title: Identifying post-translational modifications of NEMO by tandem mass spectrometry after high affinity purification.
abstract: An integral component of NF-κB signalling is NEMO, NF-κB essential modulator, a regulatory protein of the IκB kinase (IKK) complex. Post-translational modifications of NEMO, including phosphorylation, SUMOylation, and ubiquitination are critical events during stimuli induced NF-κB activation. Here we demonstrate a method to detect post-translational modifications of NEMO using cells stably expressing polyhistidine tagged NEMO which allows for high-affinity purification of NEMO following rapid denaturing lysis and characterization by MS/MS. We identified a previously uncharacterized basal phosphorylation of NEMO at Serine 387 and tested the biological significance of this phosphorylation through a somatic genetic complementation analysis using the NEMO mutants S387A, S388D, and P388I in 1.3E2 NEMO-deficient murine pre-B cells. NF-κB signalling induced by bacterial lipopolysaccharide, Interleukin-1ß or the DNA damaging agent etoposide was not perturbed by these mutations of NEMO. Thus, S387 phosphorylation of NEMO is not a general requirement to mediate efficient NF-κB signalling and therefore may have cell type and/or stimulus-specific activity in vivo.
authors.affiliation: McArdle Laboratory for Cancer Research, Department of Oncology, University of Wisconsin-Madison, 6159 Wisconsin Institute for Medical Research, 1111 Highland Avenue, Madison, WI 53705, USA; Medical Scientist Training Program, University of Wisconsin-Madison, 6159 Wisconsin Institute for Medical Research, 1111 Highland Avenue, Madison, WI 53705, USA; Cellular and Molecular Biology Program, University of Wisconsin-Madison, 6159 Wisconsin Institute for Medical Research, 1111 Highland Avenue, Madison, WI 53705, USA.
authors.forename: Shawn S
authors.initials: SS
authors.lastname: Jackson
authors.forename: Emma E
authors.initials: EE
authors.lastname: Coughlin
authors.forename: Joshua J
authors.initials: JJ
authors.lastname: Coon
authors.forename: Shigeki
authors.initials: S
authors.lastname: Miyamoto
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D057809:HEK293 Cells; D006801:Humans; D051550:I-kappa B Kinase / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology; D008070:Lipopolysaccharides / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D016328:NF-kappa B / Q000276:immunology; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D053719:Tandem Mass Spectrometry
chemical_list: C491731:IKBKG protein, human; D008070:Lipopolysaccharides; D016328:NF-kappa B; D051550:I-kappa B Kinase
keywords: MS/MS; NEMO; NF-κB; Phosphorylation; Polyhistidine purification