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Published on: April 24, 2012
An in vitro 96-well plate assay of the mitogen-activated protein kinase cascade
B Antonsson1, C J Marshall, S Montessuit
1Geneva Biomedical Research Institute, Glaxo Wellcome Research and Development S. A., 1228 Plan-les-Ouates, Geneva, Switzerland. bruno.antonsson.ch_gva@serono.com
Abstract:
Mitogen-activated protein (MAP) kinases of the extracellular signal-regulated kinase (ERK) family are activated in response to many growth and differentiation factors as well as some oncogenes. ERK activation follows phosphorylation by a class of specific upstream MAP kinase/ERK kinase (MEK) exemplified by MEK-1. Activated ERKs control many short- and long-term changes in cell function through phosphorylating a number of intracellular target substrates which include stathmin, a phosphoprotein regulating microtubule stability. We report here the development of a simple, 96-well plate, quantitative in vitro assay measuring purified ERK2 catalytic activation by a constitutive MEK-1 mutant (S218E S222E). Enzymatic activity was detected by 33P phosphorylation of purified biotinylated stathmin captured on streptavidin-coated scintillation proximity assay beads which eliminates the need for wash steps. The assay was optimized and the K0.5 value for ATP was found to be 0.9 microM and the Km for stathmin was determined to be 16 microM. The assay was also used to determine IC50 values for the protein kinase inhibitors PD98059 and staurosporine. This simple assay allows several hundred quantitative measurements of MEK1-dependent ERK2 activation to be performed in a day.
Insights
A new assay quantifies extracellular signal-regulated kinase (ERK) activation by MEK-1. This method enables high-throughput screening of kinase inhibitors, advancing cancer research.
Area of Science:
- Molecular Biology
- Cell Signaling
- Biochemistry
Background:
- Mitogen-activated protein (MAP) kinases, specifically the extracellular signal-regulated kinase (ERK) family, are crucial in cellular responses to growth factors, differentiation signals, and oncogenes.
- ERK activation is mediated by upstream kinases, such as MAP kinase/ERK kinase (MEK), and plays a role in regulating cellular functions through substrate phosphorylation, including stathmin, which impacts microtubule stability.
Purpose of the Study:
- To develop a simple, quantitative in vitro assay for measuring the catalytic activation of purified ERK2 by a constitutively active MEK-1 mutant.
- To establish a high-throughput method for assessing ERK activation and screening potential kinase inhibitors.
Main Methods:
- A 96-well plate assay was developed utilizing purified ERK2 and a constitutively active MEK-1 mutant (S218E S222E).
- Enzymatic activity was quantified by measuring the 33P phosphorylation of purified biotinylated stathmin.
- Scintillation proximity assay (SPA) beads coated with streptavidin were used to capture phosphorylated stathmin, eliminating the need for wash steps and enabling direct detection.
Main Results:
- The assay was optimized, yielding a K0.5 value for ATP of 0.9 microM and a Km for stathmin of 16 microM.
- The assay successfully determined IC50 values for known protein kinase inhibitors, PD98059 and staurosporine.
- The developed assay allows for hundreds of quantitative measurements of MEK1-dependent ERK2 activation per day, demonstrating its high-throughput capability.
Conclusions:
- A simple, robust, and quantitative in vitro assay for measuring ERK2 activation by MEK-1 has been successfully developed.
- This assay is suitable for high-throughput screening of kinase inhibitors and facilitates the study of ERK signaling pathways.
- The assay's efficiency and accuracy provide a valuable tool for research in cell signaling, cancer biology, and drug discovery.
