A 65-kappaD subunit of active NF-kappaB is required for inhibition of NF-kappaB by I kappaB.
The NF-kappaB transcription factor was affinity-purified from deoxycholate (DOC)-treated cytosol of HeLa cells and shown to contain both a 50-kappaD polypeptide (p50) with a DNA-binding specificity identical to that of nuclear NF-kappaB and a 65-kappaD protein (p65) lacking DNA binding activity. Electrophoretically purified p50, after renaturation, gave rise to a protein-DNA complex that migrated faster than that made by native NF-kappaB. Reconstitution of p50 and p65 together produced a protein that combined with DNA to form a complex with electrophoretic mobility indistinguishable from that of the complex formed by nuclear extracts and DOC-treated cytosolic fractions. Sedimentation and gel filtration analyses indicate that alone, the p50 protein exists as a dimer; two molecules of p65 bind to it to form a heterotetramer. Unlike I kappaB, the specific inhibitor of NF-kappaB, p65 displayed no inhibitor activity and was not released from NF-kappaB by DOC. p65 did not change the DNA binding specificity or the stimulatory effect of GTP on the p50 homodimer. Surprisingly, NF-kappaB could only be inactivated by I kappaB when p65 was bound. It would appear that one function of p65 is to make NF-kappaB susceptible to inhibition by I kappaB.
Baeuerle
P A
PA
Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142.
Baltimore
D
D
D002846:Chromatography, Affinity; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D003840:Deoxycholic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016328:NF-kappa B; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D016328:NF-kappa B; D014157:Transcription Factors; D003840:Deoxycholic Acid; D004247:DNA