Structural basis of RIP2 activation and signaling

Gong, Qin; Long, Ziqi; Zhong, Franklin L.; Teo, Daniel Eng Thiam; Jin, Yibo; Yin, Zehao; Boo, Zhao Zhi; Zhang, Yaming; Zhang, Jiawen; Yang, Renliang; Bhushan, Shashi; Reversade, Bruno; Li, Zongli; Wu, Bin

doi:10.1038/s41467-018-07447-9

Cited by 74 publications

(76 citation statements)

References 52 publications

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“…Recent in vitro structural data that indicate that the CARD of NOD1 forms a transient unstable complex with RIPK2 CARD to induce aggregation is consistent with our in situ data (Gong et al, 2018). NF-κB signaling thereby seems to be uncoupled from this process as mutations in NOD1 and NOD2 that interfere with NF-κB activation still triggered RIPosome formation.…”

Section: Discussionsupporting

confidence: 89%

“…Using this tool, we found that RIPosomes formed after NF-κB activation mediated by NOD1, which is contradictory to a direct role of these structures in NF-κB activation by NOD1/2, as recently proposed by others (Gong et al, 2018, Pellegrini et al, 2018. Activation of NF-κB responses by NOD1 and NOD2 is associated with membrane recruitment of NOD1 and NOD2 (Kufer, Kremmer et al, 2008, Lecine, Esmiol et al, 2007, Nakamura, Lill et al, 2014, Travassos, Carneiro et al, 2010.…”

Section: Discussionmentioning

confidence: 60%

“…The interaction of NOD1 and NOD2 with RIPK2 is mediated by heterotypic CARD:CARD interactions, involving residues in the exposed surfaces of the CARD domains of RIPK2 and NOD1/2 (Maharana, Pradhan et al, 2017, Manon, Favier et al, 2007, Mayle, Boyle et al, 2014. In vitro, this can result in stable rope-like structures, which were recently proposed to be platforms for subsequent NF-κB activation (Gong, Long et al, 2018, Pellegrini, Desfosses et al, 2018.…”

Section: Introductionmentioning

confidence: 99%

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

Ellwanger

Arnold

Briese

et al. 2019

Preprint

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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...

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Section: Discussionsupporting

confidence: 89%

Section: Discussionmentioning

confidence: 60%

Section: Introductionmentioning

confidence: 99%

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

Ellwanger

Arnold

Briese

et al. 2019

Preprint

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“…4a-b to Fig. 3c-d), which had not been highlighted by previous analyses 20,27,[29][30][31][32][33][34][35] . In ASC-CARD filaments, this bridge consists of two residues Gln185 and Tyr187, which extend from the Type IIb surface and interact with Ala168 and Asn170 on the Type IIa surface ( Fig.…”

Section: Structural Basis For the Distinct Downstream Card Preferencementioning

confidence: 51%

Structural basis for distinct inflammasome complex assembly by human NLRP1 and CARD8

Gong

Robinson²,

et al. 2020

Preprint

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Nod-like receptor (NLR) proteins activate pyroptotic cell death and IL-1 driven inflammation by assembling and activating the inflammasome complex. Closely related NLR proteins, NLRP1 and CARD8 undergo unique auto-proteolysis-dependent activation and are implicated in auto-inflammatory diseases; however, the molecular mechanisms of activation are not understood. Here we report the structural basis of how the activating domains (FIIND UPA -CARD) of NLRP1 and CARD8 self-oligomerize to trigger the assembly of distinct inflammasome complexes. Recombinant FIIND UPA -CARD of NLRP1 forms a two-layered filament, with an inner core composed of oligomerized CARD domains and the outer layer consisting of FIIND UPA rings.Biochemically, oligomerized NLRP1-CARD is sufficient to drive ASC speck formation in cultured human cells via filament formation-a process that is greatly enhanced by NLRP1-FIIND UPA , which forms ring-like oligomers in vitro. In addition, we report the cryo-EM structures of NLRP1-CARD and CARD8-CARD filaments at 3.7 Å, which uncovers unique structural features that enable NLRP1 and CARD8 to discriminate between ASC and pro-caspase-1. In summary, our findings provide unique structural insight into the mechanisms of activation for human NLRP1 and CARD8, uncovering an unexpected level of specificity in inflammasome signaling mediated by heterotypic CARD domain interactions.

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“…In recent studies, higher order intracellular signalling platforms consisting of RIPK2 and NOD receptors were described [36,57,58]. In particular, inhibition of XIAP by either siRNA or by SMAC mimetic compounds led to RIPK2-containing speck-like structures in cells, termed Riposomes [36].…”

Section: Discussionmentioning

confidence: 99%

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

Heim

Dagley

Stafford

et al. 2020

Preprint

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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 during NOD signaling and we identified a new regulatory interface on RIPK2, which dictates the crucial interaction with the E3 ligase XIAP.

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Structural basis of RIP2 activation and signaling

Cited by 74 publications

References 52 publications

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

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

Structural basis for distinct inflammasome complex assembly by human NLRP1 and CARD8

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

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