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-1Ī² 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-1Ī² 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.
Highlights
Our results revealed that PF-431396 and PF-573228 significantly inhibited IL-1Ī²secretion and pyroptosis, as measured by the release of lactate dehydrogenase (LDH) by THP-1 cells stimulated with the NLRP3 stimulators, ATP, the ionophore nigericin, and monosodium urate (MSU) crystals, and AIM2 stimulator, poly(dA:dT) (Fig. 1B and Supplementary Fig. S1A), but not affected pro-IL-1Ī²expression (Supplementary Fig. S2A)
Depletion of focal adhesion kinase (FAK), but not Pyk[2], reduced poly(dA:dT)-induced associated speck-like protein containing caspase recruitment domain (CARD) (ASC) oligomerization (Fig. 3E). These results demonstrate that Pyk[2] and FAK are required for activation of the ASC-caspase-1-IL-1Ī²axis in nigericin-stimulated NLRP3 inflammasomes
The FAK family kinases, Pyk[2] and FAK, regulate cell migration by disassembling focal adhesions to extend the leading edge and retract the trailing edge of cells[16]. This requires dynamic rearrangement of the actin cytoskeleton, which is achieved by the association of Pyk[2] and FAK with GRAF, paxillin, and other proteins, which in turn activate signaling by Rho and Rac[16]
Summary
A number of kinases [e.g., PKR5, AMPK6, Syk[7,8,9], Lyn[10], PI(3)K11, BTK12, and DAPK13] have been implicated in regulation of the NLRP3 inflammasome. Their precise mechanisms of action have not been elucidated yet. We show that Pyk[2] can bind ASC and directly phosphorylate it at Tyr[146], and that only phosphorylated ASC can form oligomers and trigger IL-1Ī²secretion These results show that Pyk[2] can regulate inflammation by directly targeting the key adaptor protein, ASC
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