Identification of a Novel Inhibitor of Nuclear Factor-κB, RelA-associated Inhibitor

Abstract

Here we report the identification and characterization of a novel protein, RelA-associated inhibitor (RAI), that binds to the NF-B subunit p65 (RelA) and inhibits its transcriptional activity. RAI gene was isolated in a yeast two-hybrid screen using the central region of p65 as bait. We confirmed the physical interaction in vitro using recombinant proteins as well as in vivo by immunoprecipitation/Western blot assay. RAI gene encodes a protein with homology to the C-terminal region of 53BP2 containing four consecutive ankyrin repeats and an Src homology 3 domain. RAI mRNA was preferentially expressed in human heart, placenta, and prostate. Despite its similarity to 53BP2, RAI did not interact with p53 in a yeast two-hybrid assay. RAI inhibited the action of NF-B p65 but not that of p53 in transient luciferase gene expression assays. Similarly, RAI inhibited the endogenous NF-B activity induced by tumor necrosis factor-␣. RAI specifically inhibited the DNA binding activity of p65 when co-transfected in 293 cells. RAI protein appeared to be located in the nucleus and colocalized with NF-B p65 that was activated by TNF-␣. These observations indicate that RAI is another inhibitor of NF-B in addition to IB proteins and may confer an alternative mechanism of regulation. Nuclear factor B (NF-B)1 is a sequence-specific DNAbinding protein complex which regulates the expression of viral genomes, including the human immunodeficiency virus, and a variety of cellular genes, particularly those involved in immune and inflammatory responses (1-4). The members of the NF-B family in mammalian cells include the proto-oncogene c-Rel, p50/p105 (NFB1), p65 (RelA), p52/p100 (NFB2), and RelB. All of these proteins share a conserved 300-amino acid region known as the Rel homology domain which is responsible for DNA binding, dimerization, and nuclear translocation of NF-B (5, 6). In most cells, Rel family members form heteroand homodimers with distinct specificities in various combinations. A common feature of the regulation of the NF-B family is their sequestration in the cytoplasm as inactive complexes with a class of inhibitory molecules known as IBs (2, 7). Treatment of cells with a variety of inducers such as phorbol esters, interleukin 1, and tumor necrosis factor-␣ (TNF-␣) results in dissociation of the cytoplasmic complexes and translocation of NF-B to the nucleus (8, 9). The dissociation of NF-B⅐IB complexes is known to be triggered by the phosphorylation and subsequent degradation of IB proteins (10 -12). This exposes the nuclear localization sequence in the remaining NF-B heterodimer, leading to nuclear translocation and subsequent binding of NF-B to DNA regulatory elements of the target genes. The p65 subunit is a major component of NF-B complexes and is responsible for trans-activation (13).Here we report the identification, cloning, and characterization of a novel RelA-binding protein, called RAI for "RelAassociated inhibitor." The RAI cDNA encodes a protein with high structural homology to the C-terminal 200 amino acid seg...

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A polymorphism in NFKB1 is associated with improved effect of interferon- maintenance treatment of patients with multiple myeloma after high-dose treatment with stem cell support

“…[13][14][15] PPP1R13L (protein phosphate 1, regulatory (inhibitory) subunit 13 like, also named RAI or iASPP) encodes an inhibitor of the RelA subunit of NF-κB. 16 It is, therefore, possible that polymorphisms affecting the activity of PPP1R13L could influence the activity of NK-κB.…”

Nuclear transcription factor-κB as a target for cancer drug development

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