Development and comparison of two nonradioactive kinase assays for I kappa B kinase

• Published in: Analytical biochemistry 2004-03, Vol.326 (1), p.106-113
• Main Authors: Sadler, Tammy M, Achilleos, Maria, Ragunathan, Shoba, Pitkin, Adam, LaRocque, James, Morin, John, Annable, Rebecca, Greenberger, Lee M, Frost, Philip, Zhang, Yixian
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - metabolism; Cell Line, Tumor; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors - pharmacology; Glutathione - metabolism; Humans; I-kappa B Kinase; IKKβ; Inhibitor; Inhibitory Concentration 50; Kinase; NF-κB; Nonradioactive; Protein-Serine-Threonine Kinases - analysis; Protein-Serine-Threonine Kinases - antagonists & inhibitors; Protein-Serine-Threonine Kinases - metabolism; Radioisotopes; Sensitivity and Specificity
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2003.11.021

In response to diverse stimuli, the transcription factor NF-κB is activated by the IKK kinase complex containing two kinases (IKKα and IKKβ) that phosphorylate IκB, an inhibitory protein of NF-κB. The phosphorylation of IκB results in ubiquitination and degradation of IκB, allowing NF-κB to translocate to the nucleus where it regulates its target genes. To elucidate the role of IKK in the NF-κB signaling pathway, we have developed and characterized two quantitative, sensitive, and nonradioactive assays for evaluating IKKβ activity: a dissociation-enhanced lanthanide fluorescence immunoassay called DELFIA and a homogeneous time-resolved fluorescence resonance energy transfer assay called LANCE. We show that the two assays have similar sensitivity and Michaelis constants ( K m ) for adenosine 5 ′-triphosphate and substrate; however, the LANCE format was far more efficient and easier to perform. Additionally, the assays were validated with the known kinase inhibitor K252a and several other kinase inhibitors, which showed that the IC 50 values of the two assays were comparable. In summary, both assays are quantitative, sensitive, reproducible, and amenable to high-throughput screening with improved waste management over radioactive assays.