Background: The nuclear factor kappa B (NF-κB) signalling pathway has been linked to several metabolic and inflammatory diseases, such as atherosclerosis. The inhibitor of NF-κB kinase regulatory subunit gamma (IKKΥ), also known as NF-κB essential modulator (NEMO), is essential for NF-κB activation. In humans, NEMO dysfunction is associated with so-called progeria syndromes, which are characterized by premature ageing, including early-onset atherosclerosis.
Results: We have previously shown that DNA-PK, an enzyme involved in DNA double-strand break (DSB) repair, associates with NEMO and triggers its phosphorylation. In this study, using NEMO immunoprecipitation followed by mass spectrometry analysis, we identified an additional NEMO phosphorylation site at serine 148. To analyze NEMO phosphorylation at serine 148, we generated phospho-specific antibodies (anti-phospho-NEMO-Ser148). Surprisingly, treatment of cells with TNFα or ionizing radiation induced NEMO-Ser148 phosphorylation in distinct time-dependent fashions. In further experiments, we identified DNA-PK and IKKβ as the kinases responsible for phosphorylating NEMO at serine 148. We confirmed these findings through mutational analysis and in vitro kinase assays. Point mutation of this serine to alanine (S148A) led to enhanced NF-κB activation upon stimulation by ionizing radiation or TNFα treatment. Consistently, transfection of cells with the NEMO-S148A mutant resulted in increased activation of the NF-κB pathway following exposure to TNFα or ionizing radiation. Blocking DNA-PK with a specific inhibitor or by using DNA-PKcs-deficient cells, we demonstrated that NEMO phosphorylation, induced by genotoxic stress, is dependent on DNA-PK. Similarly, inhibition of IKKβ blocked TNFα-induced NEMO phosphorylation. Immunohistochemical analysis of atherosclerotic tissue specimens revealed the presence of NEMO phosphorylated at serine 148, suggesting in vivo relevance.
Conclusion: Our work not only enhances the understanding of NEMO’s biological function, it also provides a framework for characterizing NEMO as a pleiotropic signal transducer. It is involved in numerous biological processes and can be phosphorylated at the same site by different kinases in a stimulus-specific manner.
