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

Anal Biochem. 1999 Mar 15;268(2):318-29. doi: 10.1006/abio.1998.3030.

Abstract

We have developed a quantitative scintillation proximity assay (SPA) that reproduces the Raf/MEK/ERK signal transduction pathway. The components of this assay include human cRaf1, MEK1, and ERK2 and a biotinylated peptide substrate for ERK2. cRaf1 was expressed as a his-tagged protein in insect cells in an active form. MEK1 and ERK2 were expressed in Escherichia coli as glutathione S-transferase (GST)-fusion proteins in their inactive forms. ERK2 was removed from the GST portion of the fusion protein by cleavage with thrombin protease. When the purified components are incubated together, cRaf-1 phosphorylates and activates MEK1, MEK1 phosphorylates and activates ERK2, and ERK2 phosphorylates the peptide, biotin-AAATGPLSPGPFA. Phosphorylation of the peptide using [gamma-33P]ATP is detected following binding to streptavidin-coated SPA beads. The assay detects inhibitors of cRaf1, MEK1, or ERK2, and has been used to screen large numbers of compounds. The specific target of inhibition was subsequently identified with secondary assays described herein.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Baculoviridae / genetics
  • Base Sequence
  • DNA Primers / genetics
  • Enzyme Inhibitors / pharmacology
  • Escherichia coli / genetics
  • Humans
  • In Vitro Techniques
  • Kinetics
  • MAP Kinase Kinase 1
  • Mitogen-Activated Protein Kinase 1 / antagonists & inhibitors
  • Mitogen-Activated Protein Kinase 1 / genetics
  • Mitogen-Activated Protein Kinase 1 / metabolism*
  • Mitogen-Activated Protein Kinase Kinases*
  • Peptides / chemistry
  • Peptides / metabolism
  • Protein-Serine-Threonine Kinases / antagonists & inhibitors
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism*
  • Protein-Tyrosine Kinases / antagonists & inhibitors
  • Protein-Tyrosine Kinases / genetics
  • Protein-Tyrosine Kinases / metabolism*
  • Proto-Oncogene Proteins c-raf / antagonists & inhibitors
  • Proto-Oncogene Proteins c-raf / genetics
  • Proto-Oncogene Proteins c-raf / metabolism*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Scintillation Counting / methods*
  • Signal Transduction
  • Substrate Specificity

Substances

  • DNA Primers
  • Enzyme Inhibitors
  • Peptides
  • Recombinant Fusion Proteins
  • Protein-Tyrosine Kinases
  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-raf
  • Mitogen-Activated Protein Kinase 1
  • MAP Kinase Kinase 1
  • MAP2K1 protein, human
  • Mitogen-Activated Protein Kinase Kinases