Analysis of mitogen-activated protein kinase activation and interactions with regulators and substrates.

Mitogen-activated protein kinase (MAPK) cascades are ubiquitous signal transduction modules in eukaryotes that are of great interest and importance. Here, we summarize some useful methods for the analysis of MAPK signaling, including methods to (1) detect MAPK activation in cells, with an emphasis on using phosphorylation-state-specific antibodies raised against mammalian phosphopeptide sequences to detect the activation of MAPKs in other species; (2) estimate the cellular concentrations of MAPKs and other proteins of interest; (3) detect and quantify the stable physical association of MAPKs with their substrates and regulators, and estimate the relevant dissociation constants; (4) delineate the MAPK-binding regions or domains of MAPK-interacting proteins, with particular emphasis on the identification and verification of MAPK-docking sites. These procedures are broadly applicable to many organisms, including both yeast and mammalian cells.