Activation by phosphorylation and purification of human c-Jun N-terminal kinase (JNK) isoforms in milligram amounts

• Published in: Protein expression and purification Vol. 75; no. 2; pp. 138 - 146
• Main Authors: Thévenin, Anastasia F., Zony, Chati L., Bahnson, Brian J., Colman, Roberta F.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.02.2011
• Subjects: Animals; c-Jun N-terminal kinases; Chromatography, Affinity; Cloning, Molecular; Enzyme Activation; Escherichia coli; Humans; JNK Mitogen-Activated Protein Kinases - genetics; JNK Mitogen-Activated Protein Kinases - isolation & purification; JNK Mitogen-Activated Protein Kinases - metabolism; Kinetics; MAP kinase; MAP Kinase Kinase 4 - genetics; MAP Kinase Kinase 4 - isolation & purification; MAP Kinase Kinase 4 - metabolism; MAP Kinase Kinase 7 - genetics; MAP Kinase Kinase 7 - isolation & purification; MAP Kinase Kinase 7 - metabolism; Phosphorylation; Protein Isoforms - genetics; Protein Isoforms - isolation & purification; Protein Isoforms - metabolism; Rats; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - isolation & purification; Recombinant Fusion Proteins - metabolism; Signal Transduction; Signaling cascade; Substrate Specificity; Threonine - metabolism; Tyrosine - metabolism; Signaling cascade; MAP kinase; Phosphorylation; c-Jun N-terminal kinases
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2010.08.002

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 coli, 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 (JNK1α1, JNK1α2 and JNK2α2) that had separately been expressed and purified from E. coli 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.