Mitogen-activated protein kinase activation assays

Johnston, P.A. et al. 2007. Development and implementation of a 384-well homogeneous fluorescence intensity high-throughput screening assay to identify mitogen-activated protein kinase phosphatase-1 dualspecificity protein phosphatase inhibitors. Assay Drug Dev. Technol. 5, 319-332. 

Nickischer, D. et al. 2006. Development and implementation of three mitogen-activated protein kinase (MAPK) signaling pathway imaging assays to provide MAPK module selectivity profiling for kinase inhibitors. Meth. Enzymol. 414, 389 118. 

Trask, O.J., Jr. et al. 2006. Assay development and case history of a 32K-biased library high-content MK2-EGFP translocation screen to identify p38 mitogen-activated protein kinase inhibitors on the Array Scan 3.1 imaging platform. Meth. Enzymol. 414, 419-439. 

US EPA (2008). Distributed Structure-Searchable Toxicity (DSSTox) Database Network, Vol. 2008. Vogt, A., and Lazo, J. S. (2007). Implementation of high-content assay for inhibitors of mitogen-activated protein kinase phosphatases. Methods 42, 268-277. 

C. Kinase Assay Using a Mitogen-Activated Protein Kinase-Specific Substrate... 

In order to determine the selectivity of each peptide sensor substrate, assays with a panel of recombinantly expressed, activated kinases can be performed (Figure 1.7b). In this case, the concentration of substrate peptide is held constant at two to three times the determined K. For example, the p38 sensor depicted in Figure 1.6 was incubated with various MAP (mitogen-activated protein) and non-MAP kinases and fluorescence emission was monitored. Figure 1.7b demonstrates the selectivity of the p38 sensor for the target kinase [23]. Following this initial screen, kinase selectivity can be addressed directly in cell lysates. Cells can be stimulated to activate the kinase of interest and kinase activity can be determined in the presence and absence of a selective inhibitor of the kinase of interest. Residual activity in the presence of inhibitor would indicate sensor cross talk with other kinases. 

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