Development of mKO2 fusion proteins for real-time imaging and mechanistic investigation of the degradation kinetics of human IκBα in living cells

Publication date: Available online 31 October 2018Source: Biochimica et Biophysica Acta (BBA) - Molecular Cell ResearchAuthor(s): Nilufar Rahimova, Hasan Babazada, Yuriko Higuchi, Fumiyoshi Yamashita, Mitsuru HashidaAbstractIn 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 Iκ...
Source: Biochimica et Biophysica Acta (BBA) Molecular Cell Research - Category: Molecular Biology Source Type: research