Identifying post-translational modifications of NEMO by tandem mass spectrometry after high affinity purification. 2013

Shawn S Jackson, and Emma E Coughlin, and Joshua J Coon, and Shigeki Miyamoto
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.

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.

UI MeSH Term Description Entries
D008070 Lipopolysaccharides Lipid-containing polysaccharides which are endotoxins and important group-specific antigens. They are often derived from the cell wall of gram-negative bacteria and induce immunoglobulin secretion. The lipopolysaccharide molecule consists of three parts: LIPID A, core polysaccharide, and O-specific chains (O ANTIGENS). When derived from Escherichia coli, lipopolysaccharides serve as polyclonal B-cell mitogens commonly used in laboratory immunology. (From Dorland, 28th ed) Lipopolysaccharide,Lipoglycans
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D009154 Mutation Any detectable and heritable change in the genetic material that causes a change in the GENOTYPE and which is transmitted to daughter cells and to succeeding generations. Mutations
D010766 Phosphorylation The introduction of a phosphoryl group into a compound through the formation of an ester bond between the compound and a phosphorus moiety. Phosphorylations
D011499 Protein Processing, Post-Translational Any of various enzymatically catalyzed post-translational modifications of PEPTIDES or PROTEINS in the cell of origin. These modifications include carboxylation; HYDROXYLATION; ACETYLATION; PHOSPHORYLATION; METHYLATION; GLYCOSYLATION; ubiquitination; oxidation; proteolysis; and crosslinking and result in changes in molecular weight and electrophoretic motility. Amino Acid Modification, Post-Translational,Post-Translational Modification,Post-Translational Protein Modification,Posttranslational Modification,Protein Modification, Post-Translational,Amino Acid Modification, Posttranslational,Post-Translational Amino Acid Modification,Post-Translational Modifications,Post-Translational Protein Processing,Posttranslational Amino Acid Modification,Posttranslational Modifications,Posttranslational Protein Processing,Protein Processing, Post Translational,Protein Processing, Posttranslational,Amino Acid Modification, Post Translational,Modification, Post-Translational,Modification, Post-Translational Protein,Modification, Posttranslational,Modifications, Post-Translational,Modifications, Post-Translational Protein,Modifications, Posttranslational,Post Translational Amino Acid Modification,Post Translational Modification,Post Translational Modifications,Post Translational Protein Modification,Post Translational Protein Processing,Post-Translational Protein Modifications,Processing, Post-Translational Protein,Processing, Posttranslational Protein,Protein Modification, Post Translational,Protein Modifications, Post-Translational
D002460 Cell Line Established cell cultures that have the potential to propagate indefinitely. Cell Lines,Line, Cell,Lines, Cell
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000595 Amino Acid Sequence The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION. Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D016328 NF-kappa B Ubiquitous, inducible, nuclear transcriptional activator that binds to enhancer elements in many different cell types and is activated by pathogenic stimuli. The NF-kappa B complex is a heterodimer composed of two DNA-binding subunits: NF-kappa B1 and relA. Immunoglobulin Enhancer-Binding Protein,NF-kappa B Complex,Nuclear Factor kappa B,Transcription Factor NF-kB,kappa B Enhancer Binding Protein,Ig-EBP-1,NF-kB,NF-kappaB,Nuclear Factor-Kappab,Complex, NF-kappa B,Enhancer-Binding Protein, Immunoglobulin,Factor NF-kB, Transcription,Factor-Kappab, Nuclear,Ig EBP 1,Immunoglobulin Enhancer Binding Protein,NF kB,NF kappa B Complex,NF kappaB,NF-kB, Transcription Factor,Nuclear Factor Kappab,Transcription Factor NF kB

Related Publications

Shawn S Jackson, and Emma E Coughlin, and Joshua J Coon, and Shigeki Miyamoto
June 1998, Current opinion in structural biology,
Shawn S Jackson, and Emma E Coughlin, and Joshua J Coon, and Shigeki Miyamoto
January 2012, International journal of computational biology and drug design,
Shawn S Jackson, and Emma E Coughlin, and Joshua J Coon, and Shigeki Miyamoto
January 2017, Methods in molecular biology (Clifton, N.J.),
Shawn S Jackson, and Emma E Coughlin, and Joshua J Coon, and Shigeki Miyamoto
March 2011, Bioinformatics (Oxford, England),
Shawn S Jackson, and Emma E Coughlin, and Joshua J Coon, and Shigeki Miyamoto
January 1988, Advances in experimental medicine and biology,
Shawn S Jackson, and Emma E Coughlin, and Joshua J Coon, and Shigeki Miyamoto
December 2009, The Biochemical journal,
Shawn S Jackson, and Emma E Coughlin, and Joshua J Coon, and Shigeki Miyamoto
April 2015, Bioinformatics (Oxford, England),
Shawn S Jackson, and Emma E Coughlin, and Joshua J Coon, and Shigeki Miyamoto
January 2006, Molecular & cellular proteomics : MCP,
Shawn S Jackson, and Emma E Coughlin, and Joshua J Coon, and Shigeki Miyamoto
December 2013, Free radical biology & medicine,
Shawn S Jackson, and Emma E Coughlin, and Joshua J Coon, and Shigeki Miyamoto
January 2022, Frontiers in cellular neuroscience,
Copied contents to your clipboard!