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A Luciferase Assay to Assess JNK1 Activation
Author(s) -
Stashko Michael,
Lubben Thomas,
Clampit Jill,
Wang Sanyi,
Sun Chaohong,
Serby Michael,
Liu Bo,
Xin Zhili,
Liu Gang,
Trevillyan James
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a54-b
Subject(s) - kinase , phosphorylation , luciferase , chemistry , enzyme activator , atpase , microbiology and biotechnology , biochemistry , enzyme , biology , transfection , gene
Activation of JNK1 has been associated with the development of insulin resistance in Type‐2 diabetes. We describe an assay to measure inhibitors of JNK1 activation, based upon a constitutive ATPase activity formed in the presence of MKK4 and MKK7. Active JNK1 is typically assayed by phosphorylation of peptide substrates such as ATF2. Monitoring inhibition of JNK1 activation must address the concern that for an inhibitor that only slows the rate of JNK1 activation, given sufficient time JNK1 will still be activated completely. A more direct approach was developed from the observation that once activated, JNK1 has a constitutive ATPase activity, but no activity in the absence of activation. Constitutive ATPase activity was monitored by a modified luciferase reaction under conditions that kept ATP above Km. Correlations with an activity assay of phosphorylation of ATF2 confirmed that the constitutive ATPase assay could be used as a surrogate to monitor JNK1 activation. Using this method, we optimized conditions for producing active JNK1. We found compounds that inhibited JNK1 activation that did not directly inhibit JNK1 kinase activity. This method may be amenable to the study of other kinases with constitutive ATPase activities, and could also provide a relatively simple, general, and non‐radioactive kinase assay method.