ERK2 Redistribution Assay

• Designed to assay compounds for their ability to modulate nuclear translocation of ERK2
• Coupled to EGFP for easy monitoring of the cellular translocation event
• Robust cell-based assay for use in high content analysis and fluorescence microscope applications

• Biologically relevant data: Compounds tested in a cellular environment
• Validated: Functionally tested cells provided with an optimized assay protocol
• Easy to use: Just plate cells, add compounds, and image

The ubiquitously expressed extracellular signal-regulated kinases (ERKs)/mitogen-activated protein kinases (MAPKs) function to transmit signals from growth factors and hormones to intracellular responses. Two major isoforms of ERK exist: ERK1 (p44) and ERK2 (p42). In resting cells, unphosphorylated ERK2 is anchored in the cytoplasm by its activator MAPK/ERK kinase 1 (MEK1). Upon relevant activation signals, MEK1 phosphorylates and activates ERK2, leading to its nuclear translocation. Activated ERK2 functions to regulate several target genes encoding proteins that control cellular processes such as cell proliferation, differentiation, and survival.

Inactivation of ERK2 is associated with its export from the nucleus. Nuclear export of ERK2 is also mediated by MEK1. For nuclear export, ERK2 binds to MEK1 that via association of its nuclear export sequence (NES) connects with the nuclear export receptor CRM1. This process can be inhibited by leptomycin B, which disrupts CRM1-mediated nuclear export. Finally, export of ERK2 from the nucleus is also regulated via action of Ser/Thr phosphatases (PPs) and dual specificity phosphatases termed MAPK phosphatases (MKPs).

Figure 1. Inhibition of nuclear accumulation of EGFP-ERK2 by U0126. Cells were treated with EGF alone (control, left panel) or with EGF + 30 μM U0126 for 60 min (right panel). Arrows indicate the EGF-induced nuclear localization that is inhibited by U0126 and that is detected by the image analysis algorithm.

Figure 2. Concentration response curves in the ERK2 assay: A) U0126 concentration response (n=16). The EC₅₀ is approximately 0.2 μM. B) EGFR inhibitor concentration response (n=8). The EC₅₀ is approximately 55 nM. Concentration response was measured in 9 point half log dilution series. Cells were incubated with test compound for 1 hr, then treated with 30 ng/ml EGF for 4 min. Cells were then fixed and nuclear translocation was measured using the Cellomics ArrayScan V^TI Reader and the Redistribution V3 BioApplication. % activity was calculated relative to the positive (30 μM U0126) and negative control (0.25% DMSO).