关键词:
IKB alpha
Kusabira-Orange 2
Ubiquitin-proteasomal degradation
PEST domain
Ankyrin repeats
摘要:
In resting cells, the nuclear factor kappa B (NF-kappa B) family of transcription factors is stabilized by complexation with the cytoplasmic inhibitor of kappa B alpha (I kappa B alpha). Extracellular stimuli, such as tumor necrosis factor alpha (TNF alpha) or bacterial lipopolysaccharide activate NF-kappa B through I kappa B alpha phosphorylation and ubiquitin-proteasomal degradation. Herein, we developed a novel biosensor, by fusing the monomeric fluorescent protein Kusabira-Orange 2 to I kappa B alpha (mKO2-I kappa B alpha), to study the dynamics and structure-activity relationship of I kappa B alpha degradation. Site-specific deletion studies on the I kappa B alpha sequence revealed that the C-terminal PEST domain is required in signal-induced proteasomal degradation of I kappa B alpha and functions independently from ankyrin repeats. Using deletion mutants, we show that I kappa B alpha ankyrin repeats do not affect I kappa B alpha degradability but affect its degradation rate. We demonstrate, by both real-time confocal microscopy and western blot analysis, that the half-life of mKO2-I kappa B alpha in response to TNF alpha is approximately 35 min, which is similar to the half-life of endogenous I kappa B alpha. Using this biosensor we also show that selective proteasome inhibitors, such as lactacystin and MG132, inhibit degradation and affect the kinetics of I kappa B alpha in a dose-dependent manner. The techniques described here can have a range of possible applications, such as facilitating studies associated with I kappa B alpha dynamics and biochemical characteristics, as well as the screening of potential proteasome inhibitors.