RRC ID 54361
Author Rahimova N, Babazada H, Higuchi Y, Yamashita F, Hashida M.
Title Development of mKO2 fusion proteins for real-time imaging and mechanistic investigation of the degradation kinetics of human IκBα in living cells.
Journal Biochim Biophys Acta Mol Cell Res
Abstract In resting cells, the nuclear factor kappa B (NF-κB) family of transcription factors is stabilized by complexation with the cytoplasmic inhibitor of kappa B alpha (IκBα). Extracellular stimuli, such as tumor necrosis factor alpha (TNFα) or bacterial lipopolysaccharide activate NF-κB through IκBα phosphorylation and ubiquitin-proteasomal degradation. Herein, we developed a novel biosensor, by fusing the monomeric fluorescent protein Kusabira-Orange 2 to IκBα (mKO2-IκBα), to study the dynamics and structure-activity relationship of IκBα degradation. Site-specific deletion studies on the IκBα sequence revealed that the C-terminal PEST domain is required in signal-induced proteasomal degradation of IκBα and functions independently from ankyrin repeats. Using deletion mutants, we show that IκBα ankyrin repeats do not affect IκBα degradability but affect its degradation rate. We demonstrate, by both real-time confocal microscopy and western blot analysis, that the half-life of mKO2-IκBα in response to TNFα is approximately 35 min, which is similar to the half-life of endogenous IκBα. Using this biosensor we also show that selective proteasome inhibitors, such as lactacystin and MG132, inhibit degradation and affect the kinetics of IκBα in a dose-dependent manner. The techniques described here can have a range of possible applications, such as facilitating studies associated with IκBα dynamics and biochemical characteristics, as well as the screening of potential proteasome inhibitors.
Volume 1866(2)
Pages 190-198
Published 2019-2-1
DOI 10.1016/j.bbamcr.2018.10.018
PII S0167-4889(18)30481-6
PMID 30391277
MeSH Ankyrins / metabolism Cell Nucleus / metabolism Cytoplasm / metabolism DNA-Binding Proteins / metabolism Fluorescent Dyes HeLa Cells Humans I-kappa B Proteins / metabolism I-kappa B Proteins / physiology Luminescent Proteins NF-KappaB Inhibitor alpha / genetics* NF-KappaB Inhibitor alpha / metabolism* NF-KappaB Inhibitor alpha / physiology* NF-kappa B / metabolism NF-kappa B / physiology Optical Imaging / methods Phosphorylation Protein Engineering / methods Proteolysis Repetitive Sequences, Nucleic Acid Signal Transduction Structure-Activity Relationship Tumor Necrosis Factor-alpha / physiology Ubiquitination
IF 4.739
Times Cited 0
DNA material pCMFlag_hsNFKBIA (RDB06668) pBS coupler 1 (RDB15126)
Human and Animal Cells HeLa(RCB0007)