The p65 subunit is responsible for the strong transcription activating potential of NF-kappa B.

Abstract

The nuclear form of the NF‐kappa B transcription factor binds to DNA as a heterodimer of a 50 kDa (p50) and 65 kDa (p65) polypeptide. The two polypeptides are encoded by different genes but share a long region of homology, the NRD motif, encompassing domains required for DNA binding and dimerization. In this study we have analysed the contribution of the two subunits to the strong transactivating potential of NF‐kappa B. Transient expression of the p65 subunit alone resulted in a potent transactivation of a CAT reporter construct under the control of two NF‐kappa B binding sites in monkey COS and mouse L cells. The strongly DNA binding p50 subunit showed only very weak, if any, induction of gene expression. Co‐expression of p50 suppressed the transactivation by p65 presumably by competitive DNA binding of transcriptionally inactive p50 dimers (KBF1). Fusion of p65 sequences to DNA binding domain of the yeast GAL4 transcription factor allowed detection of the principal transactivation domain of p65 (TA1) in the C‐terminal 30 amino acid sequence. TA1 is likely to adopt an amphipathic alpha‐helical structure which clusters serine residues on the hydrophilic surface, a structural feature conserved between human, mouse and Xenopus p65. The unique C‐terminal third of p65 contained at least one more activation domain, TA2, within a 90 amino acid sequence directly adjacent to TA1. In two mammalian cell lines, TA1 and TA2 acted separately, while in an insect cell line, the two domains were inactive after their separation. Our study suggests that the p50 subunit in NF‐kappa B might only serve a helper function in DNA binding whereas the p65 subunit is responsible for initiating transcription. Homodimers of p50 seem to have the potential of down‐regulating kappa B‐specific gene expression.

Anti-inflammatory gene therapy directed at the airway epithelium

“…9 Various homo- and heterodimer isoforms have been identified, but the heterodimer consisting of the REL proteins p50 and p65 (Rel A) appears to be most prominent in the regulation of the inflammatory response. 10 NF B is constitutively expressed in most cell types, but in unstimulated cells it is retained in an inactive form in the cytoplasm, because of its association with inhibitory proteins (I Bs), of which several are known (␣, ␤, ␥, ⑀); 11 I B␣ is the best studied member of this family. NF B is activated by a large number of stimuli, including micro-organisms, cytokines and oxidative stress.…”

cRel-TD kinase: a serine/threonine kinase binding in vivo and in vitro c-Rel and phosphorylating its transactivation domain

“…Indeed, in contrast to unspeci®c proteins, p66 remained attached to the recombinant GST-RelTD or GSTRelDD proteins after high stringency washes (Fognani, unpublished data), a property shared by the c-Jun/JNK interaction (Leppa et al, 1998). A dual site of interaction is not unique; the association between CBP/p300 and RelA occurs through a bivalent interaction, one phosphorylation-independent, one (Schmitz and Baeuerle, 1991). In the right panel, luciferase activity assayed from extracts of transiently transfected COS7 cells.…”

Interaction of the fork head domain transcription factor MPP2 with the human papilloma virus 16 E7 protein: enhancement of transformation and transactivation

“…As reporter plasmids p(Gal) 4 -tk-luc (Janknecht et al, 1993), which contains four Gal4 binding sites, and p(MBS) 3 -mintk-luc, which contains three MPP2 binding sites (HFH-11 #24, Ye et al, 1997) were used. For expression of Gal4 fusion proteins, sequences encoding E7(2-98), E7(2-41), and E7(42-98) were cloned into the poly-linker of pGallinker-HA, a derivative of pABgal-linker HA (Schmitz and Baeuerle, 1991). Gal4-Fos and Gal4-Myc(1-262) were obtained from T Kouzarides and C Dang, respectively (Kato et al, 1990;Bannister and Kouzarides, 1995).…”