Mechanism of IκBα Binding to NF-κB Dimers

Abstract

X-ray crystal structures of the NF-B⅐IB␣ complex revealed an extensive and complex protein-protein interface involving independent structural elements present in both IB␣ and NF-B. In this study, we employ a gel electrophoretic mobility shift assay to assess and quantitate the relative contributions of the observed interactions toward overall complex binding affinity. IB␣ preferentially binds to the p50/p65 heterodimer and p65 homodimer, with binding to p50 homodimer being significantly weaker. Our results indicate that the nuclear localization sequence and the region C-terminal to it of the NF-B p65 subunit is a major contributor to NF-B⅐IB␣ complex formation. Additionally, there are no contacts between the corresponding nuclear localization signal tetrapeptide of p50 and IB␣. A second set of interactions involving the acidic C-terminal/PEST-like region of IB␣ and the NF-B p65 subunit N-terminal domain also contributes binding energy toward formation of the complex. This interaction is highly dynamic and nonspecific in nature, as shown by oxidative cysteine cross-linking. Phosphorylation of the C-terminal/ PEST-like region by casein kinase II further enhances binding.The Rel/NF-B family transcription factors perform a vital role in mediating the cellular response to stress, inflammation, the immune response, and apoptosis (1-4). Mammalian Rel/ NF-B family polypeptides include p65 (RelA), p50, p52, RelB, and the proto-oncoprotein c-Rel. These subunits associate in various combinations to form homodimers and heterodimers with distinct but overlapping functions. Among the most abundant and best understood of these dimers are the p50/p65 heterodimer and the homodimers of p50 and p65 (5). The functions of other NF-B dimers are generally restricted to specific cell types.In most cells the NF-B p50/p65 heterodimer exists in the cytoplasm as a complex with the inhibitor protein IB␣. IB␣ inactivates the NF-B p50/p65 heterodimer by masking the NF-B nuclear localization sequences (6). Activation of signaling pathways by extracellular signals, such as tumor necrosis factor or interleukin-1, leads to proteolysis of IB␣, allowing active NF-B p50/p65 heterodimer to translocate into the nucleus (7).IB␣ is composed of three distinct regions: an N-terminal signal receiving domain (SRD), 1 a central ankyrin repeat-containing domain (ARD), and an acidic C-terminal/PEST-like region rich in the amino acids proline, glutamic acid, serine, and threonine (8). IB␣ is phosphorylated my multiple proteins in vivo. The C-terminal/PEST-like region in constitutively phosphorylated by casein kinase II (CKII) (9, 10), and the SRD contains sites of inducible, signaling-dependent phosphorylation (11). X-ray crystal structures of IB␣ in complex with the NF-B p50/p65 heterodimer reveal an extensive and complex protein-protein interface (12, 13). The structures reveal that an apparently flexible segment of the NF-B p65 subunit, which contains the NLS, becomes ordered upon complex formation and makes several contacts with IB␣. Interestingly, no direc...

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Temporal Control of NF-κB Activation by ERK Differentially Regulates Interleukin-1β-induced Gene Expression

“…leads to the phosphorylation and ubiquitination-dependent degradation of I-B, accompanied by NF-B translocation to the nucleus, where it binds to consensus sequences in the promoter region of target genes and activates transcription (1)(2)(3)(4)(5)(6)(7). I-B␣ is rapidly degraded after cytokine stimulation and rapidly resynthesized because of the presence of NF-B-binding motifs in the I-B␣ gene promoter (8).…”

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“…The nuclear factor kappa B/Rel family (NFκB/Rel) is a family of inducible transcription factors involved in numerous cellular processes such as immune and inflammatory responses, cell proliferation, apoptosis and development [1][2][3]. The five members of the mammalian NFκB transcription family are p65 (RelA), RelB, c-Rel, p105/p50 and p100/p52, which associate as homo-and heterodimers [4,5].…”

“…The five members of the mammalian NFκB transcription family are p65 (RelA), RelB, c-Rel, p105/p50 and p100/p52, which associate as homo-and heterodimers [4,5]. The p50/p65 heterodimer is considered the most abundant and best understood complex among the NFκB dimers and plays various roles in gene regulation [3,6]. In unstimulated cells the NFκB p50/p65 heterodimer predominantly exists in the cytoplasm as an inactive complex form with the inhibitor protein IκBα.…”