ASC phosphorylation at Y146 regulates inflammasome activation and pyroptosis

Abstract

Abstract Rationale Inflammasome assembly and activation is a complex process regulated, in part, by posttranslational modification of inflammasome proteins. ASC is a universal adaptor in virtually all inflammasome platforms and its modification is critical in regulation of inflammasome function. In particular, PTK inhibitor AG126 greatly inhibits inflammasome activation. Methods We generated stable THP-1 cells with either overexpression of ASC (YFP-ASC) or knock down of ASC (siRNA and CRISPR) followed by ASC knock in. To test the role of ASC phosphorylation in inflammasome assembly and activity, we mutated the highly conserved tyrosine 146 to Y146A. Cells were stimulated with LPS or live bacteria and inflammasome activity was measured by fluorescent microscopy, ELISA, and immunoblotting. Results Upon inflammasome activation ASC is released from the cell together with IL-1β, IL-18 and other inflammasome proteins. Inhibition of select tyrosine kinases severely reduces inflammasome activation, pyroptosis and ASC release. Elimination of the potential phosphorylation site Y146 dramatically changes visible ASC complex formation – from a single round speck to a filamentous structure. Cells with the Y146A ASC mutation show significantly abrogated inflammasome activation as compared to a wild type. Screening of a PTK inhibitor library (Selleckchem) linked several tyrosine kinases to regulating ASC phosphorylation and inflammasome activity. Conclusion ASC phosphorylation at Y146 by tyrosine kinases is an important regulator of inflammasome activity and pyroptosis.