PtdIns(3,5)P₂ Is an Endogenous Ligand of STING in Innate Immune Signaling

Cytosolic DNA exposure triggers innate immune responses through cyclic GMP-AMP (cGAMP) synthase (cGAS)^1-3. Upon binding to DNA, cGAS is activated to produce cGAMP, which functions as a second messenger that binds to stimulator of interferon genes (STING), an endoplasmic reticulum (ER)-localized signaling adaptor^3-5. STING then traffics from the ER to the Golgi, leading to activation of the kinases TBK1 and IKK and subsequent induction of interferons and other cytokines^6-10. Here we show that phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P₂] is an endogenous ligand of STING that functions together with cGAMP to induce STING activation. Proteomics analysis identified a constitutive interaction between STING and PIKfyve, an enzyme that produces PtdIns(3,5)P₂ in mammalian cells. Deletion of PIKfyve blocked STING trafficking from the ER and TBK1 activation. In vitro reconstitution revealed a strong and selective effect of PtdIns(3,5)P₂ on STING activation. Purified STING bound directly to PtdIns(3,5)P₂ in a fluorescence resonance energy transfer (FRET) assay. Consistently, PtdIns(3,5)P₂ promoted cGAMP-induced STING oligomerization by binding to a groove between STING dimers as revealed by cryo-EM (Li et al., co-submitted). Similar to PIKfyve depletion, mutation of the PtdIns(3,5)P₂-binding residues in STING blocked its trafficking and downstream signaling. These results reveal PtdIns(3,5)P₂ as a lipid ligand of STING with essential roles in innate immunity.