The Nuclear Protein IκBζ Forms a Transcriptionally Active Complex with Nuclear Factor-κB (NF-κB) p50 and the Lcn2 Promoter via the N- and C-terminal Ankyrin Repeat Motifs

Abstract

The nuclear protein IB, comprising the N-terminal transactivation domain and the C-terminal ankyrin repeat (ANK) domain composed of seven ANK motifs, activates transcription of a subset of nuclear factor-B (NF-B)-dependent innate immune genes such as Lcn2 encoding the antibacterial protein lipocalin-2. Lcn2 activation requires formation of a complex containing IB and NF-B p50, a transcription factor that harbors the DNA-binding Rel homology region but lacks a transactivation domain, on the promoter with the canonical NF-Bbinding site (B site) and its downstream cytosine-rich element. Here we show that IB productively interacts with p50 via Asp-451 in the N terminus of ANK1, a residue that is evolutionarily conserved among IB and the related nuclear IB proteins Bcl-3 and IB NS . Threonine substitution for Asp-451 abrogates direct association with the B-site-binding protein p50, complex formation with the Lcn2 promoter DNA, and activation of Lcn2 transcription. The basic residues Lys-717 and Lys-719 in the C-terminal region of ANK7 contribute to IB binding to the Lcn2 promoter, probably via interaction with the cytosinerich element required for Lcn2 activation; glutamate substitution for both lysines results in a loss of transcriptionally active complex formation without affecting direct contact of IB with p50. Both termini of the ANK domain in Bcl-3 and IB NS function in a manner similar to that of IB to interact with promoter DNA, indicating a common mechanism in which the nuclear IBs form a regulatory complex with NF-B and promoter DNA via the invariant aspartate in ANK1 and the conserved basic residues in ANK7. Nuclear factor-B (NF-B)2 plays central roles in host defense and inflammation as a homo-or heterodimer of NF-B/Rel family proteins by controlling the expression of genes for pro-inflammatory cytokines, chemokines, and antibacterial proteins (1-4). The mammalian NF-B family is composed of five structurally related polypeptides: p50, p52, p105 (the precursor of p50), p100 (the precursor of p52), p65 (also known as RelA), RelB, and c-Rel. They share the Rel homology region, which mediates dimerization, nuclear translocation, binding to specific DNA sequences known as NF-B-binding elements (B sites), and association with one of the IB family proteins (1-4). Among the members of the family, p65, RelB, and c-Rel have an ability to activate transcription by themselves via the C-terminal trans-activation domain, which is absent in the smaller p50 and p52 proteins. In resting cells, NF-B dimers are retained in the cytoplasm by associating with a member of the prototypical/cytoplasmic IB proteins including IB␣, IB␤, and IB⑀ (1-4). Cell activation with appropriate stimuli such as bacterial LPS leads to phosphorylation-induced degradation of cytoplasmic IBs and resultant liberation of NF-B dimers (1, 5, 6). The released NF-B dimers subsequently translocate to the nucleus and thus induce the expression of primary response genes via binding to B sites on their promoter/enhancer regions (1-4).The primarily induced gen...

IL‐17A synergistically enhances TLR3‐mediated IL‐36γ production by keratinocytes: A potential role in injury‐amplified psoriatic inflammation

“…However, the molecular mechanism of IκBζ‐mediated regulation on the NF‐κB–dependent IL‐36γ expression remains elusive. The cytoplasmic IκBζ members such as IκBα and IκBβ are negative regulators of NF‐κB activation and nuclear translocation, while nuclear IκBζ functions quite differently in the regulation of NF‐κB . In our study, we found that the p65 phosphorylation in response to poly(I:C)/IL‐17A was not significantly changed by IκBζ knockdown (data not shown), which were consistent with previous analyses showing that NF‐κB activation was not affected in macrophages from IκBζ‐deficient mice .…”

“…In our study, we found that the p65 phosphorylation in response to poly(I:C)/IL‐17A was not significantly changed by IκBζ knockdown (data not shown), which were consistent with previous analyses showing that NF‐κB activation was not affected in macrophages from IκBζ‐deficient mice . It was reported that IκBζ activates the expression of LCN2 by forming a regulatory complex with NF‐κB and LCN2 promoter DNA . Therefore, we postulated that the NF‐κB–dependent induction of IκBζ subsequently enhances the NF‐κB binding to the IL‐36γ promoter as a positive feedback mechanism.…”

“…*P < 0.05, **P < 0.01, ns no significant; two-way ANOVA activation and nuclear translocation, while nuclear IκBζ functions quite differently in the regulation of NF-κB. [35] In our study, we found that the p65 phosphorylation in response to poly(I:C)/IL-17A was not significantly changed by IκBζ knockdown (data not shown), which were consistent with previous analyses showing that NF-κB activation was not affected in macrophages from IκBζ-deficient mice. [36] It was reported that IκBζ activates the expression of LCN2 by forming a regulatory complex with NF-κB and LCN2 promoter DNA.…”

“…[36] It was reported that IκBζ activates the expression of LCN2 by forming a regulatory complex with NF-κB and LCN2 promoter DNA. [35] Therefore, we postulated that the NF-κB-dependent induction of IκBζ subsequently enhances the NF-κB binding to the IL-36γ promoter as a positive feedback mechanism. Future studies should aim to investigate the interaction of IκBζ, NF-κB and IL-36γ promoter.…”

Phosphorylation, Dephosphorylation, and Multiprotein Assemblies Regulate Dynamic Behavior of Neuronal Cytoskeleton: A Mini-Review

“…Ankyrin G vaccination is tested for the reduction of Aβ and lowering toxicity ( Santuccione et al, 2013 ). Ankyrin repeats are interaction sites of phosphorylation dependent dynamic assembly of proteins and nucleic acids ( Hall et al, 2018 ) including those involved in transcription regulation ( Kohda et al, 2016 ) and signaling, and present promising targets for the design of new drugs.…”