c-Jun N-terminal kinases (JNKs) are part of the mitogen-activated protein kinase (MAPK) signaling cascade They are activated through dual phosphorylation of two residues in the activation loop a threonine and a tyrosine by MAP2 kinases (MKK4 and 7) in response to various extracellular stresses such as UV or osmotic shock as well as by cytokines and growth factors Only small amounts of phosphorylated active JNKs have previously been produced because of difficulties in expressing these phosphorylated kinases in Escherichia colt which lack the appropriate upstream kinases We have now established a novel activation and purification method that allows for reproducible production of milligram amounts of active phosphorylated JNKs suitable for a variety of enzymatic biophysical and structural characterizations We utilize N-terminally His tagged MKK4 that is coexpressed in E coli with a constitutively active form of MEKK1 This phosphorylated active His-MKK4 is purified by Ni-NTA chromatography and used to phosphorylate milligram amounts of three different isoforms of human JNKs (JNK1 alpha 1 JNK1 alpha 2 and JNK2 alpha 2) that had separately been expressed and purified from E colt in their inactive forms These in vitro activated JNKs are phosphorylated on both residues (T183 Y185) in their activation loops and are active towards their substrate ATF2 (C) 2010 Elsevier Inc All rights reserved