High-throughput cellular assays for regulated posttranslational modifications.
We have developed a set of high-throughput screening (HTS)-compatible assays capable of measuring regulated, target-specific posttranslational modifications in a mammalian cell-based format. We chose the NFkappaB signal transduction cascade as a model system to validate this approach because specific target proteins in this signaling pathway undergo a multitude of posttranslational modifications in response to pathway stimulation. In this pathway, TNFalpha induces the phosphorylation, ubiquitination, and proteasomal degradation of IkappaBalpha, which leads to the release and translocation of the NFkappaB transcriptional complex into the nucleus. To measure these cellular processes, we describe the use of a stable cell line expressing a fusion of green fluorescent protein (GFP) with IkappaBalpha that can be interrogated for either ubiquitination or phosphorylation using a unique set of terbium-labeled antibodies in a time-resolved Förster resonance energy transfer (TR-FRET)-based readout. Concurrently, we have engineered a beta-lactamase-IkappaBalpha reporter cell line that can be used to quantify proteasomal degradation of IkappaBalpha in living cells. Both TR-FRET and beta-lactamase reporter technologies provide a convenient, sensitive, and robust means to interrogate the chronological steps in NFkappaB signaling in a physiologically relevant cellular context without the need to overexpress any enzyme involved in this pathway. Cellular HTS assays that interrogate such processes will provide a unique integrated approach to dissecting intermediate steps in NFkappaB activation and could serve as examples of broadly applicable pathway analysis tools for target-based drug discovery.
Robers
Matthew B
MB
Invitrogen Discovery Sciences, Madison, WI 53717, USA.
Horton
Robert A
RA
Bercher
Mark R
MR
Vogel
Kurt W
KW
Machleidt
Thomas
T
D000818:Animals; D002460:Cell Line; D002468:Cell Physiological Phenomena; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D007668:Kidney; D008322:Mammals; D051379:Mice; D000072000:NF-KappaB Inhibitor alpha; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D015533:Transcriptional Activation
D020672:I-kappa B Proteins; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; D000072000:NF-KappaB Inhibitor alpha