title: Real-time protein kinase assay.
abstract: 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. IC(50) values of inhibitors for PKC betaII 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.
authors.affiliation: Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.
authors.forename: Hongye
authors.initials: H
authors.lastname: Sun
authors.forename: Karen E
authors.initials: KE
authors.lastname: Low
authors.forename: Sam
authors.initials: S
authors.lastname: Woo
authors.forename: Richard L
authors.initials: RL
authors.lastname: Noble
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Graham
authors.forename: Sonia S
authors.initials: SS
authors.lastname: Connaughton
authors.forename: Melissa A
authors.initials: MA
authors.lastname: Gee
authors.forename: Linda G
authors.initials: LG
authors.lastname: Lee
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D020650:Combinatorial Chemistry Techniques; D020285:Cryoelectron Microscopy; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005453:Fluorescence; D008823:Micelles; D010766:Phosphorylation; D011493:Protein Kinase C / Q000032:analysis / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000378:metabolism / Q000494:pharmacology; D011494:Protein Kinases / Q000032:analysis* / Q000378:metabolism; D015203:Reproducibility of Results; D012542:Scattering, Radiation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D017186:Titrimetry
chemical_list: D008823:Micelles; D047428:Protein Kinase Inhibitors; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D011493:Protein Kinase C