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 Weel and Myt1 kinases. Our technique allows one to monitor the bisphosphorylation 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 Weel and Myt1 activity. None of the inhibitors inhibited Weel, but both staurosporine and K-252a inhibited Myt1, with IC50 values of 9.2 +/- 3.6 and 4.0 +/- 1.3 muM, respectively. (C) 2003 Elsevier Science (USA). All rights reserved.