DNA Binding and Phosphorylation Regulate the Core Structure of the NF-κB p50 Transcription Factor

Abstract

The NF-κB transcription factors are known to be extensively phosphorylated, with dynamic site-specific modification regulating their ability to dimerize and interact with DNA. p50, the proteolytic product of p105 (NF-κB1), forms homodimers that bind DNA but lack intrinsic transactivation function, functioning as repressors of transcription from κB promoters. Here, we examine the roles of specific phosphorylation events catalysed by either protein kinase A (PKA) or Chk1, in regulating the functions of p50 homodimers. LC-MS/MS analysis of proteolysed p50 following in vitro phosphorylation allows us to define Ser328 and Ser337 as PKA- and Chk1-mediated modifications, and pinpoint an additional four Chk1 phosphosites: Ser65, Thr152, Ser242 and Ser248. Native mass spectrometry (MS) reveals Chk1- and PKA-regulated disruption of p50 homodimer formation through Ser337. Additionally, we characterise the Chk1-mediated phosphosite, Ser242, as a regulator of DNA binding, with a S242D p50 phosphomimetic exhibiting a > 10-fold reduction in DNA binding affinity. Conformational dynamics of phosphomimetic p50 variants, including S242D, are further explored using ion-mobility MS (IM-MS). Finally, comparative theoretical modelling with experimentally observed p50 conformers, in the absence and presence of DNA, reveals that the p50 homodimer undergoes conformational contraction during electrospray ionisation that is stabilised by complex formation with κB DNA. Graphical Abstract ᅟ.

The regulation of sequence specific NF-κB DNA binding and transcription by IKKβ phosphorylation of NF-κB p50 at serine 80

“…p50 homodimers are important repressors of inflammatory gene expression and the stability of p50 homodimers is crucial for limiting pro-inflammatory gene expression and establishing Toll-like Receptor tolerance in macrophages (3,10). The phosphorylation of p50 at serine 337 (S337) is required for DNA binding (11), while the phosphorylation of S242 inhibits p50 homodimer DNA binding (12) .Phosphorylation of S20 promotes DNA binding, and is required for VCAM-1 expression in response to TNFα (13). p50 phosphorylation at S328 occurs in response to DNA damage and regulates the interaction of p50 with specific NF-κB binding sites to inhibit anti-apoptotic gene expression (14,15).…”

Probing the role of macromolecular crowding in cell volume regulation using fluorescence anisotropy

“…Literature also suggests that multivalent proteins form larger aggregates in response to increased MMC via self-condensation (Shin and Brangwynne, 2017; Watanabe et al ., 2018; Jalihal et al ., 2020, 2021; Keber et al ., 2021). While free EGFP depicts a non-specific weaker aggregation in the cytoplasm, NFκβ, a protein with DNA binding domain (Yan et al ., 2012; Smith et al ., 2019; Vonderach et al ., 2019), shows globular self aggregation in response to hypertonic shock in NIH/3T3 cells expressing p65-EGFP, a subunit of the NFκβ complex ( Fig S4A-i ). However, in Heclin treated NIH/3T3, NFκβ shows drastically massive, irregularly shaped aggregates ( Fig S4A-ii ) compared to the globular condensates from hypertonic shock.…”

Phospho-Site Mutations in Transcription Factor Suppressor of Hairless Impact Notch Signaling Activity During Hematopoiesis in Drosophila

“…Although novel for CSL transcription factors, phosphorylation as a means of regulating DNA-binding was previously described for other important transcription factors. This includes for example, the nuclear c-Myb oncoprotein (Lüscher et al, 1990), NF-kappaB (Vonderach et al, 2018), members of the Forkhead (Fox) family (Brent et al, 2008;Nie et al, 2013;Blane et al, 2018), as well as members of the Stat-family of transcription factors (Maiti et al, 2005). Interestingly, like CSL-mediated Notch signaling, most of these transcription factors regulate a great variety of biological processes.…”