Pyk2 activates the NLRP3 inflammasome by directly phosphorylating ASC and contributes to inflammasome-dependent peritonitis

Abstract

The inflammasome adaptor protein, ASC, contributes to both innate immune responses and inflammatory diseases via self-oligomerization, which leads to the activation of the protease, caspase-1. Here, we report that the cytosolic tyrosine kinases, FAK and Pyk2, are differentially involved in NLRP3 and AIM2 inflammasome activation. The inhibition of FAK and Pyk2 with RNA interference or chemical inhibitors dramatically abolished ASC oligomerization, caspase-1 activation, and IL-1β secretion in response to NLRP3 or AIM2 stimulation. Pyk2 is phosphorylated by the kinase Syk and relocalizes to the ASC specks upon NLRP3 inflammasome activation. Pyk2, but not FAK, could directly phosphorylate ASC at Tyr146, and only the phosphorylated ASC could participate in speck formation and trigger IL-1β secretion. Moreover, the clinical-trial-tested Pyk2/FAK dual inhibitor PF-562271 reduced monosodium urate-mediated peritonitis, a disease model used for studying the consequences of NLRP3 activation. Our results suggest that although Pyk2 and FAK are involved in inflammasome activation, only Pyk2 directly phosphorylates ASC and brings ASC into an oligomerization-competent state by allowing Tyr146 phosphorylation to participate ASC speck formation and subsequent NLRP3 inflammation.

The intra‐ and extracellular functions of ASC specks

“…Inhibition of Syk did not interfere with the recruitment of ASC by NLRP3, but substantially inhibited ASC speck formation . Pyk2 inhibitors also reduced the number of ASC specks, and cells that still assembled ASC complexes in the presence of the inhibitor exhibited specks of enlarged diameters, or filamentous structures . This indicates that phosphorylation of ASC CARD is necessary for cross‐linking of ASC PYD filaments, a mechanism that allows precise control over ASC speck assembly, and perhaps the degree of cross‐linking.…”

“…The exact tertiary structure of the ASC specks might therefore be subject to regulation. Indeed, Pyk2 phosphorylates tyrosine 146 of the ASC CARD upon inflammasome activation in a Syk‐dependent manner . While modification of the CARD may indirectly influence ASC PYD polymerization, it is more likely to modulate the function of the CARD, i.e.…”

“…41 Pyk2 inhibitors also reduced the number of ASC specks, and cells that still assembled ASC complexes in the presence of the inhibitor exhibited specks of enlarged diameters, or filamentous structures. 40 This indicates that phosphorylation of ASC CARD is necessary for cross-linking of ASC PYD filaments, a mechanism that allows precise control over ASC speck assembly, and perhaps the degree of cross-linking. Of note, the Y136 phosphorylation site is conserved in zebrafish ASC, and its mutation to phenylalanine led to filamentous ASC-mKate2 structures in the larvae upon activation.…”

Cells under stress: The mechanical environment shapes inflammasome responses to danger signals

“…Pyk2 can prime inflammasomes after LPS stimulation, and Pyk2 initiates F‐actin reorganization during cell adhesion . Additionally, the requirement for Pyk2 in macrophage podosome formation and in NLRP3 inflammasome activation may explain reduced lung injury observed in Pyk2 deficient mice following LPS‐induced ALI . Finally, a causal link between mechanotransduction and inflammasome activation was suggested by the observation that lipoxin treatment both increased tissue compliance and consequently reduced lung inflammation in a model of LPS‐induced ALI …”

“…Phosphorylation of ASC increased NLRP3 inflammasome assembly and enhanced IL‐1β production. FAK, however, does not directly phosphorylate ASC, but enhances NLRP3 and AIM2 inflammasome activation via interactions with ASC through unknown mechanisms (Fig. ).…”