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High-throughput cellular assays for regulated posttranslational modifications

Analytical Biochemistry
Publication Date
DOI: 10.1016/j.ab.2007.09.012
  • Cell-Based Assay
  • Time-Resolved Fret
  • Pathway Analysis
  • Hts
  • Phosphorylation
  • Ubiquitination
  • Proteasome
  • NfκB
  • IκBα
  • β-Lactamase
  • Green Fluorescent Protein
  • Biology
  • Engineering


Abstract 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 NFκB 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, TNFα induces the phosphorylation, ubiquitination, and proteasomal degradation of IκBα, which leads to the release and translocation of the NFκB 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 IκBα 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 β-lactamase–IκBα reporter cell line that can be used to quantify proteasomal degradation of IκBα in living cells. Both TR–FRET and β-lactamase reporter technologies provide a convenient, sensitive, and robust means to interrogate the chronological steps in NFκB 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 NFκB activation and could serve as examples of broadly applicable pathway analysis tools for target-based drug discovery.

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