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 forms higher order molecular structures in vitro, which were proposed to activate signaling. 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 depends on the CARD of NOD1 or NOD2. Complex formation upon activation was dependent on RIPK2 autophosphorylation at Y474 and influenced by phosphorylation at S176. Inhibition of activity of the cIAP protein XIAP induced spontaneous complex formation of RIPK2 but blocked NOD1-dependet NF-κB activation. Using immunoprecipitation, we identified 14-3-3 proteins as novel binding partners of non-activated RIPK2, whereas complexed RIPK2 was bound by the prohibitin proteins Erlin-1 and Erlin-2.Taken together, our work reveals novel roles of XIAP, 14-3-3 and Erlin proteins in the regulation of RIPK2 and expands our knowledge on the function of RIPK2 posttranslational modifications in NOD1/2 signaling. AUTHOR CONTRIBUTIONSCA, KE, SB and IK performed the experiments.JP performed MS analysis and data processing.TAK conceived and supervised the study.CA, KE and TAK wrote and edited the manuscript. CONFLICT OF INTERESTThe authors declare that they have no conflict of interest.A: Indirect immunofluorescence micrographs of HeLa EGFP-RIPK2 cells infected for 2 h with S. flexneri M90T. Staining for XIAP, EGFP-RIPK2, and merge with DNA-staining is shown.Co-localization is shown at higher magnification in the inlay. Scale bar = 10 µm. B: Fluorescence micrographs of HeLa EGFP-RIPK2 cells, treated for 48 h with a XIAP siRNA or a non-targeting siRNA, following infection with S. flexneri M90T for 2 h or left uninfected. Cells were treated with 1 µg/ml doxycycline for 24 h prior infection. EGFP-RIPK2 and merge with DNA-staining is shown. Scale bar = 10 µm. C: IL-8 release in the supernatants from HeLa EGFP-RIPK2 cells, treated for 48 h with a XIAP siRNA or a non-targeting siRNA, following infection with S. flexneri M90T for 6 h. Mean of n=2 with S.D. is shown. D: Immunoblot analysis of HeLa EGFP-RIPK2 cells infected with S. flexneri M90T or BS176 for the indicated time. Immunoblot was probed with anti-RIPK2 antibody, anti-XIAP, and anti-β-tubulin as loading control. E: Left panel: Fluorescence micrographs of HeLa EGFP-RIPK2 cells, transfected with 500 ng Ubi-SMAC-fl-pDS-RED, Ubi-SMAC-tr-pDS-RED, or Ubi-SMAC-AVPI-pDS-RED plasmid and treated with 1 µg/ml doxycycline for 24 h. EGFP-RIPK2, SMAC-dsRed, and merge with DNA-staining is shown. Scale bar = 10 µm. Right panel: Immunoblot analysis of cells prepared as in the left panel. Protein levels were detected using an anti-RIPK2 antibody and anti-β-tubulin as loading control.Figure 6: Phosphorylation of RIPK2 at S176 and Y474 contribute to RIPosome formation. A: Fluorescence microg...

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.…”

Grouper RIP2 inhibits Singapore grouper iridovirus infection by modulating ASC-caspase-1 interaction

“…RIP2 is able to polymerize into long filamentous structures, which serves as signaling platform to the downstream of NOD1/2. RIP2 filament formation is necessary for NOD2-dependent NF-κB signaling ( 29 , 30 ). In our paper, subcellular localization analysis signified that EcRIP2 was exhibited in cytoplasmic filament and dot aggregation patterns without SGIV infection.…”