Identifying post-translational modifications of NEMO by tandem mass spectrometry after high affinity purification.
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Jackson SS, Coughlin EE, Coon JJ, Miyamoto S
Identifying post-translational modifications of NEMO by tandem mass spectrometry after high affinity purification.
Protein Expr Purif. 2013 Nov;92(1):48-53. doi: 10.1016/j.pep.2013.08.020. Epub 2013 Sep 6.
- PubMed ID
- 24012789 [ View in PubMed]
- Abstract
An integral component of NF-kappaB signalling is NEMO, NF-kappaB essential modulator, a regulatory protein of the IkappaB kinase (IKK) complex. Post-translational modifications of NEMO, including phosphorylation, SUMOylation, and ubiquitination are critical events during stimuli induced NF-kappaB 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-kappaB 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-kappaB signalling and therefore may have cell type and/or stimulus-specific activity in vivo.