RelA control of IkappaBalpha phosphorylation: a positive feedback loop for high affinity NF-kappaB complexes.
NF-kappaB-IkappaB complex formation regulates the level and specificity of NF-kappaB activity. Quantitative analyses showed that RelA-NF-kappaB-induced IkappaBalpha binding is regulated through inhibitor retention and phosphorylation. RelA caused an increase in IkappaBalpha phosphorylation and in degradation, which was enhanced monotonically with inhibitor concentration. In vivo analysis demonstrated the RelA-induced IkappaBalpha/RelA interactions to be specific, saturable, and phosphorylation-dependent. In addition, it showed that phosphorylation regulates both the level and affinity of the complexes and demonstrated an increased average affinity to coincide with reduction in the level of complexes during cytokine-induced pathway activation. The data show that RelA regulation of NF-kappaB-IkappaBalpha complex formation is IkappaBalpha phosphorylation-dependent and that IkappaBalpha/NF-kappaB binding is dynamic and determined by concentration of the subunits. In addition, they suggest that regulation of both complex levels and affinities through phosphorylation, with effects on the system steady state, participate in selective activation of the NF-kappaB pathway.
Yang
Lin
L
Cell Biology Unit, Functional Genomics, Division of Genomic Medicine, The Medical School, University of Sheffield, Sheffield S10 2JF, United Kingdom.
Ross
Kehinde
K
Qwarnstrom
Eva E
EE
D005347:Fibroblasts / Q000166:cytology; D031541:Fluorescence Resonance Energy Transfer; D005881:Gingiva / Q000166:cytology; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism*; D007202:Indicators and Reagents / Q000378:metabolism; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics; D018613:Microscopy, Confocal; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D051996:Transcription Factor RelA
D020672:I-kappa B Proteins; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D011993:Recombinant Fusion Proteins; D051996:Transcription Factor RelA; D000072000:NF-KappaB Inhibitor alpha; D049452:Green Fluorescent Proteins