Identifying post-translational modifications of NEMO by tandem mass spectrometry after high affinity purification
Shawn S Jackson1, Emma E Coughlin, Joshua J Coon
1McArdle 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.
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.
