PtdIns(3,5)P2, produced by the constitutively STING-associated PIKfyve complex, functions as an endogenous lipid ligand that directly binds STING to promote its oligomerization, ER-to-Golgi trafficking, and TBK1 trans-autophosphorylation, thereby establishing a two-threshold regulatory mechanism where low PtdIns(3,5)P2 levels on the ER enable STING trafficking while high levels at post-Golgi compartments are required for innate immune signaling activation.

Cytosolic DNA exposure triggers innate immune responses through cyclic GMP-AMP (cGAMP) synthase (cGAS). 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. 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. 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.