The Cofilin Phosphatase Slingshot Homolog 1 (SSH1) Links NOD1 Signaling to Actin Remodeling

Bielig, Harald; Lautz, Katja; Braun, Peter; Menning, Maureen; Machuy, Nikolaus; Brügmann, Christine; Barisic, Sandra; Eisler, Stephan A.; Andree, Maria; Zurek, Birte; Kashkar, Hamid; Sansonetti, Philippe J.; Haußer, Angelika; Meyer, Thomas F.; Kufer, Thomas A.

doi:10.1371/journal.ppat.1004351

Cited by 48 publications

(52 citation statements)

References 41 publications

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“…Supporting our results, RIPK2 was identified in immunoprecipitations with 14-3-3 previously (Pozuelo Rubio, Geraghty et al, 2004) and 14-3-3 proteins might regulate RIPK2 signaling as at least 14-3-3- appeared as a candidate of a negative regulator of NOD1 and NOD2 signaling in two independent siRNA screens (Bielig, Lautz et al, 2014, Warner, Burberry et al, 2013, Warner, Burberry et al, 2014. Moreover, 14-3-3 binds RIPK3 (Zhang, Shao et al, 2009) and regulates its activity by regulating TSC2 binding (Fettweis, Di Valentin et al, 2017).…”

Section: Discussionsupporting

confidence: 85%

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: 85%

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

Ellwanger

Arnold

Briese

et al. 2019

Preprint

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“…NOD1 and NOD2, could be found at the plasma membrane in association with F actin, which is needed for downstream activation of NF-B signaling (42,51). Recently, Bielig et al have shown that the actin depolymerization factor (ADF)/cofilin phosphatase SSH1 is an essential component of the NOD1 pathway, which plays a role in activation of NF-B and cell responses to Shigella infection (52). Indeed, depletion of SSH1 mRNA resulted in reduced production of IL-8 and IL-6 following infection of HeLa cells with Shigella flexneri strain M90T.…”

Section: Discussionmentioning

confidence: 99%

Tir Triggers Expression of CXCL1 in Enterocytes and Neutrophil Recruitment during Citrobacter rodentium Infection

et al. 2015

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The hallmarks of enteropathogenic Escherichia coli (EPEC) infection are formation of attaching and effacing (A/E) lesions on mucosal surfaces and actin-rich pedestals on cultured cells, both of which are dependent on the type III secretion system effector Tir. Following translocation into cultured cells and clustering by intimin, Tir Y474 is phosphorylated, leading to recruitment of Nck, activation of N-WASP, and actin polymerization via the Arp2/3 complex. A secondary, weak, actin polymerization pathway is triggered via an NPY motif (Y454). Importantly, Y454 and Y474 play no role in A/E lesion formation on mucosal surfaces following infection with the EPEC-like mouse pathogen Citrobacter rodentium. In this study, we investigated the roles of Tir segments located upstream of Y451 and downstream of Y471 in C. rodentium colonization and A/E lesion formation. We also tested the role that Tir residues Y451 and Y471 play in host immune responses to C. rodentium infection. We found that deletion of amino acids 382 to 462 or 478 to 547 had no impact on the ability of Tir to mediate A/E lesion formation, although deletion of amino acids 478 to 547 affected Tir translocation. Examination of enterocytes isolated from infected mice revealed that a C. rodentium strain expressing Tir_Y451A/Y471A recruited significantly fewer neutrophils to the colon and triggered less colonic hyperplasia on day 14 postinfection than the wild-type strain. Consistently, enterocytes isolated from mice infected with C. rodentium expressing Tir_Y451A/Y471A expressed significantly less CXCL1. These result show that Tir-induced actin remodeling plays a direct role in modulation of immune responses to C. rodentium infection. Enteropathogenic Escherichia coli (EPEC) strains are important human pathogens causing infantile diarrhea in low-income countries (1) Recently, the Global Enteric Multicenter Study (GEMS), designed to detect the cause of pediatric diarrheal disease in sub-Saharan Africa and south Asia, found that infection with typical EPEC is associated with increased risk of fatality in infants aged 0 to 11 months (2). Citrobacter rodentium is a mouse-specific pathogen, the etiological agent of transmissible colonic hyperplasia, and a model EPEC microorganism, as both pathogens share an infection strategy and virulence factors (3, 4). Host resistance to C. rodentium infection is mediated by diverse T cell effector responses, including T cell production of interferon gamma (IFN-␥) (5, 6), interleukin 17A (IL-17A) (7,8),. Expression of the proinflammatory cytokine IL-17A leads to recruitment of neutrophils (10), and the anti-inflammatory cytokine IL-22 upregulates expression of antimicrobial peptides (such as REGIII␤ and REGIII␥) in enterocytes (9, 11).While colonizing the gut mucosa, EPEC and C. rodentium induce attaching and effacing (A/E) lesions. These are characterized by extensive remodeling of the gut epithelium leading to elongation and effacement of the brush border (BB) microvilli, intimate bacterial attachment to the enterocyte apical...

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“…These features are of particular importance in the gastrointestinal system, where chronic inflammation and tissue damage act as tumor promoters (Quante and Wang, 2008), but our understanding of the underlying regulatory network is still incomplete for many inducers. Several RNAi loss-of-function studies have identified factors involved in NF-kB pathway activation, including stimuli of the nucleotide-binding oligomerization domain-containing protein (NOD)1 and NOD2 receptors (Bielig et al, 2014;Lipinski et al, 2012;Warner et al, 2013;Yeretssian et al, 2011), Epstein-Barr virus (Gewurz et al, 2012), Neisseria gonorrhoeae (Gaudet et al, 2015), and Shigella flexneri infections (Milivojevic et al, 2017). These studies have shown that NF-kB signaling, although sharing core elements, is highly complex and diverse.…”

Section: Introductionmentioning

confidence: 99%

ALPK1- and TIFA-Dependent Innate Immune Response Triggered by the Helicobacter pylori Type IV Secretion System

et al. 2017

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Activation of transcription factor NF-κB is a hallmark of infection with the gastric pathogen Helicobacter pylori, associated with inflammation and carcinogenesis. Genome-wide RNAi screening revealed numerous host factors involved in H. pylori-, but not IL-1β- and TNF-α-dependent NF-κB regulation. Pathway analysis including CRISPR/Cas9-knockout and recombinant protein technology, immunofluorescence microscopy, immunoblotting, mass spectrometry, and mutant H. pylori strains identified the H. pylori metabolite D-glycero-β-D-manno-heptose 1,7-bisphosphate (βHBP) as a cagPAI type IV secretion system (T4SS)-dependent effector of NF-κB activation in infected cells. Upon pathogen-host cell contact, TIFA forms large complexes (TIFAsomes) including interacting host factors, such as TRAF2. NF-κB activation, TIFA phosphorylation, and TIFAsome formation depend on a functional ALPK1 kinase, highlighting the ALPK1-TIFA axis as a core innate immune pathway. ALPK1-TIFA-mediated NF-κB activation was independent of CagA protein translocation, indicating that CagA translocation and HBP delivery to host cells are distinct features of the pathogen's T4SS.

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The Cofilin Phosphatase Slingshot Homolog 1 (SSH1) Links NOD1 Signaling to Actin Remodeling

Cited by 48 publications

References 41 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

Tir Triggers Expression of CXCL1 in Enterocytes and Neutrophil Recruitment during Citrobacter rodentium Infection

ALPK1- and TIFA-Dependent Innate Immune Response Triggered by the Helicobacter pylori Type IV Secretion System

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