Anal. Chem. 2005, 77, 2043-2049
Real-Time Protein Kinase Assay Hongye Sun, Karen E. Low, Sam Woo, Richard L. Noble, Ronald J. Graham, Sonia S. Connaughton, Melissa A. Gee, and Linda G. Lee*
Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404
We report a novel, real-time fluorogenic kinase assay. The peptide substrates are synthesized with a fluorescent dye and a hydrocarbon tail. The substrate self-assembles into micelles, increasing the local concentration of the dye and quenching its fluorescence. Upon phosphorylation, the fluorescence intensity increases 4-6-fold due to micelle reorganization. Both dynamic light scattering data and cryoelectron microscope images show that the size and the shape of the phosphopeptide micelles are significantly different from micelles of substrate peptide. The system provides a robust fluorescence increase in a real-time protein kinase assay. Unlike other fluorogenic systems, the fluorophore may be distant from the serine, threonine, or tyrosine that is phosphorylated. Assays for several kinases, including PKA, PKC, p38, MAPKAP K2, akt, Erk1, and src-family kinases, have been developed. IC50 values of inhibitors for PKC βII determined with this technology are consistent with published values. The utility of this assay to high-throughput screening was demonstrated with Sigma’s LOPAC library, a collection of 640 compounds with known biological activities, and satisfactory results were obtained. Protein kinases play a key role in regulating protein function and are the second largest potential group of therapeutic targets.1-4 A total of 518 kinase genes have been identified, which corresponds to 1.7% of all human genes.5 To study and differentiate these enzymes, a simple, robust, real-time kinase assay for drug screening and mechanism studies is needed. Current kinase assays can be divided into three classes by the readout signals: radioactive detection of phosphorylated product; fluorescent or luminescent detection of product; or indirect measurement of ATP consumption, usually by luminescent detection. The scintillation proximity assay (SPA) developed by Amersham Bioscience is one example of a radioactive kinase assay. Kinase activity is measured by the incorporation of 33P-labeled ATP into a kinase substrate.6 The safety and cost of SPA are a * E-mail:
[email protected]. (1) Hunter, T. Cell 2000, 100, 1, 13-127. (2) Fletcher, L. Nat. Biotechnol. 2001, 19, 599-600. (3) Cohen, P. Nat. Rev. Drug Discovery 2002, 1, 309-315. (4) Noble, M. E. M.; Endicott, J. A.; Johnson, L. N. Science 2004, 303, 18001805. (5) Manning, G.; Whyte, D. B.; Martinez, R.; Hunter, T.; Sudarsanam, S. Science 2002, 298, 1912-1934. (6) Beveridge, M.; Park, Y. W.; Hermes, J.; Marenghi, A.; Brophy, G.; Santos, A. J. Biomol. Screening 2000, 5, 205-212. 10.1021/ac048280e CCC: $30.25 Published on Web 02/15/2005
© 2005 American Chemical Society
concern to customers, and alternative nonradioactive methods are desired. Fluorescent or luminescent kinase assays have been designed using phosphopeptide binding reagents, such as antibodies;7-10 phosphate chelators, such as metal ion complexes;11 metal ion coated particles;12 or fluorescent polymers13 as fluorescent donors or quenchers. The readout may be fluorescence, luminescence, or polarization. Antibody-based fluorescence kinase assays are especially useful for tyrosine kinases for which high-affinity antibodies are available. However, antibodies that bind phosphoserine and phosphothreonine are generally less available, which inhibits the development of antibody-based methods for the majority of known kinases. Assays that rely on chelation of a phosphate have the disadvantage that anionic compounds, such as ATP, phospholipid, and other carboxylate groups, compete with the phosphopeptide in binding the metal ions, limiting the assay to low ATP concentration and reducing the signal to background. A kinase assay based on indirect measurement of ATP consumption was recently reported.14 The ATP remaining in the reaction was detected by generation of light in a luciferase reaction. The limitations of this method include the narrow ATP dynamic range (
