Different mechanisms control signal-induced degradation and basal turnover of the NF-kappaB inhibitor IkappaB alpha in vivo.
The transcription factor NF-kappaB is sequestered in the cytoplasm by a family of IkappaB molecules. Upon cellular stimulation with diverse agents, one of these molecules, IkappaB alpha, is rapidly phosphorylated and subsequently degraded. This process triggers nuclear translocation of NF-kappaB and the successive activation of target genes. Independent of its rapid stimulation-induced breakdown, IkappaB alpha is inherently unstable and undergoes a continuous turnover. To compare the mechanisms and protein domains involved in inducible and basal degradation of IkappaB alpha in intact cells we employed a transfection strategy using tagged IkappaB alpha and ubiquitin molecules. We show that tumor necrosis factor alpha (TNFalpha) induced breakdown of IkappaB alpha but not its basal turnover coincides with ubiquitination in the amino-terminal signal response domain (SRD) of IkappaB alpha. Neither the SRD nor the carboxy-terminal PEST sequence is needed for basal turnover, which instead depends only on the core ankyrin repeat domain. Despite the differences in the requirements of protein domains and ubiquitin-conjugation for both degradation pathways, each one is mediated by the proteasome. This finding is important for understanding alternative modes of controlling NF-kappaB activity.
Krappmann
D
D
Max-Delbrück-Center for Molecular Medicine MDC, Berlin, Germany.
Wulczyn
F G
FG
Scheidereit
C
C
D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D020672:I-kappa B Proteins; D008954:Models, Biological; D009097:Multienzyme Complexes / Q000378:metabolism; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors*; D046988:Proteasome Endopeptidase Complex; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D014452:Ubiquitins / Q000378:metabolism
D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D009097:Multienzyme Complexes; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D016755:Proto-Oncogene Proteins c-jun; D011994:Recombinant Proteins; D014452:Ubiquitins; D000072000:NF-KappaB Inhibitor alpha; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex