The prion-like domain of Fused in Sarcoma is phosphorylated by multiple kinases affecting liquid- and solid-phase transitions - PubMed (original) (raw)
The prion-like domain of Fused in Sarcoma is phosphorylated by multiple kinases affecting liquid- and solid-phase transitions
Izzy Owen et al. Mol Biol Cell. 2020.
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.
Figures
FIGURE 1:
Phosphospecific antibodies recognize non-PIKK sites within FUS’s prion-like domain. (A) H4 cells with FUS knockdown by siRNA treated with DMSO or 50 nM calicheamicin (CLM) were analyzed by Western blots probed with commercially available FUS and custom phospho-FUS antibodies. (B) Quantification of percent reduction in Western blot band intensity of both FUS and phospho-FUS blots in A (n = 3). Raw data in S1A. (C, D) H4 neuroglioma cells treated with 50 nM CLM after FUS knockdown were fixed and probed with commercially available FUS and custom phosphospecific antibodies. Nuclear fluorescence signal was quantified and normalized to total fluorescence for each experiment. Figure data analyzed using Student’s t test (n = 3).
FIGURE 2:
DNA damage induces multiphosphorylation of FUS’s prion-like domain at PIKK and non-PIKK sites. (A) H4 cells treated with a dose series of calicheamicin were analyzed by Western blot probed with anti-phospho-FUS (pSer26, pSer30, pSer57, pThr71, and pSer96) and anti-FUS antibodies. (B) Recombinant DNA-PK was used to phosphorylate MBP-FUS in vitro_._ Reaction was analyzed by Western blot and probed with commercial FUS and phosphospecific antibodies.
FIGURE 3:
FUS is phosphorylated at both PIKK and non-PIKK sites following non–DNA damaging stress. (A) H4 cells treated with 500 µM sodium arsenite or 0.4 M sorbitol at various time points were analyzed by Western blot using anti-phospho-FUS (pSer26, pSer30, pSer57, pThr71, and pSer96) and anti-FUS antibodies. The time courses show that only three of the five sites analyzed are phosphorylated by non–DNA damaging stress. (B) The normalized band signal intensities from A; 95% CI error bars (n = 3). (C) H4 cells treated with either sodium arsenite or sorbitol for 1 h were analyzed using confocal microscopy. Both FUS and phospho-FUS (pSer30—representative images) are found in cytoplasmic granules. (D) H4 cells treated with either sodium arsenite or sorbitol for 1 h were analyzed using confocal microscopy. Phospho-FUS (pSer30—representative images) colocalizes with stress granule marker G3BP.
FIGURE 4:
Inhibition of PIKK-family kinases does not prevent phosphorylation of the FUS prion-like domain following osmotic or oxidative stress. (A) Phosphorylation status of FUS from H4 cells treated with or without torin 2 under varying stress conditions were analyzed by Western blot. (B) Quantification of band fluorescence normalized to total FUS; error bars represent 95% CI (n = 3). (C) Phosphorylation of FUS in H4 cells treated with or without Torin 2 under varying stress conditions. Fixed cells imaged using confocal microscopy. Cells were probed with FUS and phospho-FUS(pS30) antibodies. (D) Quantification of nuclear and cytoplasmic phospho-FUS(pS30); fluorescence error bars represent 95% CI (n = 3).
FIGURE 5:
Phosphorylated ALS-mutant FUS is present in cytoplasmic granules. (A) H4 cell transfected with GFP-FUS(R495X) for 24 h or untransfected control (*). GFP-FUS(R495X) was pulled down from cell lysates using GFP immunoprecipitation (IP). IP products were analyzed by Western blot and probed with anti-FUS (Santa Cruz) and phospho-FUS (pSer26, pSer30, pThr71, and pSer96). GFP-FUS(495X) is denoted with an arrow at roughly 100 kDa. (B) Ectopic expression of mutant FUS(R495X) in H4 cells with N- or C-terminal GFP. Anti-phospho-FUS(pSer30) antibody was used to probe for phosphorylated FUS. (C) Quantification of number of cells expressing diffuse, granular, or aggregated FUS(R495X) at 6, 8, or 24 h posttransfection (n = 3). (D) Quantification using Pearson’s coefficient of correlation of phospho-FUS (pSer26, pSer30, pSer57, pThr71, and pSer96) to the GFP-FUS(R495X) signal; error bars represent 95% CI (n = 30). (E) H4 cells transfected with GFP-FUS(495X) treated with torin 2, 6 h posttransfection. Cells were analyzed 8 h posttransfection and probed with phospho-FUS antibodies. Error bars represent 95% CI (n = 30).
FIGURE 6:
ALS-mutant FUS forms cytoplasmic droplets; phosphomimetic substitutions in the prion-like domain do not alter droplet dynamics. (A) Representative images of FUS(494)-GFP phosphomimetic constructs 24 h posttransfection. (B) Average number of large (>1 m2) or small (<1 m2) FUS(494)-GFP cytoplasmic granules per cell 24 h posttransfection; error bars represent SEM (n = 17). (C) FRAP half-times of FUS(494)-GFP 24 h posttransfection; error bars represent 95% CI (n = 30). Student’s t test was used for statistical analysis.
FIGURE 7:
Non-PIKK phosphomimetic substitution decreases FUS toxicity and prion-like aggregation in yeast. (A) Schematic of the various phosphomimetic constructs used in the lab. Solid gray circles indicate PIKK consensus sites and light gray circles indicate non-PIKK sites. The constructs are in red to indicate their use in subsequent experiments. The black-highlighted axis indicates the FUS fragment inserted into Sup35 and used in Panel E. (B) Phosphomimetic substitution in the prion-like domain rescues FUS toxicity in yeast. (C) Ectopic expression of FUS 4Ev3 and 4Ev4 analyzed by structured illumination microscopy. Cells were probed with anti-FUS. (D) Quantification of FUS signal in punctate structures compared with total FUS expression; error bars represent 95% CI. Figure data analyzed using a Student _t_-test (n = 9) (E) Sup35-FUS or Sup35-FUS 4E were expressed in yeast on SC or SC-ade media. Sup35NM was added to promote prion formation under both conditions.
FIGURE 8:
Phosphomimetic substitution reduces FUS solid-phase aggregation in vitro. (A) Differential interference microscopy of full-length and phosphomimetic variants of FUS (4Ev3, 4Ev4, or 12E). Maltose binding protein (MBP)–tagged FUS proteins were agitated for 1 day at 25°C after the addition of TEV protease. (B) Turbidity assay of full-length FUS in the presence of varying salt concentrations. Turbidity was assessed 45 min following TEV addition (n = 10). Two-way ANOVA was used for statistical analysis (*indicates significance relative to 150 mM NaCl).
References
- Armstrong R (2017). Neuronal cytoplasmic inclusions in tau, TDP-43, and FUS molecular subtypes of frontotemporal lobar degeneration share similar spatial patterns. Folia Neuropathol , 185–192. - PubMed
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