Inactivation of NF-kappa B inhibitor I kappa B alpha: ubiquitin-dependent proteolysis and its degradation product.
In most cells, the inactive dimeric NF-kappa B complexes are retained in the cytoplasm by binding to a group of inhibitory proteins. I kappa B. In response to extracellular stimuli, I kappa B is rapidly phosphorylated and degraded, thus, liberating the active NF-kappa B. To investigate the mechanisms involved, we have developed a cell-free system to study the degradation of the prototype I kappa B protein, I kappa B alpha. In this in vitro assay, ubiquitin, proteasome-containing S100 fraction and ATP are required for the proteolysis of I kappa B alpha. Both bound and free forms of I kappa B alpha isolated from intact cells can be degraded through this pathway. We also identified polyubiquitinated I kappa B alpha molecules and N-terminal truncated I kappa B alpha degradation product(s) both in vivo and in vitro. We conclude that the inactivation of I kappa B alpha occurs through a series of processes including phosphorylation, ATP-dependent ubiquitin conjugation and proteasome-mediated proteolysis.
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Biological Carcinogenesis and Development Program, SAIC-Frederick, MD, USA.
Dai
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Longo
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D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D020672:I-kappa B Proteins; D015151:Immunoblotting; D007163:Immunosorbent Techniques; D009097:Multienzyme Complexes / Q000378:metabolism; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors*; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014452:Ubiquitins / Q000494:pharmacology*
D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D020672:I-kappa B Proteins; D009097:Multienzyme Complexes; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D014452:Ubiquitins; D000072000:NF-KappaB Inhibitor alpha; D000255:Adenosine Triphosphate; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D013755:Tetradecanoylphorbol Acetate