MK2 (MAPKAPK2) Redistribution Assay

• Designed to assay compounds for their ability to modulate nuclear translocation of MK2
• 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

Mitogen-activated protein (MAP) kinase-activated protein kinase 2 (MK2/MAPKAPK2) is one of several kinases directly regulated by the p38 mitogen-activated protein (MAP) kinase (p38). The p38 signaling pathway is activated in response to environmental stressors (e.g. anisomycin and hyper-osmotic agents) and inflammatory cytokines like interleukin-1 (IL-1) and tumor necrosis factor α (TNFα). Upon activation, p38 directly phosphorylates regulatory sites of MK2 resulting in activation and nuclear export of MK2. Within the cytoplasm, MK2 promotes the production of pro-inflammatory cytokines such as TNFα, IL-6, and IL-1 by affecting cytokine mRNA translation and stability. Thus, translocation of MK2 to the cytoplasm is a measure of the inflammatory output resulting from activation of the p38 signaling cascade.

Figure 1. Translocation of EGFP-MK2 stimulated with IL-1β in response to SB203580. Cells were treated with 3 ng/mL IL-1β in the absence (left panel) or presence (right panel) of 10 µM SB203580. Arrows indicate IL-1β-mediated cytoplasmic translocation detected by the image analysis algorithm.

Figure 2. Concentration-response curve of SB203580 in the MK2 Redistribution assay optimized for response to IL-1β, TNF-α, or anisomycin (n = 8). Concentration response was measured in 9 point half log dilution series of SB203580. Cells were incubated with SB203580 for 30 min. followed by a) 30 min. incubation with IL-1β at a final conc. of 3 ng/ml (blue) b) 15 min. incubation with TNF-α at a final conc. of 30 nM (red); c) 30 min. incubation with anisomycin at a final conc. of 500 nM (green). Cells were then fixed and the nucleus to cytoplasm translocation was measured using the Cellomics ArrayScan® V^TI Reader and the RedistributionV3 BioApplication. % activity (PCTACT) was calculated relative to the positive (10 µM SB203580) and negative control (0.25% DMSO).

Figure 3. MK2 multiplex with HCS reagent kits. ArrayScan® V^TI images of untreated (top row) and IL-1β stimulated (bottom row) MK2-GFP expressing cells stained with phospho-c-jun or NFκB primary antibody from the respective Cellomics HCS Reagent kits and a secondary GαM-Dylight-549 antibody or GαR-Dylight-649 antibodies respectively. The images were obtained with a 40x objective with the following filter settings: Hoechst (XF100-Hoechst), GFP (XF100-FITC), Dylight-549 (XF93-TRITC) and Dylight-649 (XF110-Cy5, sensitive).