Site-specific phosphorylation and microtubule dynamics control Pyrin inflammasome activation

Abstract

Pyrin, encoded by the MEFV gene, is best known for its gain-of-function mutations causing familial Mediterranean fever (FMF), an autoinflammatory disease. Pyrin forms a caspase-1-activating inflammasome in response to inactivating modifications of Rho GTPases by various bacterial toxins or effectors. Pyrin-mediated innate immunity is unique in that it senses bacterial virulence rather than microbial molecules, but its mechanism of activation is unknown. Here we show that Pyrin was phosphorylated in bone marrow-derived macrophages and dendritic cells. We identified Ser-205 and Ser-241 in mouse Pyrin whose phosphorylation resulted in inhibitory binding by cellular 14-3-3 proteins. The two serines underwent dephosphorylation upon toxin stimulation or bacterial infection, triggering 14-3-3 dissociation, which correlated with Pyrin inflammasome activation. We developed antibodies specific for phosphorylated Ser-205 and Ser-241, which confirmed the stimuli-induced dephosphorylation of endogenous Pyrin. Mutational analyses indicated that both phosphorylation and signal-induced dephosphorylation of Ser-205/241 are important for Pyrin activation. Moreover, microtubule drugs, including colchicine, commonly used to treat FMF, effectively blocked activation of the Pyrin inflammasome. These drugs did not affect Pyrin dephosphorylation and 14-3-3 dissociation but inhibited Pyrin-mediated apoptosis-associated Speck-like protein containing CARD (ASC) aggregation. Our study reveals that site-specific (de)phosphorylation and microtubule dynamics critically control Pyrin inflammasome activation, illustrating a fine and complex mechanism in cytosolic immunity.

Keywords: FMF; Pyrin; Rho toxins; inflammasome; phosphorylation.

Fig. 1.

TcdA activates the Pyrin inflammasome in mouse BMDM cells. (A) TcdA could modify RhoA as TcdB does. Lysates of TcdA- or TcdB-treated DC2.4 cells were subjected to in vitro ADP ribosylation reaction by purified C3 toxin. Anti-RhoA immunoblotting shows that RhoA resisted further modification by C3 toxin after TcdA treatment, as it did after TcdB treatment. (B–D), Assays of inflammasome activation by TcdA in comparison with TcdB, IbpA_Fic1, and FliC (from Pseudomonas aeruginosa) in primary BMDM (priBMDM) cells from wild-type C57BL/6 or Mefv^−/− mice. TcdA and TcdB were added directly to the cell medium. IbpA_Fic1 and FliC were delivered by the LFn-PA system. The supernatants of BMDMs following stimulation were collected and used for the assays. Anti–caspase-1 immunoblotting of the supernatants is shown with anti-actin immunoblots serving as the loading control (B). Lactate dehydrogenase (LDH) release-based cell death (C) and ELISA of IL-1β release (D) are expressed as mean values ± SD from three technical replicates. Pro-CASP1, the caspase-1 precursor; p20, the mature caspase-1. Data shown are representative of three independent experiments.

Fig. 2.

Association of Pyrin with 14-3-3 and dissociation of the complex during activation of the Pyrin inflammasome. (A) Schematic representation of Flag-Pyrin_ΔN used for immuno-purification of the Pyrin complex and subsequent experiments in this study. (B) DC2.4 and iBMDM cells stably expressing Flag-Pyrin_ΔN were stimulated with wild-type TcdB or catalytically inactive mutant TcdB (TcdB^m). Cell lysates were subjected to immuno-purification using the anti-Flag M2 resin. The samples were analyzed by SDS/PAGE followed by silver staining. The single and double asterisks indicate two protein bands coeluted with Pyrin_ΔN in the presence of TcdB^m. (C and D) Coimmunoprecipitation assays of Pyrin and 14-3-3 association. Flag-Pyrin_ΔN and the indicated HA-tagged 14-3-3 isoforms were cotransfected into 293T cells (C). To examine the stimulus-dependent interaction of Pyrin with endogenous 14-3-3ε, DC2.4 cells stably expressing Flag-Pyrin_ΔN were stimulated with TcdB or other indicated toxins or were infected with B. cenocepacia (B. c.) (D). Total cell lysates (Input) and the anti-Flag M2 immunoprecipitates (Flag IP) were blotted with antibodies against the indicated epitope or 14-3-3ε. –, empty vector; Mock, nonstimulation control. All data shown are representative of three independent experiments.

Fig. S1.

Mass spectrometry identification of 14-3-3 as the Pyrin-binding protein and the expression of different 14-3-3 isoforms in primary BMDM cells. (A) Mass spectrometry identification data for the two protein bands that were coeluted with Flag-6xMyc-Pyrin_ΔN purified from stably expressing DC2.4 cells shown in Fig. 2A. The two proteins disappeared upon stimulation of the cells with TcdB. (B) Quantitative real-time PCR (qRT-PCR) analyses of 14-3-3 expression in primary BMDMs. The mRNA level of 14-3-3 was normalized to that of Mefv. Sequences of the qRT-PCR primers used for the seven different 14-3-3 isoforms are available upon request.

Fig. S2.

Yeast two-hybrid assay of the interaction between Pyrin and 14-3-3. Pyrin (wild type or the S205A/S241A double mutant) was cloned into the pGAD-GH bait vector; 14-3-3 (different isoforms or the indicated point mutations) was cloned into the pGBK-T7 prey vector. TAK1 in the prey vector was included as the negative control. The chart on the right summarizes the interaction results.

Fig. 3.

Phosphorylation of Pyrin mediates its interaction with 14-3-3. (A and B) Coimmunoprecipitation assays of Pyrin and 14-3-3ε interaction in 293T cells. (A) Flag-Pyrin_ΔN was expressed into 293T cells. HA-tagged 14-3-3ε or its phospho-binding–deficient mutants were cotransfected with Flag-Pyrin_ΔN. (B) Cell lysates were subjected to anti-Flag immunoprecipitation. Cells were stimulated with TcdB before immunoprecipitation, or the immunoprecipitates were treated with the indicated recombinant phosphatases. PP_E. coli and PP_Y are two λ phosphatase-like phosphatases from E. coli and Y. enterocolitica, respectively. (C and D) Phos-tag gel analyses of Pyrin phosphorylation and its response to TcdB or λ phosphatase treatment. (C) DC2.4 cells expressing full-length Pyrin (Upper) or Pyrin_ΔN (Lower) were assayed. (D) Flag-tagged 14-3-3ε was cotransfected with HA-tagged full-length Pyrin into 293T cells. In C and D, live cells were stimulated with TcdB; λ phosphatase was added into the cell lysates before analyses in C. Flag-14-3-3ε also was immunoprecipitated and analyzed by immunoblotting as indicated. (E and F) Phos-tag gel analyses of Pyrin phosphorylation and its effect on caspase-1 activation and14-3-3ε interaction. DC2.4 cells expressing full-length Pyrin (E) or Pyrin_ΔN (F) were stimulated with the indicated Rho-modifying toxins or were infected with B. cenocepacia (wild type or the Δhcp mutant strain). Cell supernatants were analyzed by anti–caspase-1 immunoblotting (E), and cell lysates were also subjected to anti-Flag immunoprecipitation (F). The phos-tag gel analyses were performed with total cell lysates. The total lysates (Input) and the immunoprecipitates (Flag IP) were analyzed by immunoblotting using indicated antibodies. Pro-CASP1, the caspase-1 precursor; p20, the mature caspase-1. All data shown are representative of three independent experiments.

Fig. 4.

Dephosphorylation of Pyrin on Ser-205 and Ser-241 correlates with activation of the Pyrin inflammasome.(A and B) Assays of Pyrin (89–410) truncation and serine mutants of Pyrin_ΔN in signal-induced dephosphorylation and 14-3-3ε disassociation. DC2.4 cells stably expressing Flag-Pyrin (89–410) (A) and 293T cells expressing Flag-Pyrin_ΔN (or the indicated serine mutants) (B) were stimulated with TcdB or with the other indicated Pyrin inflammasome stimuli. (C) Flag-Pyrin_ΔN or its phosphorylation-site mutants were transiently expressed in 293T cells or stably expressed in DC2.4 cells. Total cell lysates were either directly subjected to immunoblotting analyses or were treated first with λ phosphatase. (D and E) Assays of toxin-induced Ser-241/205 dephosphorylation and 14-3-3ε disassociation from Pyrin. DC2.4 cells stably expressing Flag-Pyrin (D) or intact primary BMDMs (E) were stimulated with the indicated Rho-modifying toxins. (F and G) Assays of human Pyrin phosphorylation on Ser-242/Ser-208 and effects of point mutations of serine residues. 293T cells transiently transfected with indicated human Pyrin_ΔN (hPyrin_ΔN) point mutations were treated with or without TcdB. The point mutants analyzed in F are mutants found in FMF patients. Exogenous Flag-tagged Pyrin was immunoprecipitated using anti-Flag antibody (A, B, F, and G), and endogenous Pyrin was immunoprecipitated using a monoclonal anti-Pyrin antibody (E). The immunoprecipitates and total lysates were analyzed by anti-Flag, anti-Pyrin, anti–14-3-3ε, or the phospho-specific antibodies (Ab_pS241 and Ab_pS205) immunoblotting as indicated. Ab_pS241 and Ab_pS205 denote rabbit phospho-S241– and phospho-S205–specific monoclonal antibodies, respectively. The total lysates also were subjected to the phos-tag gel immunoblotting (A, D, and E). All data shown are representative of three independent experiments.

Fig. S3.

Identification of Pyrin phosphorylation sites by mass spectrometry. Flag-Pyrin (89–410) was affinity-purified from the stable DC2.4 cell line and was subjected to mass spectrometry analysis. (A) Red-highlighted sequences are tryptic peptides identified from the spectra obtained. The sequence coverage is about 95%, among which three serine residues, Ser-188, Ser-205, and Ser-241, were found to be phosphorylated. (B–D) The MS/MS spectra of tryptic peptides containing Ser-188, Ser-205, and Ser-241 are shown in B, C, and D respectively. The β- and γ-type product ions are marked in the spectra and are illustrated along with the corresponding peptide sequence above spectra. Data shown are representative of two independent experiments.

Fig. S4.

Mutation screen of candidate serine residues in Pyrin that are important for 14-3-3 binding. 293T cells transiently transfected with constructs of the single serine mutation in Flag-Pyrin_ΔN were subjected to anti-Flag immunoprecipitation. Anti-Flag and anti–4-3-3ε immunoblots of the total cell lysates (Input) and the immunoprecipitates (Flag IP) are shown. Ser-188, Ser-205, and Ser-241 are the phospho-serine residues identified from mass spectrometry analyses in Fig. S3. Other serine residues conserved in human and mouse Pyrin were also included in the assay. Endogenous 14-3-3ε was examined. The experiments were performed three times; representative results are shown.

Fig. S5.

Validation of the rabbit monoclonal anti-Pyrin antibody. Lysates of DC2.4 cells or DC2.4 cells expressing Flag-Pyrin (Left) and primary mouse BMDMs (priBMDM) (Right) were subjected to immunoblotting analyses using the rabbit monoclonal antibody developed using recombinant mouse Pyrin (89–410) as the antigen and anti-actin antibody as the loading control. The BMDMs were derived from wild-type or Mefv^−/− mice and were either left untreated (Mock) or stimulated with LPS or TNF-α.

Fig. S6.

The aspartate/glutamate substitutions of Ser-205 and Ser-241 do not functionally mimic the phosphorylated serine. (A and B) Flag-Pyrin_ΔN or the indicated single mutation stably expressed in DC2.4 cells (A) or transiently expressed in 293T cells (B) was subjected to anti-Flag immunoprecipitation. Cells were either left untreated or were stimulated with TcdB. Immunoblots of the precipitates (Flag IP) and the total cell lysates (Input) using the indicated antibodies are shown. Phosphorylation of Pyrin was also examined by the phos-tag gel immunoblotting (A). (C and D) Effects of aspartate substitution of Ser-205 and Ser-241 in Pyrin on TcdB-induced activation of the Pyrin inflammasome. DC2.4 cells stably expressing wild-type Flag-Pyrin or its S205D or S241D mutant were left untreated or were stimulated with TcdB. Cell supernatants were analyzed by anti–caspase-1 immunoblotting; Pro-CASP1, the caspase-1 precursor; p20, the mature caspase-1. (C) Total cell lysates were blotted with anti-actin, anti-ASC, or anti-Pyrin antibody or were subjected to crosslinking before anti-ASC immunoblotting. (D) LDH release-based cell death is expressed as mean values ± SD from three technical replicates. Data shown are representative of three independent experiments.

Fig. 5.

Phosphorylation and dephosphorylation of Pyrin on Ser-205/241 are important for toxin-induced activation of the Pyrin inflammasome. (A and B) Tet-on expression of Pyrin phosphorylation-negative mutants induces pyroptotic cell death. Wild-type Pyrin or the Pyrin S205A or S241A mutant was stably transfected into DC2.4 cells under a tetracycline-inducible promoter. Doxycycline (Dox) was added to induce Pyrin expression. Immunoblotting analyses of Pyrin expression are shown in A. (C and D) Assays of phosphorylation-site mutants on TcdB-induced Pyrin inflammasome activation. Viable DC2.4 cells stably expressing wild-type or the indicated serine mutants of Pyrin were subjected to TcdB stimulation. Cell supernatants were analyzed by anti–caspase-1 immunoblotting. Pro-CASP1, the caspase-1 precursor; p20, the mature caspase-1. Total cell lysates were subjected to immunoblotting analyses as indicated (C), in which cross-linking and phos-tag gel electrophoresis were performed to examine ASC oligomerization and the phosphorylation status of Pyrin, respectively. LDH release-based cell death shown in B and D is expressed as mean values ± SD from three technical replicates. All data shown are representative of three independent experiments.

Fig. 6.

Microtubule dynamics controls the activation of the Pyrin inflammasome downstream of Pyrin dephosphorylation. (A–D) Specific inhibition of Pyrin inflammasome activation by the microtubule-targeting drugs colchicine, vinblastine, and BAL27862. Primary BMDMs were preincubated with the indicated drugs and then were stimulated with TcdA, TcdB, IbpA_Fic1, or other inflammasome agonists. IbpA_Fic1 and FliC were delivered into the cells by the LFn-PA system. LPS plus nigericin (LPS+Nig) was used to stimulate NLRP3 inflammasome activation. (E and F) Effects of microtubule-targeting drugs on TcdB-induced 14-3-3 dissociation from Pyrin and Pyrin dephosphorylation. DC2.4 cells stably expressing Pyrin_ΔN (E) or full-length Pyrin (F) were pretreated with the indicated compounds and then were stimulated with TcdB. The interaction between Pyrin_ΔN and endogenous 14-3-3ε was assayed by anti-Flag immunoprecipitation (E), and the phosphorylation status of Pyrin was examined by the phospho-specific antibody blotting (Ab_pS241 and Ab_pS205) as well as by the phos-tag gel immunoblotting analysis (F). (G) Effects of microtubule-targeting drugs on TcdB-induced ASC aggregation downstream of Pyrin activation. Primary BMDM cells pretreated with colchicine, vinblastine, or BAL27862 were stimulated with TcdA, TcdB, or LPS plus nigericin. Shown are the anti-ASC (endogenous) immunofluorescence images of stimulated cells. The percentages of cells developing the ASC foci structure are marked on the images (mean values ± SD). (Scale bars: 20 μm.) Cell supernatants were analyzed by anti–caspase-1 immunoblotting in A, D, and F; Pro-CASP1, the caspase-1 precursor; p20, the mature caspase-1. ELISA of IL-1β release (B) and LDH release-based cell death (C) are expressed as mean values ± SD from three technical replicates. All data shown are representative of three independent experiments.

Fig. S7.

Microtubule-disrupting or -stabilizing drugs inhibit the activation of the Pyrin inflammasome. (A and B) Effects of the microtubule-targeting drugs colchicine, vinblastine, and BAL27862 on activation of the Pyrin inflammasome. Primary BMDM cells were preincubated with indicated drugs and then were stimulated with TcdA, TcdB, or IbpA_Fic1 (A and B) or with LPS plus the indicated concentration of nigericin (B). IbpA_Fic1 was delivered into the cells by the LFn-PA system. (C and D) Comparison of the activity of colchicine, vinblastine, and BAL27862 on microtubule disruption and their inhibition of Pyrin inflammasome activation. Primary BMDMs were pretreated with the indicated compounds at different concentrations and then were stimulated with TcdA. Anti-tubulin fluorescence images are shown in C. (E and F) Assays of the effect of the microtubule-stabilizing drug paclitaxel on TcdB-induced activation of the Pyrin inflammasome. DC2.4 cells stably expressing full-length Pyrin were pretreated with paclitaxel or the indicated microtubule-disrupting compounds. ELISA of IL-1β release (A) and LDH release-based cell death (A, B, and E) are expressed as mean values ± SD from three technical replicates. Cell supernatants (B, D, and F) were analyzed by anti–caspase-1 immunoblotting. Pro-CASP1, the caspase-1 precursor; p20, the mature caspase-1. Total cell lysates were blotted with anti-actin (B, D, and F) or with anti-Pyrin antibody and the rabbit phospho-S241– and phospho-S205–specific antibodies (Ab_pS241 and Ab_pS205) (F). Pyrin phosphorylation was also examined by phos-tag gel immunoblotting. Data shown are representative of three independent experiments.

Fig. 7.

A model of Pyrin inflammasome activation by Rho-modifying toxins.