Real-time protein kinase assay.

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.

Cell-based high-throughput screening assay system for monitoring G protein-coupled receptor activation using beta-galactosidase enzyme complementation technology.

A novel cell-based functional assay to directly monitor G protein-coupled receptor (GPCR) activation in a high-throughput format, based on a common GPCR regulation mechanism, the interaction between beta-arrestin and ligand-activated GPCR, is described. A protein-protein interaction technology, the InteraX trade mark system, uses a pair of inactive beta-galactosidase (beta-gal) deletion mutants as fusion partners to the protein targets of interest. To monitor GPCR activation, stable cell lines expressing both GPCR- and beta-arrestin-beta-gal fusion proteins are generated. Following ligand stimulation, beta-arrestin binds to the activated GPCR, and this interaction drives functional complementation of the beta-gal mutant fragments. GPCR activation is measured directly by quantitating restored beta-gal activity. The authors have validated this assay system with two functionally divergent GPCRs: the beta2-adrenergic amine receptor and the CXCR2 chemokine-binding receptor. Both receptors are activated or blocked with known agonists and antagonists in a dose-dependent manner. The beta2-adrenergic receptor cell line was screened with the LOPAC trade mark compound library to identify both agonists and antagonists, validating this system for high-throughput screening performance in a 96-well microplate format. Hit specificity was confirmed by quantitating the level of cAMP. This assay system has also been performed in a high-density (384-well) microplate format. This system provides a specific, sensitive, and robust methodology for studying and screening GPCR-mediated signaling pathways.