Duckett C S, Perkins N D, Kowalik T F, Schmid R M, Huang E S, Baldwin A S, Nabel G J
Department of Internal Medicine, Howard Hughes Medical Institute, University of Michigan Medical Center, Ann Arbor 48109-0650.
Mol Cell Biol. 1993 Mar;13(3):1315-22. doi: 10.1128/mcb.13.3.1315-1322.1993.
Inducible expression of human immunodeficiency virus (HIV) is regulated by a cellular transcription factor, nuclear factor kappa B (NF-kappa B). NF-kappa B 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 kappa B 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 kappa B site. p49 has a approximately 18-fold-lower affinity for the HIV kappa B site (KD = 69.1 pM) than does the approximately 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 approximately 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 I kappa B-alpha(MAD-3). Recombinant I kappa B-alpha(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.
人类免疫缺陷病毒（HIV）的诱导性表达受一种细胞转录因子——核因子κB（NF-κB）调控。NF-κB由不同的亚基组成；已分离出五个独立基因，即NFKB1（p105）、NFKB2（p100）、RelA（p65）、c-rel和relB，它们编码与κB DNA元件结合的相关蛋白质。我们之前发现，NFKB2（p49/p52）与RelA（p65）协同作用，在Jurkat T白血病细胞中刺激HIV增强子。在此，我们研究NFKB2对HIV转录调控的生化基础。通过Scatchard分析，我们确定了同二聚体p49和异二聚体p49/p65与HIV κB位点结合的解离常数。与源自p105的约50 kDa蛋白NFKB1（p50）（KD = 3.9 pM）相比，p49对HIV κB位点的亲和力低约18倍（KD = 69.1 pM）。相反，异二聚体NFKB2（p49）/RelA（p65）对该位点的亲和力比单独的p49高约6倍（KD = 11.8 pM）。与这些发现一致，添加预先形成的异二聚体NFKB2（p49）/RelA（p65）蛋白可使体外转录增强18倍。HIV增强子的转录激活也受最近克隆的IκB-α（MAD-3）调控。重组IκB-α（MAD-3）抑制p65、p49/p65和p50/p65的DNA结合活性，但刺激NFKB2（p49）或NFKB1（p50）的结合。在瞬时转染的Jurkat T白血病细胞中，MAD-3的共表达也抑制了p49/p65对HIV报告质粒的功能激活。这些数据表明，RelA（p65）促进了NFKB2亚基与HIV增强子的结合，并且这种NFKB2（p49）/p65异二聚体复合物介导了受MAD-3调控的转录激活。
