Fused in Sarcoma (FUS) is a ubiquitously expressed protein that can phase-separate from nucleoplasm and cytoplasm into distinct liquid-droplet structures. It is predominately nuclear and most of its functions are related to RNA and DNA metabolism. Excessive persistence of FUS within cytoplasmic phase-separated assemblies is implicated in the diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Phosphorylation of FUS's prion-like domain (PrLD), by nuclear PIKK-family kinases following DNA damage, was previously shown to alter FUS's liquid-phase and solid-phase transitions in cell models and in vitro. However, proteomic data suggest FUS's PrLD is phosphorylated at numerous additional si... More
Fused in Sarcoma (FUS) is a ubiquitously expressed protein that can phase-separate from nucleoplasm and cytoplasm into distinct liquid-droplet structures. It is predominately nuclear and most of its functions are related to RNA and DNA metabolism. Excessive persistence of FUS within cytoplasmic phase-separated assemblies is implicated in the diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Phosphorylation of FUS's prion-like domain (PrLD), by nuclear PIKK-family kinases following DNA damage, was previously shown to alter FUS's liquid-phase and solid-phase transitions in cell models and in vitro. However, proteomic data suggest FUS's PrLD is phosphorylated at numerous additional sites and it is unknown if other non-PIKK and non-nuclear kinases might be influencing FUS's phase transitions. Here we evaluated disease mutations and stress conditions that increase FUS accumulation into cytoplasmic phase-separated structures. We observed that cytoplasmic liquid-phase structures contain FUS phosphorylated at novel sites, which occured independently of PIKK-family kinases. We engineered phosphomimetic substitutions within FUS's PrLD and observed that mimicking a few phosphorylation sites strongly inhibited FUS solid-phase aggregation, while minimally altering liquid-phase condensation. These effects occured independent of the exact location of the phosphomimetic substitutions, suggesting that modulation of PrLD phosphorylation may offer therapeutic strategies that are specific for solid-phase aggregation observed in disease.
