Dimerization of NF-KB2 with RelA(p65) regulates DNA binding, transcriptional activation, and inhibition by an IκB-α (MAD-3)

Colin S. Duckett, Neil D. Perkins, Timothy F. Kowalik, Roland M. Schmid, Eng Shang Huang, Albert S. Baldwin, Gary J. Nabel

Research output: Contribution to journalArticle

Abstract

Inducible expression of human immunodeficiency virus (HIV) is regulated by a cellular transcription factor, nuclear factor κB (NF-κB). NF-κB is composed of distinct subunits; five independent genes, NFKB1(p105), NFKB2(p100), RelA(p65), c-rel and relB, that encode related proteins that bind to κB DNA elements have been isolated. We have previously found that NFKB2(p49/p52) acts in concert with RelA(p65) to stimulate the HIV enhancer in Jurkat T-leukemia cells. Here we examine the biochemical basis for the transcriptional regulation of HIV by NFKB2. Using Scatchard analysis, we have determined the dissociation constants of homodimeric p49 and heterodimeric p49/p65 for binding to the HIV kB site. p49 has a ∼18-fold-lower affinity for the HIV kB site (KD = 69.1 pM) than does the ∼50-kDa protein NFKB1(p50) derived from p105 (KD = 3.9 pM). In contrast, the affinity of heterodimeric NFKB2(p49)/RelA(p65) for this site is ∼-6-fold higher (KD = 11.8 pM) than that of p49 alone. Consistent with these findings, in vitro transcription was stimulated 18-fold by the addition of preformed, heterodimeric NFKB2(p49)/RelA(p65) protein. Transcriptional activation of the HIV enhancer was also subject to regulation by recently cloned IκB-α(MAD-3). Recombinant IκB-α(MAD-3) inhibited the DNA binding activity of p65, p49/p65, and p50/p65 but stimulated the binding of NFKB2(p49) or NFKB1(p50). Functional activation of an HIV reporter plasmid by p49/p65 in transiently transfected Jurkat T-leukemia cells was also inhibited by coexpression of MAD-3. These data suggest that binding of the NFKB2 subunit to the HIV enhancer is facilitated by RelA(p65) and that this NFKB2(p49)/p65 heterodimeric complex mediates transcriptional activation which is subject to regulation by MAD-3.

Original languageEnglish (US)
Pages (from-to)1315-1322
Number of pages8
JournalMolecular and Cellular Biology
Volume13
Issue number3
StatePublished - Mar 1993
Externally publishedYes

ASJC Scopus subject areas

  • Cell Biology
  • Genetics
  • Molecular Biology

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    Duckett, C. S., Perkins, N. D., Kowalik, T. F., Schmid, R. M., Huang, E. S., Baldwin, A. S., & Nabel, G. J. (1993). Dimerization of NF-KB2 with RelA(p65) regulates DNA binding, transcriptional activation, and inhibition by an IκB-α (MAD-3). Molecular and Cellular Biology, 13(3), 1315-1322.