A scintillation proximity-assay for the Raf/MEK/ERK Kinase cascade: High-throughput screening and identification of selective enzyme inhibitors

We have developed a quantitative scintillation proximity assay (SPA) that r eproduces the Raf/MEK/ERK signal transduction pathway. The components of th is assay include human cRaf1, MEK1, and ERK2 and a biotinylated peptide sub strate for ERK2. cRaf1 was expressed as a his-tagged protein in insect cell s in an active form. MEK1 and ERK2 were expressed in Escherichia coli as gl utathione S-transferase (GST)-fusion proteins in their inactive forms. ERK2 was removed from the GST portion: of the fusion protein by cleavage with t hrombin protease. When the purified components are incubated together, cRaf -1 phosphorylates and activates MEK1, MEK1 phosphorylates and activates ERK 2, and ERK2 phosphorylates the peptide, biotin-AAATGPLSPGPFA. Phosphorylati on of the peptide using [gamma-P-33]ATP is detected following binding to st reptavidin-coated SPA beads,The assay detects : inhibitors of cRaf1, MEK1, or ERK2, and has been used to screen large numbers of compounds, The specif ic target of inhibition was subsequently identified with secondary assays d escribed herein. (C) 1999 Academic Press.