Determination of ERK Activity: Antiphospho-ERK Antibodies, In Vitro Phosphorylation, and In-Gel Kinase Assay

The mitogen-activated protein kinases (MAPKs) are a family of protein serine/threonine kinases that operate within specific signaling pathways called MAPK cascades (for reviews see Chapter 1 and references therein). Each MAPK cascade is composed of up to six tiers of protein kinases, which activate each other, and thus participate in the amplification and specificity determination of the transmitted signals. Activation of the protein kinase components of the cascade is carried out by phosphorylation, which for enzymes at a given tier of the cascade occurs at a common phosphorylation site, such as the Thr-Xaa-Tyr motif for MAPKs. Eventually the signals are transmitted to several regulatory proteins that essentially govern all stimulated cellular processes including proliferation, differentiation, and response to stress.