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Abstract

The Ras, Raf, mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) signaling cascade is critically involved in cellular signaling with activating mutations in Ras and Raf present in many human tumors. Each constituent of this pathway is considered an important target for pharmaceutical intervention. The terminal kinase ERK is known to phosphorylate p90RSK among myriad substrates, yet robust plate-based high-throughput cellular assays monitoring such activity are not commercially available. In this study, we have utilized the Meso Scale Discovery platform to develop a plate-based assay to monitor the level of phosphorylation of p90RSK. This method is highly robust and can be used to evaluate a large number of inhibitors of ERK, MEK, or Raf in a variety of cellular backgrounds. Furthermore, this assay can be used to quantify the level of phospho-p90RSK in tumor lysates to function as a valuable pharmacodynamic readout.