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Dynamic phosphorylation of RelA on Ser42 and Ser45 in response to TNFα stimulation regulates DNA binding and

Francesco Lanucara1, Connie Lam2, Jelena Mann3

  • 1Centre for Proteome Research, Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool L69 7ZB, UK.

Open Biology
|July 29, 2016
PubMed

Insights

The study reveals that tumor necrosis factor alpha (TNFα) triggers dynamic, multisite phosphorylation of the RelA/p65 protein, uncovering new regulatory mechanisms in NF-κB signaling. These findings suggest complex control of transcription via distinct NF-κB proteoforms.

Area of Science:

  • Molecular Biology
  • Cell Signaling
  • Proteomics

Background:

  • The Nuclear Factor kappa B (NF-κB) signaling pathway regulates gene transcription through a complex network of proteins.
  • Phosphorylation of NF-κB subunits, particularly RelA/p65, is crucial for controlling pathway dynamics and gene expression.
  • Understanding the precise phosphorylation events on RelA/p65 is key to deciphering NF-κB-mediated transcriptional control.

Purpose of the Study:

  • To identify and characterize novel phosphorylation sites on the RelA/p65 protein induced by tumor necrosis factor alpha (TNFα).
  • To investigate the functional consequences of RelA/p65 phosphorylation on DNA binding and transcriptional activity.
  • To explore the role of dynamic phosphorylation patterns in regulating NF-κB-mediated transcription.

Main Methods:

  • Utilized both non-targeted discovery and quantitative selected reaction monitoring-targeted proteomics to analyze RelA/p65 phosphorylation.
  • Employed computational modeling of crystal structures to predict the functional roles of identified phosphorylation sites.
  • Performed quantitative analyses to determine phosphorylation stoichiometry and assess promoter-specific DNA binding.

Main Results:

  • Identified dynamic, multisite phosphorylation of RelA/p65 at previously unknown residues in response to TNFα.
  • Observed preferential early phosphorylation of Ser45 over Ser42 upon TNFα stimulation.
  • Demonstrated differential roles of pSer42 and pSer45 in promoter-specific DNA binding and IL-6 promoter transcription regulation.

Conclusions:

  • RelA/p65 phosphorylation is a dynamic, multisite event critical for NF-κB pathway regulation.
  • Distinct phosphorylation patterns on RelA/p65 contribute to functionally different proteoforms.
  • Transcriptional control by NF-κB is likely mediated by a combinatorial code of protein modifications rather than single events.
Keywords:
NF-κBRelAphosphorylationproteomicsquantificationtranscription

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