Identifying post-translational modifications of NEMO by tandem mass spectrometry after high affinity purification.
Protein Expr Purif
; 92(1): 48-53, 2013 Nov.
Article
em En
| MEDLINE
| ID: mdl-24012789
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.
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Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Processamento de Proteína Pós-Traducional
/
Quinase I-kappa B
Tipo de estudo:
Prognostic_studies
Limite:
Animals
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Humans
Idioma:
En
Revista:
Protein Expr Purif
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2013
Tipo de documento:
Article