Mapping of the inducible IkappaB phosphorylation sites that signal its ubiquitination and degradation.
Extracellular stimuli that activate the transcription factor NF-kappaB cause rapid phosphorylation of the IkappaBalpha inhibitor, which retains NF-kappaB in the cytoplasm of nonstimulated cells. Phosphorylation of IkappaBalpha is followed by its rapid degradation, the inhibition of which prevents NF-kappaB activation. To determine the relationship between these events, we mapped the inducible phosphorylation sites of IkappaBalpha. We found that two residues, serines 32 and 36, were phosphorylated in response to either tumor necrosis factor, interleukin-1, or phorbol ester. Substitution of either serine blocks or slows down induction of IkappaBalpha degradation. Substitutions of the homologous sites in IkappaBbeta, serines 19 and 23, also prevent inducible IkappaBbeta degradation. We suggest that activation of a single IkappaB kinas e or closely related IkappaB kinases is the first cr itical step in NF-kappaB activation. Once phosphorylated, IkappaB is ubiquitinated. Unlike wild-type IkappaBalpha, the phosphorylation-defective mutants do not undergo inducible polyubiquitination. As substitution of a conserved lysine residue slows down the ubiquitination and degradation of IkappaBalpha without affecting its phosphorylation, polyubiquitination is required for inducible IkappaB degradation.
DiDonato
J
J
Department of Pharmacology, Program in Biomedical Science, University of California, San Diego School of Medicine, La Jolla, USA.
Mercurio
F
F
Rosette
C
C
Wu-Li
J
J
Suyang
H
H
Ghosh
S
S
Karin
M
M
D016475:3T3 Cells; D000818:Animals; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D051550:I-kappa B Kinase; D020672:I-kappa B Proteins; D007375:Interleukin-1 / Q000494:pharmacology; D008239:Lysine / Q000235:genetics; D051379:Mice; D009154:Mutation; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010449:Peptide Mapping; D010766:Phosphorylation; D011233:Precipitin Tests; D011480:Protease Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000235:genetics; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014452:Ubiquitins / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; C083487:I kappa B beta protein; D020672:I-kappa B Proteins; D007375:Interleukin-1; D016328:NF-kappa B; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; D011480:Protease Inhibitors; D014409:Tumor Necrosis Factor-alpha; D014452:Ubiquitins; D000072000:NF-KappaB Inhibitor alpha; D012694:Serine; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; C496564:Chuk protein, mouse; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human; C496562:Ikbkb protein, mouse; C496566:Ikbke protein, mouse; D008239:Lysine