The prion-like domain of Fused in Sarcoma is phosphorylated by multiple kinases affecting liquid- and solid-phase transitions

Mol Biol Cell. 2020 Nov 1;31(23):2522-2536. doi: 10.1091/mbc.E20-05-0290. Epub 2020 Sep 2.

Abstract

Fused in Sarcoma (FUS) is a ubiquitously expressed protein that can phase-separate from nucleoplasm and cytoplasm into distinct liquid-droplet structures. It is predominantly 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 and frontotemporal dementia. Phosphorylation of FUS's prion-like domain (PrLD) by nuclear phosphatidylinositol 3-kinase-related kinase (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 that FUS's PrLD is phosphorylated at numerous additional sites, and it is unknown if other non-PIKK and nonnuclear kinases might be influencing FUS's phase transitions. Here we evaluate 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 occurred independent 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 occurred 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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Cell Nucleus / metabolism
  • Cytoplasm / metabolism
  • DNA Damage
  • Humans
  • Mutation
  • Phase Transition / drug effects*
  • Phosphatidylinositol 3-Kinases / metabolism
  • Phosphorylation
  • Prions / genetics
  • Prions / metabolism*
  • Protein Aggregation, Pathological
  • Protein Processing, Post-Translational
  • Proteomics
  • RNA-Binding Protein FUS / metabolism*
  • RNA-Binding Protein FUS / physiology

Substances

  • FUS protein, human
  • Prions
  • RNA-Binding Protein FUS