Ser-26 and -30 in the FUS prionlike domain are phosphorylated following DNA-damaging stress
Of the three drugs identified by Deng and colleagues to cause FUS phosphorylation, only calicheamicin has been established to directly cause DNA damage (Zein et al., 1988; Deng et al., 2014). Staurosporine is a promiscuous kinase inhibitor (Karaman et al., 2008), and calyculin-A is an inhibitor of protein phosphatases 1/2A (Ishihara et al., 1989). When HEK293T or H4 cells were treated with several DNA-damaging agents (e.g., etoposide, doxorubicin, camptothecin) or okadaic acid (phosphatase inhibitor similar to calyculin-A), we saw no characteristic band shift in FUS (Figure 2A). Phosphorylated ATM was used to confirm these treatments caused a DNA-damage response (Figure 2A). Even when dosage or treatments were extended and Western blot exposures were increased, there were no definitive FUS band shifts observed, although band shadowing suggested some FUS might be running at a slightly higher molecular weight (Supplemental Figure S1B).
FIGURE 2:  Custom anti-FUS(pSer26) and anti-FUS(pSer30) antibodies are specific to phosphorylated FUS. (A, B) Lysates of HEK293T cells treated with various reagents (DMSO, 50 nM calicheamicin, 100 nM calyculin-A, 200 μM etoposide, 10 μM camptothecin, 2 μM doxorubicin, or 100 nM okadaic acid) for 3 h at 37°C were analyzed by Western blotting with commercial FUS or with anti-FUS(pSer26) or anti-FUS(pSer30) antibodies, respectively; n = 2. (C) Phosphorylated and unphosphorylated synthetic peptides—corresponding to regions within FUS’s prionlike domain—were serially diluted, spotted on nitrocellulose, and immunoprobed with custom antibodies: anti-FUS(pSer26) or anti-FUS(pSer30); n = 2. (D) FUS was knocked down using siRNA in H4 cells then treated with calicheamicin (or DMSO; negative control) to induce phosphorylation. Western blots using anti-FUS(pSer26), anti-FUS(pSer30), and commercial FUS antibodies revealed specificity of the phosphoantibodies to the FUS protein; n = 4. (E) Densitometry analysis of the percentage of signal reduction with FUS knockdown compared with the control; error bars represent 95% confidence intervals.   We concluded that calicheamicin, calyculin-A, and staurosporine were extreme in their effects on total cellular FUS and hypothesized that the other drugs might still cause FUS PrLD phosphorylation, but in a small subpopulation of FUS, or at lower frequencies on individual proteins, not revealed by a discernible band shift (Figure 2B). For these reasons, we generated polyclonal antibodies against FUS PrLD peptides encompassing phosphorylated Ser-26 or Ser-30 (Figure 2C). These peptides were chosen because phospho-Ser-26 and phospho-Ser-30 were repeatedly identified by our mass spectrometry experiments. Immunoblotting of phosphorylated and unphosphorylated synthetic peptides with the anti-FUS(pSer26) and anti-FUS(pSer30) antibodies indicated their specificity (Figure 2C). In Western blots with calyculin-A– or calicheamicin-treated HEK293T cell lysates, the antibodies recognized a protein species at the same position as commercial FUS antibody (Figure 2B). The phospho-specific antibodies did not recognize species from untreated controls. In all Western blots, there was a direct relationship between FUS protein band shift and phospho-specific antibody recognition (Figures 2 and 3 and Supplemental Figure S1, A, C, D, and F). Small interfering RNA (siRNA) knockdown was performed to ensure the custom antibodies were specific to FUS (Figure 2, D and E, and Supplemental Figure S3).
Because FUS-linked pathology presents in neurons, we chose to continue all experiments in the H4 cell type as it is neuronal in origin. Administering a dose series of calicheamicin to H4 cells revealed that at lower concentrations anti-FUS(pSer26) and anti-FUS(pSer30) recognize a subpopulation of FUS prior to an increase in apparent molecular weight by Western blot (Figure 3A). This confirmed that phosphorylation could occur at lower levels without an obvious band shift. We next looked specifically for low levels of phosphorylation after treating H4 cells with etoposide, camptothecin, doxorubicin, bleomycin, UV radiation, and ionizing radiation (IR). These treatments are known to induce DNA damage through different mechanisms (Povirk, 1996; Liu et al., 2000; Rastogi et al., 2010; Yang et al., 2014a; Montecucco et al., 2015; Xu and Her, 2015; Mavragani et al., 2017). However, each results in the production of double-strand DNA breaks that induce the DNA damage response pathway involving both ATM and DNA-PK (Shrivastav et al., 2008). All treatments caused FUS phosphorylation without a pronounced concomitant band shift (Figure 3, B and C; and in HEK293T cells shown in Supplemental Figure S1, C, D, and F). Of note, low concentrations of calyculin-A (5 vs. 100 nM) and UV radiation reproducibly caused observable phosphorylation at Ser-30 but not Ser-26. In control experiments with synthetic peptides, anti-FUS(pSer26) appeared more sensitive in epitope recognition than anti-FUS(pSer30), and its specificity was not diminished by diphosphorylation at both positions 26 and 30 (Figure 2C and Supplemental Figure S1E). For these reasons, we concluded differential phosphorylation is occurring in cells and is not a probing artifact.
FIGURE 3:  DNA-damaging conditions result in phosphorylation of Ser-26 and -30 of FUS. (A) Lysates of H4 cells treated with increasing amounts of calicheamicin were analyzed by Western blotting with commercial FUS and anti-FUS(pSer26) or anti-FUS(pSer30) antibodies; n = 2. (B, C) Lysates of H4 cells subjected to DNA-damaging conditions (0.5 nM calicheamicin, 5 nM calyculin-A, 200 μM etoposide, 10 μM camptothecin, 2 μM doxorubicin, 100 nM BLM, 90 mJ UV, or 20 Gy IR) were analyzed by Western blotting with commercial FUS and anti-FUS(pSer26) or anti-FUS(pSer30) antibodies; n = 3 for each treatment except IR in which n = 2. (D) Lysates of H4 cells treated with and without DNA-PK inhibitor (NU7441) prior to calicheamicin treatment were analyzed by Western blotting with commercial FUS and anti-FUS(pSer26) or anti-FUS(pSer30) antibodies; n = 3.   As mentioned above, DNA-PK was previously implicated in the phosphorylation of FUS following DNA-damaging stress (Deng et al., 2014). When we treated cells with the DNA-PK inhibitor NU7441 followed by calicheamicin, the FUS band shift was completely eliminated, and the phospho-specific antibodies no longer recognized any FUS species (Figure 3D). This corroborates DNA-PK’s involvement in FUS PrLD phosphorylation.