kinases to increase the understanding of intracellular signal transduction.
Department of Microbiology and Cancer Center, University of Virginia, Health Science Center, Charlottesville, VA 22908, USA.
N Vinay
NV
Kumar
Scott T
ST
Eblen
Michael J
MJ
Weber
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D019556:COS Cells; D002474:Cell-Free System / Q000737:chemistry / Q000378:metabolism; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D007553:Isotope Labeling; D013058:Mass Spectrometry; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D004676:Myelin Basic Protein / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D015202:Protein Engineering / Q000379:methods*; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000032:analysis / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D013379:Substrate Specificity; D014157:Transcription Factors / Q000378:metabolism; D014170:Transformation, Genetic; D044767:Ubiquitin-Protein Ligases / Q000032:analysis / Q000378:metabolism; D050807:ets-Domain Protein Elk-1
D004268:DNA-Binding Proteins; D004676:Myelin Basic Protein; D011518:Proto-Oncogene Proteins; D014157:Transcription Factors; D050807:ets-Domain Protein Elk-1; D004815:Epidermal Growth Factor; D000255:Adenosine Triphosphate; C117617:UBR5 protein, human; D044767:Ubiquitin-Protein Ligases; D011494:Protein Kinases; D038744:Ribosomal Protein S6 Kinases, 90-kDa; C069330:ribosomal protein S6 kinase, 90kDa, polypeptide 3; D019950:Mitogen-Activated Protein Kinase 1