Site-specific phosphorylation and microtubule dynamics control Pyrin inflammasome activation

Significance

Pyrin, encoded by the MEFV gene, is causative for familial Mediterranean fever (FMF), an autoinflammatory disease. Pyrin responds to bacterial modifications/inactivation of Rho GTPases by assembling an inflammasome complex for activating caspase-1. Pyrin is a unique immune sensor because it senses bacterial virulence rather than recognizing microbial products. We found that Pyrin is phosphorylated on two serine sites which keep Pyrin inactive through binding by 14-3-3 proteins. Toxin stimulation and bacterial infection trigger Pyrin dephosphorylation and 14-3-3 dissociation, allowing Pyrin inflammasome activation. Colchicine, a microtubule-disrupting drug used to treat FMF, inhibits Pyrin activation downstream of dephosphorylation and 14-3-3 dissociation. These findings not only help us understand FMF pathogenesis/treatment but also shed mechanistic insights into cytosolic immunity.

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.