Development of a Nonradioactive, Time-Resolved Fluorescence Assay for the Measurement of Jun N-Terminal Kinase Activity

Abstract

Activated transcription factor AP-1 is composed of c-Jun homodimers or c-Jun/c-Fos heterodimers and mediates expression of several gene products that have been implicated in disease pathogenesis. Activation of AP-1 is dependent on phosphorylation of c-Jun by Jun N-terminal kinase (JNK). Therefore, identification of inhibitors of JNK-mediated phosphorylation of c-Jun may lead to a novel class of therapeutics. A nonradioactive, high-through-put, time-resolved fluorescence assay was developed to measure and identify inhibitors of JNK activity. This assay utilized a lanthanide (europium)-labeled antibody that was specific for N-terminally phosphorylated c-Jun. The optimized europium-based assay was approximately 15-fold more sensitive compared to a similar 32P-based JNK assay. Compounds that were identified as inhibitors of JNK using the europium-based assay also inhibited JNK activity in the 32P-based assay with similar IC50 values. The europium-based JNK assay eliminates the contamination problems associated with the use of radioactivity. The sensitivity and safety of the europium-based assay make it amenable to robotics that will significantly increase screening throughput. © 1997, Sage Publications. All rights reserved.