Functional characterization of the NF-kappa B p65 transcriptional activator and an alternatively spliced derivative.

Abstract

The NF-KB transcription factor complex is composed of two proteins, designated p50 and p65, both having considerable homology to the product of the rel oncogene. We present evidence that the p65 subunit is a potent transcriptional activator in the apparent absence of the p50 subunit, consistent with in vitro results demonstrating that p65 can interact with DNA on its own. To identify the minimal activation domain, chimeric fusion proteins between the DNA binding domain of the yeast transcriptional activator protein GAL4 and regions of the carboxy terminus of p65 were constructed, and their transcriptional activity was assessed by using a GAL4 upstream activation sequence-driven promoter-chloramphenicol acetyltransferase fusion. This analysis suggests that the boundaries of the activation domain lie between amino acids 415 and 550. Moreover, single amino acid changes within residues 435 to 459 greatly diminished activation. Similar to other activation domains, this region contains a leucine zipper-like motif as well as an overall net negative charge. To identify those residues essential for DNA binding, we made use of a naturally occurring derivative of p65, lacking residues 222 to 231 (hereafter referred to as p65A), and produced via an alternative splice site. Gel mobility shift analysis using bacterially expressed p65, p65A, and various mutants indicates that residues 222 to 231 are important for binding to KB DNA. Coimmunoprecipitation analysis suggests that these residues likely contribute to the multimerization function required for homomeric complex formation or heteromeric complex formation with p50 in that no association of p65A with itself or with p50 was evident. However, p65A was able to form weak heteromeric complexes with p65 that were greatly reduced in their ability to bind DNA. On the basis of these findings, we suggest that subtle changes within the proposed multimerization domain can elicit different effects with the individual Rel-related proteins and that a potential role of p65A may be to negatively regulate NF-KB function through formation of nonfunctional heteromeric complexes.The NF-KB transcription factor complex is a pleiotropic activator that participates in the induction of numerous cellular and viral genes (4, 32). The active complex is composed of two subunits, designated p50 and p65 (3, 18). The genes encoding p50 (19, 29) and p65 (37, 45) have recently been cloned, and the amino termini of both proteins have been shown to have considerable homology to the rel oncogene product. The rel oncogene, originally identified in the avian retrovirus Rev T, causes lymphoid cell tumors in birds (41). It is now clear that the Rel family of proteins, of which the Drosophila maternal morphogen dorsal is a member (55), possess transcriptional regulatory properties (8,16,23,28,42,58), as best exemplified by the NF-KB transcription factor complex.NF-KB exists in both an active and an inactive state. The inactive complex is present in the cytosol as a complex bound to an inhibitor protein termed IKB...

Copine-I represses NF-κB transcription by endoproteolysis of p65

“…More work is needed to understand how truncated p65 protein acts as a repressor. The RHD of p65 interacts with p50 in a region located between residues 222 and 232 (Ruben et al, 1992). Since copine-I cleaves p65 across residues 31-51, the 55 kDa form of p65 still contains its p50-interacting domain.…”

Rel/NF-κB transcription factors and IκB inhibitors: Evolution from a unique common ancestor

“…cRel, RelA and RelB paralogous genes diverged with similar evolutionary speed, although the RelB gene seems to have slightly accelerated. Despite their highly conserved features, they strikingly di er in their dimerization speci®city since RelB proteins are unable to homodimerize (Dobrzanski et al, 1993;Ruben et al, 1992b;Ryseck et al, 1992;Suzuki et al, 1995). The third group of subfamily I proteins contains Dorsal and Dif which present the RHD-TA organization and bind DNA as homodimers only.…”

Transcriptional regulatory effects of lymphoma-associated NFKB2/lyt10 protooncogenes

“…The Lyt10-Ca-expression plasmid, pHCMV lyt10Ca, was kindly provided by Dr Riccardo Dalla-Favera (Columbia University, New York, NY, USA). The pCMV-p65RelA expression vector (Ruben et al, 1992) was a kind gift of Dr C Rosen.…”