title: High yield purification of JNK1β1 and activation by in vitro reconstitution of the MEKK1→MKK4→JNK MAPK phosphorylation cascade.
abstract: The c-Jun N-terminal kinase (JNK) pathway forms part of the mitogen-activated protein kinase (MAPK) signaling pathways comprising a sequential three-tiered kinase cascade. Here, an upstream MAP3K (MEKK1) phosphorylates and activates a MAP2K (MKK4 and MKK7), which in turn phosphorylates and activates the MAPK, JNK. The C-terminal kinase domain of MEKK1 (MEKK-C) is constitutively active, while MKK4/7 and JNK are both activated by dual phosphorylation of S/Y, and T/Y residues within their activation loops, respectively. While improvements in the purification of large quantities of active JNKs have recently been made, inadequacies in their yield, purity, and the efficiency of their phosphorylation still exist. We describe a novel and robust method that further improves upon the purification of large yields of highly pure, phosphorylated JNK1β1, which is most suitable for biochemical and biophysical characterization. Codon harmonization of the JNK1β1 gene was used as a precautionary measure toward increasing the soluble overexpression of the kinase. While JNK1β1 and its substrate ATF2 were both purified to >99% purity as GST fusion proteins using GSH-agarose affinity chromatography and each cleaved from GST using thrombin, constitutively-active MEKK-C and inactive MKK4 were separately expressed in E. coli as thioredoxin-His(6)-tagged proteins and purified using urea refolding and Ni(2+)-IMAC, respectively. Activation of JNK1β1 was then achieved by successfully reconstituting the JNK MAPK activation cascade in vitro; MEKK-C was used to activate MKK4, which in turn was used to efficiently phosphorylate and activate large quantities of JNK1β1. Activated JNK1β1 was thereafter able to phosphorylate ATF2 with high catalytic efficiency.
authors.affiliation: Protein Structure-Function Research Unit, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg 2050, South Africa.
authors.forename: Gavin R
authors.initials: GR
authors.lastname: Owen
authors.forename: Ikechukwu
authors.initials: I
authors.lastname: Achilonu
authors.forename: Heini W
authors.initials: HW
authors.lastname: Dirr
mesh_terms: D051698:Activating Transcription Factor 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002846:Chromatography, Affinity; D003062:Codon; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D048728:MAP Kinase Kinase Kinase 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D048768:MAP Kinase Kinase Kinase 4 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020935:MAP Kinase Signaling System; D008958:Models, Molecular; D010766:Phosphorylation
chemical_list: C497105:ATF2 protein, human; D051698:Activating Transcription Factor 2; D003062:Codon; D048031:JNK Mitogen-Activated Protein Kinases; D048728:MAP Kinase Kinase Kinase 1; D048768:MAP Kinase Kinase Kinase 4; C482576:MAP3K1 protein, human; C482779:MAP3K4 protein, human