Phosphorylation of NLRC4 is critical for inflammasome activation
- 12 August 2012
- journal article
- Published by Springer Science and Business Media LLC in Nature
- Vol. 490 (7421), 539-542
- https://doi.org/10.1038/nature11429
Abstract
NLRC4 is a cytosolic member of the NOD-like receptor family that is expressed in innate immune cells. It senses indirectly bacterial flagellin and type III secretion systems, and responds by assembling an inflammasome complex that promotes caspase-1 activation and pyroptosis. Here we use knock-in mice expressing NLRC4 with a carboxy-terminal 3×Flag tag to identify phosphorylation of NLRC4 on a single, evolutionarily conserved residue, Ser 533, following infection of macrophages with Salmonella enterica serovar Typhimurium (also known as Salmonella typhimurium). Western blotting with a NLRC4 phospho-Ser 533 antibody confirmed that this post-translational modification occurs only in the presence of stimuli known to engage NLRC4 and not the related protein NLRP3 or AIM2. Nlrc4(-/-) macrophages reconstituted with NLRC4 mutant S533A, unlike those reconstituted with wild-type NLRC4, did not activate caspase-1 and pyroptosis in response to S. typhimurium, indicating that S533 phosphorylation is critical for NLRC4 inflammasome function. Conversely, phosphomimetic NLRC4 S533D caused rapid macrophage pyroptosis without infection. Biochemical purification of the NLRC4-phosphorylating activity and a screen of kinase inhibitors identified PRKCD (PKCδ) as a candidate NLRC4 kinase. Recombinant PKCδ phosphorylated NLRC4 S533 in vitro, immunodepletion of PKCδ from macrophage lysates blocked NLRC4 S533 phosphorylation in vitro, and Prkcd(-/-) macrophages exhibited greatly attenuated caspase-1 activation and IL-1β secretion specifically in response to S. typhimurium. Phosphorylation-defective NLRC4 S533A failed to recruit procaspase-1 and did not assemble inflammasome specks during S. typhimurium infection, so phosphorylation of NLRC4 S533 probably drives conformational changes necessary for NLRC4 inflammasome activity and host innate immunity.Keywords
This publication has 23 references indexed in Scilit:
- Innate immune recognition of bacterial ligands by NAIPs determines inflammasome specificityNature, 2011
- Resurrection of a clinical antibody: Template proteogenomic de novo proteomic sequencing and reverse engineering of an anti‐lymphotoxin‐α antibodyProteomics, 2010
- Redundant roles for inflammasome receptors NLRP3 and NLRC4 in host defense againstSalmonellaThe Journal of Experimental Medicine, 2010
- Absent in melanoma 2 is required for innate immune recognition of Francisella tularensisProceedings of the National Academy of Sciences of the United States of America, 2010
- The AIM2 inflammasome is essential for host defense against cytosolic bacteria and DNA virusesNature Immunology, 2010
- The AIM2 inflammasome is critical for innate immunity to Francisella tularensisNature Immunology, 2010
- Innate immune detection of the type III secretion apparatus through the NLRC4 inflammasomeProceedings of the National Academy of Sciences of the United States of America, 2010
- Structural Basis of Protein Kinase C Isoform FunctionPhysiological Reviews, 2008
- Staurosporine‐related compounds, K252a and UCN‐01, inhibit both cPKC and nPKCFEBS Letters, 1993
- The protein kinase inhibitor staurosporine, like phorbol esters, induces the association of protein kinase C with membranesBiochemical and Biophysical Research Communications, 1988