title: Dynamic phosphorylation of RelA on Ser42 and Ser45 in response to TNFα stimulation regulates DNA binding and transcription.
abstract: The NF-κB signalling module controls transcription through a network of protein kinases such as the IKKs, as well 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.
authors.affiliation: Centre for Proteome Research, Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool L69 7ZB, UK.
authors.forename: Francesco
authors.initials: F
authors.lastname: Lanucara
authors.affiliation: Systems Microscopy Centre, Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, UK.
authors.forename: Connie
authors.initials: C
authors.lastname: Lam
authors.affiliation: Fibrosis Laboratory, Liver Group, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne NE2 4HH, UK.
authors.forename: Jelena
authors.initials: J
authors.lastname: Mann
authors.affiliation: MRC Human Nutrition Research, University of Cambridge, Cambridge CB2 1GA, UK.
authors.forename: Tom P
authors.initials: TP
authors.lastname: Monie
authors.affiliation: Systems Microscopy Centre, Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, UK.
authors.forename: Stefano A P
authors.initials: SA
authors.lastname: Colombo
authors.affiliation: Centre for Proteome Research, Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool L69 7ZB, UK.
authors.forename: Stephen W
authors.initials: SW
authors.lastname: Holman
authors.affiliation: Systems Microscopy Centre, Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, UK.
authors.forename: James
authors.initials: J
authors.lastname: Boyd
authors.affiliation: Centre for Proteome Research, Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool L69 7ZB, UK.
authors.forename: Manohar C
authors.initials: MC
authors.lastname: Dange
authors.affiliation: Fibrosis Laboratory, Liver Group, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne NE2 4HH, UK.
authors.forename: Derek A
authors.initials: DA
authors.lastname: Mann
authors.affiliation: Systems Microscopy Centre, Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, UK.
authors.forename: Michael R H
authors.initials: MR
authors.lastname: White
authors.affiliation: Centre for Proteome Research, Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool L69 7ZB, UK claire.eyers@liverpool.ac.uk.
authors.forename: Claire E
authors.identifier: 0000-0002-3223-5926
authors.initials: CE
authors.lastname: Eyers
mesh_terms: D002460:Cell Line; D018360:Crystallography, X-Ray; D004247:DNA / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D015850:Interleukin-6 / Q000235:genetics*; D008958:Models, Molecular; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011487:Protein Conformation; D040901:Proteomics / Q000379:methods; D012694:Serine / Q000378:metabolism*; D051996:Transcription Factor RelA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: C508600:IL6 protein, human; D015850:Interleukin-6; C498143:RELA protein, human; C439524:TNF protein, human; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D012694:Serine; D004247:DNA
keywords: NF-κB; RelA; phosphorylation; proteomics; quantification; transcription