Phosphoprotein Phosphatase Activity Positively Regulates Oligomeric Pyrin to Trigger Inflammasome Assembly in Phagocytes

Pyrin is a pattern-recognition receptor in phagocytes that triggers capase-1 inflammasome assembly in response to bacterial toxins and effectors that inactivate RhoA. Pyrin contains oligomerization domains and is negatively regulated by phosphorylation of two residues, S205 and S241 (murine) or S208 and S242 (human), via the kinases PKN1/2, which are activated by RhoA. Familial Mediterranean Fever (FMF) is caused by phagocyte production of pyrin gain of function variants, which have a lower threshold for inflammasome assembly upon RhoA-PKN axis inhibition. Inactivation of the RhoA-PKN axis removes negative regulation but a phosphoprotein phosphatase (PPP) is needed to positively regulate pyrin. No PPP that dephosphorylates pyrin has been identified, oligomerization of murine pyrin has not been studied, and the phosphorylation status of oligomeric pyrin is unknown. We used murine macrophages and FMF patient’s monocytes combined with the use of bacterial agonists and chemical inhibitors, native PAGE, phospho-specific antibodies and siRNA knockdowns to determine if a PPP positively regulates oligomeric pyrin. Results with broadly-specific inhibitors indicate that PPP activity is required to dephosphorylate murine and human pyrin in wild type or FMF patient’s phagocytes. Findings from native PAGE show that murine pyrin forms oligomers that are phosphorylated on S205 prior to RhoA inactivation. Inhibitors cause reduced mobility of murine pyrin on native PAGE and hyperphosphorylation of S242 in human pyrin, suggesting a PPP constitutively counterbalances PKN to keep the second site hypophosphorylated. Data from siRNA knockdown experiments implicate PP2A in dephosphorylation of S205 and positive regulation of pyrin in response to RhoA inactivation. Key points Murine pyrin is oligomeric and phosphorylated on S205 prior to inflammasome assembly PPP activity positively regulates pyrin inflammasome assembly in mice and humans The alpha and beta subunits of PP2A dephosphorylate murine pyrin S205 in macrophages