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

Journal Article · Tue Jan 15 04:00:00 EST 2019 · Journal of the American Society for Mass Spectrometry

DOI:https://doi.org/10.1007/S13361-018-1984-0· OSTI ID:22923189

Vonderach, Matthias ^[1]; Byrne, Dominic P. ^[2]; Barran, Perdita E. ^[3]; Eyers, Patrick A. ^[2]

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1. University of Liverpool, Centre for Proteome Research, Department of Biochemistry, Institute of Integrative Biology (United Kingdom)
2. University of Liverpool, Department of Biochemistry, Institute of Integrative Biology (United Kingdom)
3. The University of Manchester, Michael Barber Centre for Collaborative Mass Spectrometry, Manchester Institute of Biotechnology (United Kingdom)

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{sub c}) 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{sub c}- and Chk1-mediated modifications, and pinpoint an additional four Chk1 phosphosites: Ser65, Thr152, Ser242 and Ser248. Native mass spectrometry (MS) reveals Chk1- and PKA{sub c}-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. .

• OSTI ID: 22923189
• Journal Information: Journal of the American Society for Mass Spectrometry, Journal Name: Journal of the American Society for Mass Spectrometry Journal Issue: 1 Vol. 30; ISSN 1044-0305; ISSN JAMSEF
• Country of Publication: United States
• Language: English

Related Subjects

74 ATOMIC AND MOLECULAR PHYSICS
DNA
ION MOBILITY
IONIZATION
MASS SPECTROSCOPY
MOLECULES
PHOSPHORYLATION
PHOSPHOTRANSFERASES
PROMOTERS
SIMULATION
TRANSCRIPTION FACTORS