Development and comparison of two nonradioactive kinase assays for I kappa B kinase.
In response to diverse stimuli, the transcription factor NF-kappaB is activated by the IKK kinase complex containing two kinases (IKKalpha and IKKbeta) that phosphorylate IkappaB, an inhibitory protein of NF-kappaB. The phosphorylation of IkappaB results in ubiquitination and degradation of IkappaB, allowing NF-kappaB to translocate to the nucleus where it regulates its target genes. To elucidate the role of IKK in the NF-kappaB signaling pathway, we have developed and characterized two quantitative, sensitive, and nonradioactive assays for evaluating IKKbeta 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 (Km) 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.
Sadler
Tammy M
TM
Department of Oncology and ImmunoInflammatory Research, Wyeth Research, Pearl River, NY 10965, USA.
Achilleos
Maria
M
Ragunathan
Shoba
S
Pitkin
Adam
A
LaRocque
James
J
Morin
John
J
Annable
Rebecca
R
Greenberger
Lee M
LM
Frost
Philip
P
Zhang
Yixian
Y
D000255:Adenosine Triphosphate / Q000378:metabolism; D045744:Cell Line, Tumor; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D051550:I-kappa B Kinase; D020128:Inhibitory Concentration 50; D017346:Protein Serine-Threonine Kinases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011868:Radioisotopes; D012680:Sensitivity and Specificity
D004791:Enzyme Inhibitors; D011868:Radioisotopes; D000255:Adenosine Triphosphate; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human; D005978:Glutathione