title: A fluorescence polarization assay for native protein substrates of kinases.
abstract: Protein phosphorylation is the mediator of many important cellular processes of signal transduction and cell regulation. Phosphorylation often occurs on multiple sites within a single protein, whereby the results of individual phosphorylations are not well defined. This is partially due to the lack of tools for analyzing specific phosphorylation states in a quantitative manner. We have developed a high-throughput, rapid, and quantitative method for the determination of the phosphorylation status of peptides and, more importantly, native protein substrates of kinases using a competitive fluorescence-based approach. We have applied our method to measuring the phosphorylation activity of the Wee1 and Myt1 kinases. Our technique allows one to monitor the bis-phosphorylation status of the Cdk2 protein using an antibody specific for bis-phosphorylated Cdk2 and a fluorescently labeled bis-phosphorylated Cdk2 peptide. We have used this assay to screen a library of 16 general kinase inhibitors against Wee1 and Myt1 activity. None of the inhibitors inhibited Wee1, but both staurosporine and K-252a inhibited Myt1, with IC(50) values of 9.2+/-3.6 and 4.0+/-1.3 microM, respectively.
authors.affiliation: Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.
authors.forename: Kolbrún
authors.initials: K
authors.lastname: Kristjánsdóttir
authors.forename: Johannes
authors.initials: J
authors.lastname: Rudolph
mesh_terms: D000595:Amino Acid Sequence; D002227:Carbazoles / Q000494:pharmacology; D018797:Cell Cycle Proteins; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005454:Fluorescence Polarization / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D026121:Indole Alkaloids; D008565:Membrane Proteins; D009687:Nuclear Proteins; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: D002227:Carbazoles; D018797:Cell Cycle Proteins; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D026121:Indole Alkaloids; D008565:Membrane Proteins; D009687:Nuclear Proteins; D010455:Peptides; D047428:Protein Kinase Inhibitors; C049985:staurosporine aglycone; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; C499349:WEE1 protein, human; C490679:PKMYT1 protein, human; D017346:Protein Serine-Threonine Kinases; D019311:Staurosporine