A fluorescence polarization assay for native protein substrates of kinases.
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.
Kristjánsdóttir
Kolbrún
K
Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.
Rudolph
Johannes
J
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
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