A regulatory region on RIPK2 is required for XIAP binding and NOD signaling activity

Signaling via the intracellular pathogen receptors nucleotide‐binding oligomerization domain‐containing proteins NOD1 and NOD2 requires receptor interacting kinase 2 (RIPK2), an adaptor kinase that can be targeted for the treatment of various inflammatory diseases. However, the molecular mechanisms of how RIPK2 contributes to NOD signaling are not completely understood. We generated FLAG‐tagged RIPK2 knock‐in mice using CRISPR/Cas9 technology to study NOD signaling mechanisms at the endogenous level. Using cells from these mice, we were able to generate a detailed map of post‐translational modifications on RIPK2. Similar to other reports, we did not detect ubiquitination of RIPK2 lysine 209 during NOD2 signaling. However, using site‐directed mutagenesis we identified a new regulatory region on RIPK2, which dictates the crucial interaction with the E3 ligase XIAP and downstream signaling outcomes. Synopsis Polyubiquitination on multiple lysine residues of RIPK2 is required for anti‐bacterial responses mediated by the pathogen recognition receptor NOD2. This study identifies a regulatory region on the C‐lobe of the kinase domain that controls binding of the E3 ligase XIAP, RIPK2 ubiquitination and downstream signalling events. RIPK2 is phosphorylated and ubiquitinated on multiple residues in the kinase, intermediate and CARD domain during NOD2 signaling. Mutagenesis of single modified sites has little impact on RIPK2 function. K209 and I212 form a regulatory region on the C‐lobe of the kinase that controls XIAP binding and NOD2 signaling. Graphical Abstract Polyubiquitination on multiple lysine residues of RIPK2 is required for anti‐bacterial responses mediated by the pathogen recognition receptor NOD2. This study identifies a regulatory region on the C‐lobe of the kinase domain that controls binding of the E3 ligase XIAP, RIPK2 ubiquitination and downstream signalling events.