An enzyme-linked immunosorbent assay for the Raf/MEK1/MAPK signaling cascade

Anal Biochem. 2001 Jul 1;294(1):48-54. doi: 10.1006/abio.2001.5151.

Abstract

The Ras-MAPK signaling cascade transmits mitogenic stimuli from growth factor receptors and activated Ras to the cell nucleus. Inappropriate Ras activation is associated with approximately 30% of all human cancers. The kinase components of the Ras-MAPK signaling cascade are attractive targets for pharmaceutical intervention. Therefore, we have developed a high-throughput, nonradioactive ELISA method to monitor Raf and MEK1 kinase activity. In this assay system activated Raf phosphorylates and activates MEK1, which in turn phosphorylates MAPK. Antibodies that specifically detect phosphorylated MAPK (vs. nonphosphorylated MAPK) made enzyme-linked immunosorbent assay (ELISA) development possible. This assay detects inhibitors of Raf and/or MEK1 and has been used to screen large numbers of random compounds. The specific target of inhibition in the Raf/MEK1/MAPK ELISA can be subsequently identified by secondary assays which directly measure Raf phosphorylation of MEK1 or MEK1 phosphorylation of MAPK.

MeSH terms

  • Enzyme-Linked Immunosorbent Assay / methods*
  • HeLa Cells
  • Humans
  • MAP Kinase Kinase 1
  • MAP Kinase Signaling System*
  • Mitogen-Activated Protein Kinase Kinases / metabolism*
  • Mitogen-Activated Protein Kinases / metabolism*
  • Phosphorylation
  • Protein-Serine-Threonine Kinases / metabolism*
  • Proto-Oncogene Proteins c-raf / metabolism*

Substances

  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-raf
  • Mitogen-Activated Protein Kinases
  • MAP Kinase Kinase 1
  • MAP2K1 protein, human
  • Mitogen-Activated Protein Kinase Kinases