Activation by phosphorylation and purification of human c-Jun N-terminal kinase (JNK) isoforms in milligram amounts |
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| Authors: | Anastasia F Thévenin Chati L Zony Brian J Bahnson Roberta F Colman |
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| Institution: | 1. Combinatorial Chemistry Unit, Barcelona Science Park, University of Barcelona, 08028 Barcelona, Spain;2. MRC Centre for Inflammation Research, Queen''s Medical Research Institute, University of Edinburgh, EH16 4TJ Edinburgh, United Kingdom;3. Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas. (CIBERNED), Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, 08028 Barcelona, Spain;4. Laboratori de Medicina Computacional, Unitat de Bioestadística, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain;5. CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Barcelona Science Park, 08028 Barcelona, Spain;6. Institute for Research in Biomedicine, 08028 Barcelona, Spain;1. Department of Biochemistry and Molecular Biology, College of Medicine, Howard University, 520 W Street, NW, Washington, DC 20059, USA;2. Regulatory RNAs and Cancer Section, Genetics Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA;1. Departments of Pediatrics and Genetics, Stanford University, Stanford, California, USA;1. Research Unit, Department of Internal Medicine, University Hospital Zurich, Switzerland;2. Center of Competence Multimorbidity and University Research Priority Program “Dynamics of Healthy Aging”, University of Zurich, Switzerland;3. Biozentrum, University of Basel, Switzerland;4. Zurich Center for Integrative Human Physiology, University of Zurich, Switzerland;1. Department of Physiology & Pharmacology, Oregon Health & Science University, Portland, OR 97239, USA;2. Casey Eye Institute, Oregon Health & Science University, Portland, OR 97239, USA |
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| Abstract: | 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. |
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