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.

融合在肉瘤中（FUS）是一种普遍表达的蛋白质，可以从核质和细胞质中相分离成不同的液滴结构。它主要是核的，其大部分功能与RNA和DNA代谢有关。FUS在细胞质相分离的组件中过度持久存在与肌萎缩性侧索硬化症（ALS）和额颞痴呆（FTD）有关。DNA损伤后，PISK家族核激酶将FUS的病毒样结构域（PrLD）磷酸化，先前已证明可改变FUS在细胞模型和体外的液相和固相转变。但是，蛋白质组学数据表明FUS的PrLD在许多其他位点被磷酸化，尚不清楚其他非PIKK和非核激酶是否可能影响FUS的相变。在这里，我们评估了增加FUS积累到细胞质相分离结构中的疾病突变和应激条件。我们观察到，胞质液相结构包含FUS磷酸化在新的站点，独立于PIKK家族激酶发生。我们在FUS的PrLD中设计了模拟磷酸酯的取代基，并观察到模仿一些磷酸化位点会强烈抑制FUS固相聚集，同时最小程度地改变液相缩合。这些效应的发生与拟磷酸酯取代的确切位置无关，这表明PrLD磷酸化的调节可能提供特定于疾病中观察到的固相聚集的治疗策略。