Large Scale Screening Assay for the Phosphorylation of Mitogen-Activated Protein Kinase in Cells

Abstract

Mitogen-activated protein (MAP) kinases are serine/threonine kinases that are activated by phosphorylation and are involved in the cellular response to various physiologic stimuli and stress conditions. Because MAP kinases play an important role in cellular functioning, a screening assay to determine the phosphorylation of MAP kinase upon various conditions was desirable. Therefore, we have developed a cellular enzyme-linked immunosorbent assay (Cell-ELISA), in which the phosphorylated forms of p42MAPK and p44MAPK are detected. We show that in this Cell-ELISA, MAP kinase becomes phosphorylated in a dose- and time-dependent manner under proliferative or stress conditions. This dose- and time-dependent phosphorylation agrees with observations using classical gel-electrophoresis and Western blotting techniques. Furthermore, we show that our assay is applicable to different cell types and that serum-starvation is not required for detection of an increase in MAP kinase phosphorylation. From these experiments, it is concluded that the Cell-ELISA is a reliable and fast method for quantitative detection of the phosphorylation, and thus the activation, of MAP kinase. This assay is applicable for a large-scale screening of the effectivity of biological or chemical compounds that modulate the cellular response to physiologic stimuli or stress through phosphorylation and activation of MAP kinase.