IPS-1 plays an essential role in dsRNA-induced stress granule formation by interacting with PKR and promoting its activation

The formation of cytoplasmic stress granules and the innate immune response are two distinct cellular stress responses. Our study investigated the involvement of four innate immune proteins retinoic- acid-inducible gene I (RIG-I, also known as DDX58), melanoma differentiation-associated gene 5 (MDA5, also known as IFIH1), IFN-beta promoter stimulator (IPS-1, also known as MAVS) and protein kinase regulated by dsRNA (PKR, also known as EIF2AK2) in the formation of stress granules. Knockdown of IPS-1 or PKR significantly decreased the formation of stress granules induced by double-stranded (ds)RNA. IPS-1 depletion markedly attenuated the phosphorylation of PKR and eIF2 alpha that was triggered by dsRNA, and IPS-1 facilitated the in vitro autophosphorylation of PKR. In IPS-1-depleted cells, the dsRNA-mediated dimerization of PKR through its dsRNA-binding domains was significantly abrogated, suggesting that IPS-1 might be involved in PKR dimerization. By co-immunoprecipitation and pulldown assays, our data demonstrate that IPS-1 directly binds to PKR through the IPS-1 caspase activation and recruitment domain (CARD), suggesting that the effect of IPS-1 on the formation of stress granules might be exerted through interacting with PKR and mediating its activation. PKR was recruited into stress granules upon activation, whereas the majority of IPS-1 protein formed clusters on mitochondrial membranes. Our work provides the first evidence that the innate signaling molecule IPS-1 plays an essential role in stress granule formation.