The transcription factor NF-kappaB coordinates the activation of numerous genes in response to pathogens and pro-inflammatory cytokines, and is, therefore, vital in the development of acute and chronic inflammatory diseases. NF-kappaB is activated by phsophorylation of its inhibitory subunit, IkappaB-alpha, on serine residues 32 and 36 by cytokine-activated IKB kinases (IKKs); this phosphorylation precedes rapid degradation of IkappaB. IKK-alpha and IKK-beta isozymes are found in large complexes of relative molecular mass 700,000-900,000 (M(r) 70K-90K), but little is known about other components that organize and regulate these complexes. IKK-alpha was independently discovered as a NF-kappaB-inducing kinase (NIK)-associated protein in a yeast two-hybrid screen, and IKK-beta was also identified by homology screening. It is, however, unknown whether NIK is part of the IKK complex. Here we isolate large, interleukin-1-inducible IKK complexes that contain NIK, IKK-alpha, IKK-beta, IkappaB-alpha, NF-kappaB/RelA and a protein of M(r) 150K. This latter component is a new protein, termed IKK-complex-associated protein (IKAP), which can bind NIK and IKKs and assemble them into an active kinase complex. We show that IKAP is a scaffold protein and a regulator for three different kinases involved in pro-inflammatory cytokine signalling.

This study demonstrates distinct virus-inducible enhanson properties for three regions of the human beta interferon (IFN-,B) promoter; maximum vrus inducibility required syngerism among all three enhansons. Expression of the ERF-1 transcription factor differentially increased the expression of plasmids containing (AAGTGA)4 or PRDM (-94 to -78) motifs but was inefficient in the induction of the intact IFN-0 promoter.The human T-cell (11,14,38,41). The contribution of an individual enhanson to overall enhancer activity may vary in a cellspecific manner, reflecting the relative abundance and/or activity of specific transcription factors. Four distinct classes of enhansons have been described. Class A enhansons display strict spacing requirements and exhibit enhancer activity when tandem repeats of the motif are oligomerized. Class B enhansons exhibit no enhancer activity when multimerized on their own but can generate enhancer activity when juxtaposed with class A motifs (to form a proto-enhancer) and then oligomerized. Class C enhansons possess intrinsic proto-enhancer activity and, when oligomerized, form a functional enhancer element without strict spacing requirements between enhansons. Class D enhansons, exemplified by steroid response elements, display enhancer activity once the receptor is bound (14,31,36,41).Virus-induced activation of beta interferon (IFN-P) transcription is mediated by the interaction of regulatory proteins with enhanson elements in the IFN-1 promoter. In particular, a hexameric sequence, AAGTGA, permutations of which are present throughout the IFN-P promoter between -107 and -65 relative to the mRNA start site, can function as a virus-inducible or constitutive enhancer when present in tandem repeats (12,20). Multimers of the AAGTGA hexamer generate the sequence GAAAGT, which * Corresponding author.is thought to represent a high-affinity site for two DNAbinding proteins, 18,19,26,39). Recently, different types of (GAAANN)4 sequences mediating virus inducibility have been described, indicating that different hexameric sequences are not equivalent and that other IRF-like proteins are involved in alpha interferon and IFN-,B induction (37).The natural IFN-P promoter contains an interferon regulatory element (located at -77 to -37) which includes two positive regulatory domains (PRDI and PRDII) and one negative regulatory domain, as defined by mutational analysis and protein-DNA interactions (21,22,33,48,49). The PRDI domain (-77 to -64) is thought to interact with the IRF proteins (19,26,39,48), as well as with other constitutive and inducible factors (33,37,48). The PRDII domain (5'-GGGAAATTCC-3'; -64 to -55) binds NF-KB, a cellular factor involved in the transcriptional activation of several viral and cellular genes (3,6,30,32,40,43 (16,28,34,46

The relationship between transcription of alpha and beta interferon (IFN-alpha and IFN-beta) genes and the interaction of IFN promoter-binding transcription factors has been examined in monoblastoid U937 cells following priming with recombinant IFN-alpha 2 (rIFN-alpha 2) and Sendai virus induction. Pretreatment of U937 cells with rIFN-alpha 2 prior to Sendai virus infection increased the mRNA levels of IFN-alpha 1, IFN-alpha 2, and IFN-beta as well as the final yield of biologically active IFN. Analysis of nuclear protein-IFN promoter DNA interactions by electrophoretic mobility-shift assays demonstrated increased factor binding to IFN-alpha 1 and IFN-beta regulatory domains, although no new induction-specific complexes were identified. On the basis of competition electrophoretic mobility-shift assay results, factors interacting with the IFN-alpha 1 and IFN-beta promoters appear to be distinct DNA-binding proteins. U937 factor binding was localized to the P2 domain (-64 to -55) of the IFN-beta regulatory element, a sequence motif with 80% homology to the recognition site of transcription factor NF-kappa B. Protein-DNA interactions within the IFN-beta P2 domain were, in fact, specifically competed by either excess homologous P2 fragment or the human immunodeficiency virus enhancer element which contains two duplicated NF-kappa B recognition sites. Hybrid promoter-chloramphenicol acetyltransferase fusion plasmids, containing either the IFN-beta regulatory element or the human immunodeficiency virus enhancer element linked to the simian virus 40 promoter, were analyzed for virus and phorbol ester inducibility in epithelial and lymphoid cells, respectively. In the 293 cell line, both plasmids were constitutively expressed but not virus inducible, while in Jurkat cells, chloramphenicol acetyltransferase activity from these plasmids was induced by tumor-promoting agent treatment. These experiments suggest that induction of IFN gene expression may be controlled in part by transcription regulatory proteins binding to an NF-kappa B-like site within the IFN-beta promoter.

No abstract

No abstract