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

Francesco Lanucara, Connie Lam, Jelena Mann, Tom P. Monie, Stefano A P Colombo, Stephen W. Holman, James Boyd, Manohar C. Dange, Derek A. Mann, Michael R H White, Claire E. Eyers

Research output: Contribution to journalArticlepeer-review

Abstract

The NF-κB signalling module controls transcription through a network of protein kinases such as the IKKs, aswell as inhibitory proteins (IκBs) and transcription factors including RelA/p65. Phosphorylation of the NF-κB subunits is critical for dictating system dynamics. Using both non-targeted discovery and quantitative selected reaction monitoring-targeted proteomics, we show that the cytokine TNFα induces dynamic multisite phosphorylation of RelA at a number of previously unidentified residues. Putative roles for many of these phosphorylation sites on RelA were predicted by modelling of various crystal structures. Stoichiometry of phosphorylation determination of Ser45 and Ser42 revealed preferential early phosphorylation of Ser45 in response to TNFα. Quantitative analyses subsequently confirmed differential roles for pSer42 and pSer45 in promoter-specific DNA binding and a role for both of these phosphosites in regulating transcription from the IL-6 promoter. These temporal dynamics suggest that RelA-mediated transcription is likely to be controlled by functionally distinct NF-κB proteoforms carrying different combinations of modifications, rather than a simple 'one modification, one effect' system.

Original languageEnglish
Article number160055
JournalOpen Biology
Volume6
Issue number7
Early online date27 Jul 2016
DOIs
Publication statusPublished - 2016

Keywords

  • NF-κB
  • Phosphorylation
  • Proteomics
  • Quantification
  • RelA
  • Transcription

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