Degradation of IkappaBalpha is limited by a postphosphorylation/ubiquitination event.
Regulation of IkappaBalpha during activation was examined using EGFP. Single cell analysis showed that both localisation- and cytokine-induced degradation of IkappaBalpha are dependent on expression levels. Cells expressing higher levels of the inhibitor demonstrated an increase in nuclear IkappaBalphaEGFP with a pronounced enhancement in the nuclear/cytoplasmic ratio. Enhancing the levels of the endogenous IkappaBalpha by relA transfection caused significant reduction in IL-1-mediated degradation of the fusion protein. Similarly, IkappaBalphaEGFP-transfected cells showed an inverse correlation between the level of the fusion protein and IL-1-mediateddegradation. Comparing absolute levels demonstrated a biphasic response, with reduction in cells expressing over 15-fold that of endogenous levels. Further experiments using Western analysis showed a positive correlation between both phosphorylation and ubiquitination of IkappaBalphaEGFP, and the level the inhibitor. In contrast, and in agreement with the singlecell analysis, while IL-1 stimulation caused the expected degradation at lower levels of the fusion protein,breakdown of IkappaBalphaEGFP was totally inhibited at the higher transfection levels. The data show that turnover of IkappaBalpha is saturable and suggest that limitation of the pathway by enhanced inhibitor expression is regulated through a post phosphorylation/ubiquitination event, at the level of degradation.
Yang
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L
Division of Molecular and Genetic Medicine, University of Sheffield, Sheffield S10 2JF, United Kingdom.
Chen
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H
Qwarnstrom
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E
D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D020672:I-kappa B Proteins; D007375:Interleukin-1 / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics; D000072000:NF-KappaB Inhibitor alpha; D010766:Phosphorylation / Q000187:drug effects; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D014162:Transfection; D014452:Ubiquitins / Q000378:metabolism*
D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D007375:Interleukin-1; D008164:Luminescent Proteins; C000606204:NFKBIA protein, human; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; D000072000:NF-KappaB Inhibitor alpha