IκBβ Interacts with the Retinoid X Receptor and Inhibits Retinoid-dependent Transactivation in Lipopolysaccharide-treated Cells

Abstract

To elucidate the molecular action of the NFB inhibitor IB␤, we isolated a number of IB␤ interactors using the yeast two-hybrid system. These include the retinoid X receptor (RXR), whose interaction with IB␤ is significantly stimulated by the RXR ligand 9-cis-retinoic acid, as shown in the yeast system as well as the glutathione S-transferase pull down assays. RXR is a nuclear protein, whereas IB␤ accumulates in the nucleus only in cells stimulated with lipopolysaccharide or other inducers that result in prolonged activation of NFB. Consistent with this, cotransfection with IB␤ specifically repressed the 9-cis-RA-induced transcriptional activities of RXR in an lipopolysaccharide-dependent manner. These results suggest a novel IB␤-mediated antagonism between the signaling pathways of NFB and RXR.The transcription factor NFB is important for the inducible expression of a wide variety of cellular and viral genes (1, 2). NFB is composed of homo-and heterodimeric complexes of members of the Rel/NFB family of polypeptides. In vertebrates, this family comprises p50, p65 (RelA), c-Rel, p52, and RelB. These proteins share a 300-amino acid region, known as the Rel homology domain, which binds to DNA and mediates homo-and heterodimerization. This domain also is the target of the IB␤ inhibitors, which include IB␣, IB␤, IB␥, Bcl-3, p105, and p100 (3). In the majority of cells, NFB exists in an inactive form in the cytoplasm, bound to the inhibitory IB proteins. Treatment of cells with various inducers results in the degradation of IB proteins. The bound NFB is released and translocates to the nucleus, where it activates appropriate target genes. IB␣ is degraded in response to all of the known inducers of NFB, whereas IB␤ is degraded only when cells are stimulated with inducers such as lipopolysaccharide (LPS) 1 and interleukin-1 that cause persistent activation of NFB (4). Following degradation of the initial pool of IB␤ in response to LPS or interleukin-1, newly synthesized IB␤ accumulates in the nucleus as an unphosphorylated protein that forms a stable complex with NFB and prevents it from binding to newly synthesized IB␣ (5-7).To understand the molecular action of IB␤, we exploited the yeast two-hybrid system (8) to isolate a series of cDNAs encoding proteins that specifically interact with IB␤. Interestingly, these include retinoid X receptor (RXR), a member of the nuclear hormone receptors that comprise a large family of liganddependent transcription factors, bind as homodimers or heterodimers to their cognate DNA elements, and regulate genes involved in critical aspects of cell proliferation, differentiation, and homeostasis (9). Herein, we show that the RXR-IB␤ interactions are stimulated by the RXR ligand 9-cis-RA and that cotransfection with IB␤ specifically represses the 9-cis-RAinduced transcriptional activities of RXR in an LPS-dependent manner. These results are consistent with a novel IB␤-mediated antagonism between the signaling pathways of NFB and RXR. EXPERIMENTAL PROCEDURESPlasmids-Polymerase chain reaction...

Per-Arnt-Sim domain-dependent association of cAMP-phosphodiesterase 8A1 with IκB proteins

“…However, the possibility of an involvement of the PDE8A1-I B association in the NF-B pathway cannot be ruled out. Several proteins have been reported to physically associate with I B, including protein kinase A (28), cyclin-dependent kinase 4 (29), Tax protein (30), B-Ras (31), and retinoid X receptor (32). The I B binding by each of these proteins has certain impact on NF-B pathway.…”

Interactions between Activating Signal Cointegrator-2 and the Tumor Suppressor Retinoblastoma in Androgen Receptor Transactivation

“…Specifically bound proteins were eluted from beads with 40 mM reduced glutathione in 50 mM Tris (pH 8.0) and analyzed by SDS-PAGE and autoradiography as described previously (20).…”

“…Total amounts of expression vectors were kept constant by adding pcDNA3. Transfections and luciferase assays were done as described (20), and the results were normalized to the LacZ expression. Similar results were obtained in more than two similar experiments.…”

β-Arrestin inhibits NF-κB activity by means of its interaction with the NF-κB inhibitor IκBα

“…Thus, proteins that bind to I B are critical regulators of NF-B activity. Several proteins have been shown to interact with I B, including the retinoic acid receptor (19), the catalytic subunit of protein kinase A (20), and B-ras proteins (21). Here, we identify ␤-arrestin as a binding partner of I B␣ and characterize its effect on NF-B signaling.…”