MEK1/2 (MAP/ERK) Cellular Phosphorylation Assay (intracellular kinase activity assay) for compound screening and profiling in intact cells
MEK (Map/Erk kinase) 1/2 are essential components of the mitogen-activated protein (MAP) kinase signal transduction pathway and act as a MAP kinase. The downstream targets of MEK1/2 are extracellular signal-regulated kinases (ERK) 1 and 2. MEK1/2 and ERK1/2 act as an integration point in multiple biochemical signaling pathways and are involved in cellular processes like cell cycle progression, proliferation, migration/adhesion, differentiation, transcription regulation, and development. Activation of ERK1/2 occurs via phosphorylation at Tyr204/187 and Thr202/185 by upstream kinases MEK1/2 and allows activated ERK1/2 to enter the nucleus. About 160 substrates of the protein-serin/threonine kinases ERK1/2 have been discovered, among them many nuclear transcription factors but also cytosolic proteins. In about one-third of human cancers, the activity of the Ras-Raf-MEK-ERK cascade is increased which provides interesting targets for therapeutic strategies.
MEK1: MAP2K1, MAPKK1, MKK1, PRKMK1
MEK2: MAP2K2, MAPKK2, MKK2, PRKMK2
The human pancreatic cancer cell line PANC-1 endogenously expresses high levels of ERK1/2. In response to inhibition of MEK1/2, phosphorylation of the downstream target ERK1/2 at Thr202/Tyr204 (ERK1) and Thr185/Tyr187 (ERK2) is reduced. Quantification of phosphorylated ERK1/2 is assessed using the AlphaLISA Technology. In this homogeneous assay system, an ERK1/2 kinase-specific and an anti-phospho-ERK Thr202/Tyr204-specific antibody immobilized to acceptor, and donor beads are used. The assay is validated based on known inhibitors of ERK1/2 activation (see Fig. 1).
Phosphorylation of ERK1 at residues Thr202 and Tyr204 or ERK2 at residues Thr185 and Tyr187 as a readout of intracellular kinase activity of MEK1/2 via AlphaLISA technology.
More information can be found on our website Cellular Phosphorylation Assay Services.
Reference compound IC50 with phospho-ERK1/2 readout
The well-described inhibitors Trametinib and Selumetinib potently inhibit MEK1/2 activity and the downstream kinase ERK1/2 in the cellular phosphorylation assay. All compounds were included in the validation process and the cellular phospho-ERK1/2 assay generated highly reproducible IC50 values. The graphs show representative results.