Assays of protein kinases using exogenous substrates.
In studies of the regulation of specific biochemical events by reversible phosphorylation, assaying the protein kinases themselves can often lead to significant progress in understanding the mechanistic details of a system under study. This unit describes assays for a variety of protein kinases that require different conditions to detect and measure their activities--cyclic nucleotide-dependent kinases, protein kinase C and isoforms, casein kinases, Ca(2+)/calmodulin-dependent kinases, and tyrosine kinase. The unit is not meant to be a catalog of individual protein kinase assays; however, the general principles of these assays should apply to most if not all known protein kinases. In-gel assays for specific kinase activity are also described. Support protocols describe production of crude cell lysates as a source of enzyme activity, and methods of evaluating assays, including trichloroacetic acid (TCA) precipitation to measure incorporation of radioactivity, adsorption of the labeled material onto P81 phosphocellulose paper, or electrophoretic separation and autoradiography.
Carter
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The Salk Institute for Biological Studies, La Jolla, California, USA.
D000327:Adsorption; D000595:Amino Acid Sequence; D001681:Biological Assay / Q000379:methods*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D047388:Casein Kinases / Q000378:metabolism; D002364:Caseins / Q000378:metabolism; D002457:Cell Extracts; D011232:Chemical Precipitation; D017867:Cyclic Nucleotide-Regulated Protein Kinases / Q000378:metabolism; D004586:Electrophoresis; D007527:Isoenzymes / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry; D013379:Substrate Specificity; D014238:Trichloroacetic Acid
D002364:Caseins; D002457:Cell Extracts; D007527:Isoenzymes; D010455:Peptides; D011506:Proteins; D014238:Trichloroacetic Acid; D011494:Protein Kinases; D017867:Cyclic Nucleotide-Regulated Protein Kinases; D047388:Casein Kinases; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases