XIAP controls RIPK2 signaling by preventing its deposition in speck-like structures

Abstract

The receptor interacting serine/threonine kinase 2 (RIPK2) is essential for linking activation of the pattern recognition receptors NOD1 and NOD2 to cellular signaling events. Recently, it was shown that RIPK2 can form higher order molecular structures in vitro. Here, we demonstrate that RIPK2 forms detergent insoluble complexes in the cytosol of host cells upon infection with invasive enteropathogenic bacteria. Formation of these structures occurred after NF-κB activation and depended on the caspase activation and recruitment domain of NOD1 or NOD2. Complex formation upon activation required RIPK2 autophosphorylation at Y474 and was influenced by phosphorylation at S176. We found that the E3 ligase X-linked inhibitor of apoptosis (XIAP) counteracts complex formation of RIPK2, accordingly mutation of the XIAP ubiquitylation sites in RIPK2 enhanced complex formation. Taken together, our work reveals novel roles of XIAP in the regulation of RIPK2 and expands our knowledge on the function of RIPK2 posttranslational modifications in NOD1/2 signaling.

A regulatory region on RIPK 2 is required for XIAP binding and NOD signaling activity

“…Notably, RIPK2 K209R was the only form of RIPK2 which seemed to present in a second, higher molecular weight band after stimulation, similar to recently described Riposomes (Ellwanger et al , 2019). To test the impact of each individual modified site on the ubiquitination pattern on RIPK2 during NOD signaling, cells were subjected to UBA pulldowns and analyzed by Western blot.…”

“…In recent studies, higher order intracellular signaling platforms consisting of RIPK2 and NOD receptors were described (Gong et al , 2018; Pellegrini et al , 2018; Ellwanger et al , 2019). In particular, inhibition of XIAP by either siRNA or by SMAC mimetic compounds led to RIPK2‐containing speck‐like structures in cells, termed Riposomes (Ellwanger et al , 2019). Here, we described two mutations that perturbed the interaction between RIPK2 and XIAP.…”

The diverse roles of RIP kinases in host-pathogen interactions

“…RIPK2 signaling may also be regulated via the formation of RIPosomes, which are high molecular weight cytoplasmic complexes comprised of RIPK2 [ 199 , 200 ]. Ellwanger et al [ 199 ] showed RIPosomes form post-NF-κB activation, and suggest that sequestration of RIPK2 in these complexes may act to dampen RIPK2 signaling. Intriguingly, RIPosomes form in the cytosol of epithelial cells upon invasion with S. flexneri , suggesting the pathogen may actively prevent RIPK2 signaling via an unknown process.…”

“…Intriguingly, RIPosomes form in the cytosol of epithelial cells upon invasion with S. flexneri , suggesting the pathogen may actively prevent RIPK2 signaling via an unknown process. Given that inhibition of XIAP was shown to promote deposition of RIPK2 in RIPosomes, it may be that Shigella encodes a virulence factor that targets XIAP for cleavage or degradation, or actively inhibits XIAP-designated sites of ubiquitylation on RIPK2 to inhibit inflammatory signaling [ 199 ].…”

NOD Signaling and Cell Death

“…Recent studies also re-raised questions about the importance of the regulatory autophosphorylation sites S176 and Y474. Overexpression of RIPK2 mutants in HeLa cells showed that wild-type RIPK2 and the S176A mutant resulted in similar amounts of cytokines following infections with S. flexneri , while the S176E mutant resulted in reduced cytokine levels (Ellwanger et al, 2019). In contrast, cytokine production was completely abolished in cells expressing the RIPK2 Y474F mutant.…”