Conservation of transcriptional activation functions of the NF-kappa B p50 and p65 subunits in mammalian cells and Saccharomyces cerevisiae.

Abstract

The NF-KB transcription factor complex is composed of a 50-kDa (p50) and a 65-kDa (p65) subunit. Both subunits bind to similar DNA motifs and elicit transcriptional activation as either homo-or heterodimers. By using chimeric proteins that contain the DNA binding domain of the yeast transcriptional activator GALA and subdomains of p65, three distinct transcriptional activation domains were identified. One domain was localized to a region of 42 amino acids containing a potential leucine zipper structure, consistent with earlier reports. Two other domains, both acidic and rich in prolines, were also identified. Of perhaps more significance, the same minimal activation domains that were functional in mammalian cells were also functional in the yeast Saccharomyces cerevisiae. Coexpression of the NF-KcB inhibitory molecule, IKB, reduced the transcriptional activity of p65 significantly, suggesting the ability of IKB to function in a similar manner in S. cerevisiae. Surprisingly, while the conserved rel homology domain of p65 demonstrated no transcriptional activity in either mammalian cells or S. cerevisiae, the corresponding domain in p50 was a strong transcriptional activator in S. cerevisiae. The observation that similar domains elicit transcriptional activation in mammalian cells and S. cerevisiae demonstrates strong conservation of the transcriptional machinery required for NF-cB function and provides a powerful genetic system to study the transcriptional mechanisms of these proteins.Nuclear factor KB (NF-KB), originally described as a factor involved in tissue-specific control of the mouse K light-chain enhancer (65,66), is now known to be involved in the inducible expression of a wide range of genes (2, 6, 44). The KB DNA binding site has been identified in the regulatory elements of cytokine, cytokine receptor, major histocompatibility complex class I and II antigens, inflammatory and acute-phase response genes, and several viral enhancer elements (2, 6, 44). DNA binding activity appears to be constitutive in mature B cells (66) and in activated macrophages (24). However, in most other cells NF-KB is complexed with an inhibitory molecule (IKB) in the cytoplasm (3,4,21). Sequestration in the cytoplasm is thought to involve masking of the nuclear localization signal (8). Upon activation by a variety of stimuli, including mitogens, cytokines, and various viral proteins (2, 6, 44), NF-KB shuttles to the nucleus, a process thought to involve phosphorylation of IKB (21,68). Initial studies demonstrated that IKB inhibits binding of the p65 homodimer and the p5O/p65 heterodimer through interaction with p65 (5, 21, 54, 78). However, recent studies suggest that both subunits can interact with IKB (8).Biochemical studies have shown that the major form of NF-KB is composed of a heterodimer of a 50-kDa (p50) and a 65-kDa (p65) subunit (5). Both subunits of NF-KB have been cloned, and sequence analyses demonstrate strong similarity in the amino-terminal 300 amino acids to the rel proto-oncogene product (22,38,54,58), origi...

NF-?B inhibition accelerates apoptosis of bovine neutrophils

“…Both family members contain at least one transcription activation domain which makes them responsible for gene induction [25]. Since these observations were obtained in the human species, the need exists to demonstrate the presence of both proteins in their bovine counterpart.…”

Mutational analysis of the transcription activation domain of RelA: identification of a highly synergistic minimal acidic activation module.

“…Instead, these workers suggested that a pattern of aromatic and large hydrophobic amino acids plays a critical role in mediating activation of transcription by VP16. In this study, we sought to test the generality of this observation by functional dissection of the highly potent transcription activation domain of the RelA(p65) subunit of the inducible cellular transcription factor NF-KB (1,40). This 135-amino-acid (aa) activation domain has previously been shown to contain at least three discrete activation subdomains and has characteristics of both an acidic and a proline-rich transcriptional activator (31,41). Here, we provide evidence suggesting that this domain likely contains at least three discrete acidic activation modules (AAMs) centered on three phenylalanine residues.…”

Identification of a tumor-associated mutant form of the NF-κB RelA gene with reduced DNA-binding and transactivating activities

“…Immuno¯uorescence analysis was performed using an antibody speci®c for the C-terminal region of RelA domain, which has been localized between amino acids 416 and 550 Moore et al, 1993;Blair et al, 1994). This domain is highly conserved throughout species; 86% and 37% amino acids of human p65 protein are identical to those of mouse and chicken RelA respectively (Nolan et al, 1991;Ikeda et al, 1993).…”

“…This domain is highly conserved throughout species; 86% and 37% amino acids of human p65 protein are identical to those of mouse and chicken RelA respectively (Nolan et al, 1991;Ikeda et al, 1993). Three independent and cooperating acidic activating modules (AAMs) have been identi®ed in this region (Moore et al, 1993;Blair et al, 1994). In particular, each module consists of bulky, mainly acidic hydrophobic amino acids, surrounding a phenylalanine residue; mutation of phenylalanines 444, 473 and 541 reduces or abrogates the transcriptional activity of RelA (Blair et al, 1994).…”

“…In particular, each module consists of bulky, mainly acidic hydrophobic amino acids, surrounding a phenylalanine residue; mutation of phenylalanines 444, 473 and 541 reduces or abrogates the transcriptional activity of RelA (Blair et al, 1994). Furthermore, phenylalanine 444 is contained within a leucin zipper-like (LZ) motif (aa 443 to 451), a domain involved in protein-protein interactions; disruption of this motif by means of site directed mutagenesis completely abolishes the transcriptional activity of RelA Moore et al, 1993). Our analysis indicates that RelA 494D has less transactivating ability (about 50%) than normal RelA; however, the amino acidic substitution within the TA domain does not seem to alter its intrinsic transactivating potential, as shown by the ability of the Gal4-RelA 494D fusion protein to fully activate a reporter plasmid containing Gal4-responsive elements.…”