Critical role for lysines 21 and 22 in signal-induced, ubiquitin-mediated proteolysis of I kappa B-alpha.
The NF-kappa B transcription factor induces rapid transcription of many genes in response to a variety of extracellular signals. NF-kappa B is readily activated from normally inhibited cytoplasmic stores by induced proteolytic degradation of I kappa B-alpha, a principal inhibitor of this transcription factor. Following the inhibitor's degradation, NF-kappa B is free to translocate to the nucleus and induce gene transcription. The I kappa B-alpha inhibitor is targeted for degradation by signal-induced phosphorylation of two closely spaced serines in its NH2 terminus (Ser32 and Ser36). Proteolytic degradation appears to be carried out by proteasomes which can recognize ubiquitinated intermediates of the I kappa B-alpha inhibitor. We provide evidence which supports a ubiquitin-mediated mechanism. Amino acid substitutions of two adjacent potential ubiquitination sites in the NH2 terminus of I kappa B-alpha (Lys21 and Lys22) almost completely block the rapid, signal-induced degradation of the mutant protein, while they do not interfere with induced phosphorylation. The mutant I kappa B-alpha also does not permit signal-induced activation of NF-kappa B bound to it. The data suggest that ubiquitination at either of the two adjacent lysines (21 and 22) is required for degradation following induced phosphorylation at nearby serines 32 and 36. Such dependence on ubiquitination of specific sites for protein degradation is unusual. This mechanism of degradation may also apply to I kappa B-beta, an inhibitor related to and functionally overlapping with I kappa B-alpha, as well as to cactus, an I kappa B homolog of Drosophila.
Baldi
L
L
Laboratory of Immunoregulation, NIAID, National Institutes of Health, Bethesda, Maryland 20892-1876, USA.
Brown
K
K
Franzoso
G
G
Siebenlist
U
U
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D020672:I-kappa B Proteins; D008239:Lysine / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D000072000:NF-KappaB Inhibitor alpha; D014411:Neoplastic Stem Cells; D011518:Proto-Oncogene Proteins; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D014407:Tumor Cells, Cultured; D014452:Ubiquitins / Q000378:metabolism*
D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; D011518:Proto-Oncogene Proteins; D014452:Ubiquitins; D000072000:NF-KappaB Inhibitor alpha; D008239:Lysine