期刊
ANALYTICAL BIOCHEMISTRY
卷 372, 期 2, 页码 189-197出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2007.09.012
关键词
cell-based assay; time-resolved FRET; pathway analysis; HTS; phosphorylation; ubiquitination; proteasome; NF kappa B; I kappa B alpha; beta-lactamase; green fluorescent protein
We have developed a set of high-throughput screening (HTS)-compatible assays capable of measuring regulated, target-specific post-translational modifications in a mammalian cell-based format. We chose the NF kappa B signal transduction cascade as a model system to validate this approach because specific target proteins in this signaling pathway undergo a multitude of post-translational modifications in response to pathway stimulation. In this pathway, TNF alpha induces the phosphorylation, ubiquitination, and proteasomal degradation of I kappa B alpha, which leads to the release and translocation of the NF kappa B 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 I kappa B alpha that can be interrogated for either ubiquitination or phosphorylation using a unique set of terbium-labeled antibodies in a tune-resolved Forster resonance energy transfer (TR-FRET)-based readout. Concurrently, we have engineered a (3-lactamase-I kappa B alpha reporter cell line that can be used to quantify proteasomal degradation of I kappa B alpha in living cells. Both TR-FRET and beta-lactamase reporter technologies provide a convenient, sensitive, and robust means to interrogate the chronological steps in NF kappa B 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 NF kappa B activation and could serve as examples of broadly applicable pathway analysis tools for target-based drug discovery. (C) 2007 Elsevier Inc. All rights reserved.
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