NLRP3 tyrosine phosphorylation is controlled by protein tyrosine phosphatase PTPN22
Marianne R. Spalinger, Stephanie Kasper, Claudia Gottier, Silvia Lang, Kirstin Atrott, Stephan R. Vavricka, Sylvie Scharl, Petrus M. Gutte, Markus G. Grütter, Hans-Dietmar Beer, Emmanuel Contassot, Andrew C. Chan, Xuezhi Dai, David J. Rawlings, Florian Mair, Burkhard Becher, Werner Falk, Michael Fried, Gerhard Rogler, Michael Scharl
Marianne R. Spalinger, Stephanie Kasper, Claudia Gottier, Silvia Lang, Kirstin Atrott, Stephan R. Vavricka, Sylvie Scharl, Petrus M. Gutte, Markus G. Grütter, Hans-Dietmar Beer, Emmanuel Contassot, Andrew C. Chan, Xuezhi Dai, David J. Rawlings, Florian Mair, Burkhard Becher, Werner Falk, Michael Fried, Gerhard Rogler, Michael Scharl
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Research Article Gastroenterology Immunology

NLRP3 tyrosine phosphorylation is controlled by protein tyrosine phosphatase PTPN22

Abstract

Inflammasomes form as the result of the intracellular presence of danger-associated molecular patterns and mediate the release of active IL-1β, which influences a variety of inflammatory responses. Excessive inflammasome activation results in severe inflammatory conditions, but physiological IL-1β secretion is necessary for intestinal homeostasis. Here, we have described a mechanism of NLRP3 inflammasome regulation by tyrosine phosphorylation of NLRP3 at Tyr861. We demonstrated that protein tyrosine phosphatase non-receptor 22 (PTPN22), variants in which are associated with chronic inflammatory disorders, dephosphorylates NLRP3 upon inflammasome induction, allowing efficient NLRP3 activation and subsequent IL-1β release. In murine models, PTPN22 deficiency resulted in pronounced colitis, increased NLRP3 phosphorylation, but reduced levels of mature IL-1β. Conversely, patients with inflammatory bowel disease (IBD) that carried an autoimmunity-associated PTPN22 variant had increased IL-1β levels. Together, our results identify tyrosine phosphorylation as an important regulatory mechanism for NLRP3 that prevents aberrant inflammasome activation.

Authors

Marianne R. Spalinger, Stephanie Kasper, Claudia Gottier, Silvia Lang, Kirstin Atrott, Stephan R. Vavricka, Sylvie Scharl, Petrus M. Gutte, Markus G. Grütter, Hans-Dietmar Beer, Emmanuel Contassot, Andrew C. Chan, Xuezhi Dai, David J. Rawlings, Florian Mair, Burkhard Becher, Werner Falk, Michael Fried, Gerhard Rogler, Michael Scharl

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Figure 6

NLRP3 is tyrosine phosphorylated at Tyr861.

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NLRP3 is tyrosine phosphorylated at Tyr861. (A) THP-1 cells expressing c...
(A) THP-1 cells expressing control or PTPN22-targeting shRNA were treated with control or NLRP3-targeting siRNA constructs and incubated for 12 hours with upLPS before activation with MSU (6 hours). NLRP3 or PTPN22 was precipitated from the cell lysates and analyzed for tyrosine phosphorylation and PTPN22 coprecipitation or NLRP3 coprecipitation, respectively. (B) Purified NLRP3 was incubated in phosphatase buffer for 60 minutes in the presence or absence of purified WT-PTPN22 (WT), altered-function PTPN22 (619W), loss-of-function PTPN22 (263Q), or purified PTPN2. (C) Multiple sequence alignment of NLRP3 sequences from the indicated species using FASTA.2 (http://www.ebi.ac.uk) reveals conservation of the tyrosine at position 861. (D) HEK293T cells were transfected with WT NLRP3 or a NLRP3 construct where Tyr861 was replaced by a phenylalanine (Y>F). NLRP3 was immunoprecipitated and analyzed for the presence of pTyr. Lys, lysates. (E and F) Nlrp3–/– BMDCs were left nontransfected or transfected with WT NLRP3; Y>F NLRP3; or NLRP3 where Tyr858 was replaced with a glutamine to mimic constitutive phosphorylation (Y>E) or by an alanine (control to glutamine substitution [Y>A]) and activated with MSU. Lysates (E) and supernatants (F) were analyzed for the indicated proteins. (G) BMDCs were derived from ASC-, caspase-1–, NLRP3-, or PTPN22-deficient mice or mice expressing the autoimmunity-associated PTPN22 variant (619W); pretreated for 12 hours with upLPS; and activated with MSU. NLRP3 was immunoprecipitated and analyzed for pTyr and PTPN22. All data are representative of 1 of 3–5 independent experiments. Numbers below the blots show results of densitometry.