Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Jun 5;214(6):1725-1736.
doi: 10.1084/jem.20160933. Epub 2017 May 2.

NLRP3 inflammasome assembly is regulated by phosphorylation of the pyrin domain

Andrea Stutz  1 , Carl-Christian Kolbe  1 , Rainer Stahl  1 , Gabor L Horvath  1 , Bernardo S Franklin  1 , Olivia van Ray  1 , Rebecca Brinkschulte  1   2 , Matthias Geyer  1   2 , Felix Meissner  3 , Eicke Latz  4   5   6   7
Affiliations

NLRP3 inflammasome assembly is regulated by phosphorylation of the pyrin domain

Andrea Stutz et al. J Exp Med. .

Abstract

NLRP3 is a cytosolic pattern recognition receptor that senses microbes and endogenous danger signals. Upon activation, NLRP3 forms an inflammasome with the adapter ASC, resulting in caspase-1 activation, release of proinflammatory cytokines and cell death. How NLRP3 activation is regulated by transcriptional and posttranslational mechanisms to prevent aberrant activation remains incompletely understood. Here, we identify three conserved phosphorylation sites in NLRP3 and demonstrate that NLRP3 activation is controlled by phosphorylation of its pyrin domain (PYD). Phosphomimetic residues in NLRP3 PYD abrogate inflammasome activation and structural modeling indicates that phosphorylation of the PYD regulates charge-charge interaction between two PYDs that are essential for NLRP3 activation. Phosphatase 2A (PP2A) inhibition or knock-down drastically reduces NLRP3 activation, showing that PP2A can license inflammasome assembly via dephosphorylating NLRP3 PYD. These results propose that the balance between kinases and phosphatases acting on the NLRP3 PYD is critical for NLRP3 activation.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
NLRP3 is phosphorylated. (A) Mass spectrometry workflow for the analysis of NLRP3. (B) Summed peptide intensities per amino acid display quantitative evidences for overlapping peptides. 92% of the NLRP3 sequence is covered by identified and quantified peptides using a combination of trypsin, chymotrypsin, and Glu-C. Representative of at least two different experiments. (C) Identification of phosphorylated NLRP3 peptides by mass spectrometry. MS2 fragmentation spectra illustrating the coverage of y-ion and b-ion series. Phosphorylated amino acids are indicated with (ph). Spectra are representative of at least two different experiments. (D) ClustalW alignment of NLRP3 orthologs surrounding the phosphorylated residues (highlighted in red). "*" indicates fully conserved residue: strongly similar and "." weakly similar properties. See also Fig. S1.
Figure 2.
Figure 2.
Phosphorylated residues between PYD/NOD and in LRR region show little influence on NLRP3 activation. (A) Images and quantification of HEK293 FlpIn cells expressing ASC-mTurquoise transfected with NLRP3-mCitrine WT, the indicated NLRP3 mutants or mCitrine-HA (as a control). Bar, 20 µm. Images are representative of four independent experiments. n = 4 ± SEM. *, P < 0.05; **, P < 0.01 (ANOVA with Holm-Sidak). (B) NLRP3-deficient immortalized macrophages (iMOs) were reconstituted with point-mutated NLRP3-mCitrine. (C) Quantification of TNF by ELISA in NLRP3-deficient iMOs reconstituted with NLRP3-mCitrine WT or indicated mutations after 3-h LPS priming. n = 3 ± SEM. (D) Immunoblot of NLRP3 deficient iMOs reconstituted with NLRP3-mCitrine WT or indicated mutations after stimulation with nigericin (1 h) or left untreated. Immunoblots are representative of two independent experiments.
Figure 3.
Figure 3.
Phosphorylation of S5 inhibits NLRP3 activation. (A–C) Quantification of IL-1β and TNF by ELISA in NLRP3-deficient iMOs reconstituted with NLRP3-mCitrine WT or indicated mutations after 3 h LPS priming (for [A] TNF) and stimulated with (B) lethal toxin (6 h) or (C) nigericin (1 h). (A–C) n = 3 ± SEM; (C) *, P < 0.05 (ANOVA with Holm-Sidak). (D) Immunoblot of NLRP3-deficient iMOs reconstituted with NLRP3-mCitrine WT or indicated mutations after stimulation with nigericin (1 h) or left untreated. Immunoblots are representative of three independent experiments. (E) Co-immunoprecipitation (IP) of ASC with NLRP3-mCitrine. NLRP3-deficient iMOs reconstituted with NLRP3-mCitrine WT or indicated mutations were primed with LPS for 2 h and left untreated (none) or stimulated with nigericin (1 h). NLRP3 was immunoprecipitated using anti-GFP antibodies. Asterisk denotes unspecific bands. Immunoblots are representative of two independent experiments.
Figure 4.
Figure 4.
Phosphorylation interferes with a polybasic cluster in the NLRP3 PYD. (A) Electrostatic surface representation of NLRP3 PYD. (left) Nonphosphorylated PYD (PDB accession no. 3QF2); (right) surface charge changes with phosphorylated serine 5 modeled. (B) Mutations introduced in the N/C terminal region of NLRP3 PYD. (C–E) Quantification of IL-1β and TNF by ELISA in NLRP3 deficient iMOs reconstituted with NLRP3-mCitrine WT or PYD-N-terminal (R7A, K9A, and R12A) or PYD-C-terminal (K86A, R89A, and K93A) mutations after 3 h LPS priming (for [C] TNF) and stimulated with (D) lethal toxin (6 h) or (E) nigericin (1 h). (C–E), n = 3 ± SEM. (F) Immunoblot of NLRP3-deficient iMOs reconstituted with NLRP3-mCitrine WT or indicated mutations after 2 h LPS priming and left untreated (none) or stimulated with nigericin (1 h). Immunoblots are representative of two independent experiments.
Figure 5.
Figure 5.
S5 is located in a PYD-PYD interaction interface. (A) Model of a NLRP3 PYD filament based on the crystal structure of NLRP3 PYD (PDB 3QF2) and the EM structure of ASC filaments (PDB 3J63). (B, top) Sequence of NLRP3 PYD with residues participating in interface 1a highlighted in green, the residues of interface 1b colored in orange. Interacting residues of the interfaces 1a and 1b are derived from a sequence alignment with ASC PYD in fibrillary state (PDB 3J63). (middle) Ribbon representation of two interacting NLRP3 PYD. Residues are color coded as above. (bottom) Electrostatic surface representation of NLRP3 PYD. (C) Structure-based sequence alignment of inflammasome-forming human PYDs. Helices are indicated above the alignment and shaded in gray in the individual sequences. The residues potentially participating in interface 1a are marked by a red dot above the alignment, the residues of interface 1b are marked by an orange dot. (D) Images and quantification of HEK293T cells transfected with constructs expressing NLRP3-PYD(1–99)-mCitrine WT or the indicated NLRP3 mutants. Bar, 20 µm. Images are representative of five independent experiments. n = 5 ± SEM; **, P < 0.01; ***, P < 0.001 (ANOVA with Holm-Sidak). (E) Images and quantification of HEK293T cells transfected with constructs expressing NLRP3-PYD(1–99)-mCitrine WT or PYD-N-terminal (R7A, K9A, and R12A) or PYD-C-terminal (K86A, R89A, and K93A) mutations. Bar, 20 µm. Images are representative of three independent experiments. n = 3 ± SEM; *, P < 0.05; **, P < 0.01 (ANOVA with Holm-Sidak).
Figure 6.
Figure 6.
NLRP3 PYD gets dephosphorylated with involvement of protein phosphatase 2A. (A and B) Quantification of (A) TNF and (B) IL-1β by ELISA in NLRP3-deficient iMOs reconstituted with NLRP3-FLAG after 15 min pretreatment with okadaic acid (OKA) or left untreated (none), followed by 2 h LPS priming (for TNF) and stimulated with nigericin (1 h). (A and B) n = 2 ± SEM. (C) Ion intensities for the peptide containing the phosphorylated S3 (acTphSVRCKL; m/z = 493.23046; z = 2), normalized to the nonphosphorylated counterpart (acTSVRCKL; m/z = 453.24729; z = 2) are plotted at the indicated retention times for LPS-treated and LPS+OKA-treated samples. Representative of two independent experiments. (D) Ratio of the apex of phosphorylated to nonphosphorylated peptide intensities (mean ± SEM of replicates, representative of two independent experiments). (E) Immunoblot of NLRP3-deficient iMOs reconstituted with NLRP3-FLAG WT and transfected with the indicated siRNAs for 40 h. Immunoblots are representative of three independent experiments. (F and G) Same as E, but showing mRNA levels (fold of control, normalized to Hprt expression) for (F) Ppp2ca and (G) Ppp2cb. (F and G) n = 4 ± SEM. ***, P < 0.001; ****, P < 0.0001 (ANOVA with Holm-Sidak). (H and I) Quantification of IL-1β (H) and TNF (I) by ELISA in NLRP3-deficient iMOs reconstituted with NLRP3-FLAG WT, transfected with the indicated siRNAs for 40 h, and then primed for 2 h with LPS (for TNF) and stimulated with nigericin (1 h). (H and I) n = 3 ± SEM. *, P < 0.05; **, P < 0.01 (ANOVA with Holm-Sidak).
Figure 7.
Figure 7.
Model for licensing through dephosphorylation. In unprimed cells, NLRP3 is phosphorylated at S5, resulting in electrostatic repulsion of pyrin domains. Dephosphorylation involving PP2A licenses NLRP3 for activation and is required before inflammasome assembly can occur.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bae J.Y., and Park H.H.. 2011. Crystal structure of NALP3 protein pyrin domain (PYD) and its implications in inflammasome assembly. J. Biol. Chem. 286:39528–39536. 10.1074/jbc.M111.278812 - DOI - PMC - PubMed
    1. Baker N.A., Sept D., Joseph S., Holst M.J., and McCammon J.A.. 2001. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl. Acad. Sci. USA. 98:10037–10041. 10.1073/pnas.181342398 - DOI - PMC - PubMed
    1. Bauernfeind F.G., Horvath G., Stutz A., Alnemri E.S., MacDonald K., Speert D., Fernandes-Alnemri T., Wu J., Monks B.G., Fitzgerald K.A., et al. . 2009. Cutting edge: NF-kappaB activating pattern recognition and cytokine receptors license NLRP3 inflammasome activation by regulating NLRP3 expression. J. Immunol. 183:787–791. 10.4049/jimmunol.0901363 - DOI - PMC - PubMed
    1. Corpet F. 1988. Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res. 16:10881–10890. 10.1093/nar/16.22.10881 - DOI - PMC - PubMed
    1. Cox J., and Mann M.. 2008. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26:1367–1372. 10.1038/nbt.1511 - DOI - PubMed

Publication types