Phosphorylation at Ser182 and Thr186 blocks GSDMD pyroptotic activity

Pyroptosis, mediated by gasdermin D (GSDMD), is a form of programmed cell death highly associated with inflammatory and immune diseases. While its activation is well-studied, the precise negative regulatory mechanisms remain to be fully elucidated. Here, we sought to investigate the regulatory role of phosphorylation on GSDMD activity. We identified that phosphorylation at murine Ser182/Thr186 (Ser181/Ser185 in humans) constitutes an inhibitory regulatory mechanism for the cytolytic activity of the GSDMD N-terminal fragment (GD-NT). Mechanistically, using structural modeling and site-directed mutagenesis, we found that phosphorylation at Ser182/Thr186 did not affect the auto-inhibition of full-length GSDMD or its cleavage by caspase-11. However, it severely impaired the pyroptotic activity of GD-NT by affecting its membrane localization and oligomerization. Consequently, the double phosphomimetic mutant of GD-NT (S182D/T186D) was non-lytic to cells. Thus, we define Ser182/Thr186 as critical post-cleavage checkpoint sites for inhibiting pyroptosis. This discovery enriches the regulatory network of GSDMD and provides potential therapeutic targets for modulating inflammatory diseases.