Simple SummaryThe extracellular-signal-regulated kinase 2 (ERK2) plays a key role in the Ras-Raf-MEK-ERK signal transduction cascade and is involved in the regulation of many cellular processes. The ERK2 signaling converts extracellular stimuli into cell proliferation and, when deregulated, can promote oncogenic transformation. The ERK2 missense variants, carrying a single amino acid substitution in the protein sequence, have been identified in cancer tissues. This study reports a comprehensive biochemical and biophysical study of the ERK2 wild-type and variants in the common docking site present in cancer. A detailed analysis, from a molecular point of view, of the variants clarifies the impact of a single nucleotide variation on protein structure, stability and function, is essential to design alternative therapeutic strategies, and is a preliminary step to personalized medicine.AbstractThe extracellular-signal-regulated kinase 2 (ERK2), a mitogen-activated protein kinase (MAPK) located downstream of the Ras-Raf-MEK-ERK signal transduction cascade, is involved in the regulation of a large variety of cellular processes. The ERK2, activated by phosphorylation, is the principal effector of a central signaling cascade that converts extracellular stimuli into cells. Deregulation of the ERK2 signaling pathway is related to many human diseases, including cancer. This study reports a comprehensive biophysical analysis of structural, function, and stability data of pure, recombinant human non-phosphorylated (NP-) and phosphorylated (P-) ERK2 wild-type and missense variants in the common docking site (CD-site) found in cancer tissues. Because the CD-site is involved in interaction with protein substrates and regulators, a biophysical characterization of missense variants adds information about the impact of point mutations on the ERK2 structure–function relationship. Most of the P-ERK2 variants in the CD-site display a reduced catalytic efficiency, and for the P-ERK2 D321E, D321N, D321V and E322K, changes in thermodynamic stability are observed. The thermal stability of NP-ERK2 and P-ERK2 D321E, D321G, and E322K is decreased with respect to the wild-type. In general, a single residue mutation in the CD-site may lead to structural local changes that reflects in alterations in the global ERK2 stability and catalysis.