Generation and characterization of a stable MK2-EGFP cell line and subsequent development of a high-content imaging assay on the Cellomics ArrayScan platform to screen for p38 mitogen-activated protein kinase inhibitors.
This chapter describes the generation and characterization of a stable MK2-EGFP expressing HeLa cell line and the subsequent development of a high-content imaging assay on the Cellomics ArrayScan platform to screen for p38 MAPK inhibitors. Mitogen-activated protein kinase activating protein kinase-2 (MK2) is a substrate of p38 MAPK kinase, and p38-induced phosphorylation of MK-2 induces a nucleus to cytoplasm translocation (Engel et al., 1998; Neininger et al., 2001; Zu et al., 1995). Through a process of heterologous expression of a MK2-EGFP fusion protein in HeLa cells using retroviral infection, antibiotic selection, and flow sorting, we were able to isolate a cell line in which the MK2-EGFP translocation response could be robustly quantified on the Cellomics ArrayScan platform using the nuclear translocation algorithm. A series of assay development experiments using the A4-MK2-EGFP-HeLa cell line are described to optimize the assay with respect to cell seeding density, length of anisomycin stimulation, dimethyl sulfoxide tolerance, assay signal window, and reproducibility. The resulting MK2-EGFP translocation assay is compatible with high-throughput screening and was shown to be capable of identifying p38 inhibitors. The MK2-EGF translocation response is susceptible to other classes of inhibitors, including nonselective kinase inhibitors, kinase inhibitors that inhibit upstream kinases in the p38 MAPK signaling pathway, and kinases involved in cross talk between different modules (ERKs, JNKs, and p38s) of the MAPK signaling pathways. An example of mining "high-content" image-based multiparameter data to extract additional information on the effects of compound treatment of cells is presented.
Williams
Rhonda Gates
RG
Sphinx RTP Laboratories, Eli Lilly and Company, Research Triangle Park, NC, USA.
Kandasamy
Ramani
R
Nickischer
Debra
D
Trask
Oscar J
OJ
Laethem
Carmen
C
Johnston
Patricia A
PA
Johnston
Paul A
PA
D001331:Automation; D001681:Biological Assay / Q000295:instrumentation* / Q000379:methods*; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D020935:MAP Kinase Signaling System; D008856:Microscopy, Fluorescence; D020411:Oligonucleotide Array Sequence Analysis; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D015203:Reproducibility of Results; D015398:Signal Transduction; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*; D000099190:MAP-Kinase-Activated Kinase 2
D049452:Green Fluorescent Proteins; D047908:Intracellular Signaling Peptides and Proteins; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D000099190:MAP-Kinase-Activated Kinase 2