ATM-dependent Phosphorylation of Nemo SQ Motifs Is Dispensable for Nemo-mediated Gene Expression Changes in Response to DNA Double-Strand Breaks

• Published in: The Journal of immunology (1950) Vol. 213; no. 5; pp. 628 - 640
• Main Authors: Glynn, Rebecca A, Hayer, Katharina E, Bassing, Craig H
• Format: Journal Article
• Language: English
• Published: United States 01-09-2024
• Subjects: Amino Acid Motifs; Animals; Ataxia Telangiectasia Mutated Proteins - genetics; Ataxia Telangiectasia Mutated Proteins - metabolism; DNA Breaks, Double-Stranded; Etoposide - pharmacology; Gene Expression Regulation; I-kappa B Kinase - genetics; I-kappa B Kinase - metabolism; Intracellular Signaling Peptides and Proteins - genetics; Intracellular Signaling Peptides and Proteins - metabolism; Mice; Mice, Knockout; NF-kappa B - metabolism; Phosphorylation
• ISSN: 0022-1767; 1550-6606; 1550-6606
• DOI: 10.4049/jimmunol.2300139

In response to DNA double-strand breaks (DSBs), the ATM kinase activates NF-κB factors to stimulate gene expression changes that promote survival and allow time for cells to repair damage. In cell lines, ATM can activate NF-κB transcription factors via two independent, convergent mechanisms. One is ATM-mediated phosphorylation of nuclear NF-κB essential modulator (Nemo) protein, which leads to monoubiquitylation and export of Nemo to the cytoplasm where it engages the IκB kinase (IKK) complex to activate NF-κB. Another is DSB-triggered migration of ATM into the cytoplasm, where it promotes monoubiquitylation of Nemo and the resulting IKK-mediated activation of NF-κB. ATM has many other functions in the DSB response beyond activation of NF-κB, and Nemo activates NF-κB downstream of diverse stimuli, including developmental or proinflammatory stimuli such as LPSs. To elucidate the in vivo role of DSB-induced, ATM-dependent changes in expression of NF-κB-responsive genes, we generated mice expressing phosphomutant Nemo protein lacking consensus SQ sites for phosphorylation by ATM or related kinases. We demonstrate that these mice are viable/healthy and fertile and exhibit overall normal B and T lymphocyte development. Moreover, treatment of their B lineage cells with LPS induces normal NF-κB-regulated gene expression changes. Furthermore, in marked contrast to results from a pre-B cell line, primary B lineage cells expressing phosphomutant Nemo treated with the genotoxic drug etoposide induce normal ATM- and Nemo-dependent changes in expression of NF-κB-regulated genes. Our data demonstrate that ATM-dependent phosphorylation of Nemo SQ motifs in vivo is dispensable for DSB-signaled changes in expression of NF-κB-regulated genes.