Signal-dependent degradation of IkappaBalpha is mediated by an inducible destruction box that can be transferred to NF-kappaB, bcl-3 or p53.
Activation of the transcription factor NF-kappaB in response to a variety of stimuli is governed by the signal-induced proteolytic degradation of NF-kappaB inhibitor proteins, the IkappaBs. We have investigated the sequence requirements for signal-induced IkappaBalpha phosphorylation and proteolysis by generating chimeric proteins containing discrete sub-regions of IkappaBalpha fused to the IkappaBalpha homologue Bcl-3, the transcription factor NF-kappaB1/p50 and the tumour suppressor protein p53. Using this approach we show that the N-terminal signal response domain (SRD) of IkappaBalpha directs their signal-dependent phosphorylation and degradation when transferred to heterologous proteins. The C-terminal PEST sequence from IkappaBalpha was not essential for induced proteolysis of the chimeric proteins. A deletion analysis conducted on the SRD identified a 25 amino acid sub-domain of IkappaBalpha that is necessary and sufficient for the degradative response in vivo and for recognition by TNFalpha-dependent IkappaBalpha kinase in vitro . The results obtained should prove instrumental in the further characterization of IkappaB-specific kinases, as well as the E2 and E3 enzymes responsible for IkappaBalpha ubiquitination. Furthermore, they suggest a novel strategy for generating conditional mutants, by targetting heterologous proteins for transient elimination by the IkappaBalpha pathway.
Wulczyn
F G
FG
Max-Delbrück-Center for Molecular Medicine, MDC, Robert-Rössle-Strasse 10, 13122 Berlin, Germany.
Krappmann
D
D
Scheidereit
C
C
D000595:Amino Acid Sequence; D000081242:B-Cell Lymphoma 3 Protein; D001483:Base Sequence; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D020672:I-kappa B Proteins; D008969:Molecular Sequence Data; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016366:Open Reading Frames; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
D000081242:B-Cell Lymphoma 3 Protein; C000631344:BCL3 protein, human; D017931:DNA Primers; D004268:DNA-Binding Proteins; C083487:I kappa B beta protein; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53; D000072000:NF-KappaB Inhibitor alpha