DNA-PK–dependent phosphorylation of the PrLD does not promote FUS cytoplasmic localization
We asked whether PrLD multiphosphorylation has any effect on the change in localization of FUS induced by osmotic stress. H4 cells were treated with calicheamicin to induce phosphorylation and then subjected to osmotic stress with sorbitol. Using immunofluorescence microscopy, accumulation of total FUS in the cytoplasm appeared slightly inhibited in the calicheamicin-treated cells relative to control cells over time (Figure 5A). The FUS band shift in the Western blot suggested that FUS was multiphosphorylated under these conditions (Figure 5B). Next, using the phospho-specific anti-FUS(pSer30) antibody, the experiment was repeated with an extended end point. As before, the addition of calicheamicin prior to sorbitol resulted in a modest, but statistically significant, reduction of FUS in the cytoplasm (Figure 5, C and E). However, phosphorylated FUS was only observed in the nucleus of calicheamicin-treated cells—never in the cytoplasm. Likewise, we found that adding calicheamicin after sorbitol resulted in the rapid appearance of phosphorylated FUS only in the nucleus and not in the cytoplasm (Figure 5, C and E). This is consistent with DNA-PK’s nuclear localization and activity (Anderson and Lees-Miller, 1992). Western blot controls confirming FUS phosphorylation are shown in Figure 5D, and extended immunofluorescence control images are shown in Supplemental Figure S6. Similar nuclear-only localization of phospho-FUS was observed using the anti-FUS(pSer26) antibody (Supplemental Figure S7).
FIGURE 5:  Calicheamicin-induced phosphorylated FUS is maintained in the nucleus following osmotic stress. (A) H4 cells treated with 0.4 M sorbitol, with and without 50 nM calicheamicin pretreatment, were fixed for immunocytochemical analysis with a commercial FUS antibody; nuclear and cytoplasmic fluorescence signals at the 45-min time point were quantified to determine the percentage of total FUS in the cytoplasm per cell (right panel); error bars represent 95% confidence intervals; n = 3. (B) Lysates were analyzed by Western blotting; n = 2. (C) H4 cells were fixed for immunocytochemical analysis with commercial FUS and anti-FUS(pSer30) antibodies following sequential addition of sorbitol and/or calicheamicin (60 min sorbitol before 60 min calicheamicin, or 60 min calicheamcin before 60 min sorbitol, without removal of the first treatment); n = 3. (D) Lysates were analyzed by Western blotting; n = 3. (E) Quantification of nuclear and cytoplasmic fluorescence signals was done to determine the percentage of total FUS in the cytoplasm per cell and the percentage of total phospho-FUS in the nucleus per cell (N.D. = not determined); error bars represent 95% confidence intervals.   We next asked how inhibition of DNA-PK could alter the balance between nuclear and cytoplasmic FUS. When H4 cells were treated with the DNA-PK inhibitor NU7441, we observed no effect on FUS localization. However, if cells were subjected to calicheamicin after treatment with the inhibitor, the majority of FUS localized to the cytoplasm (Figure 6, A and B). Figure 6C shows Western blots confirming the efficacy of calicheamicin and inhibitor. The data suggest that neither DNA-PK activity nor PrLD phosphorylation at positions 26 and 30 is sufficient to drive FUS cytoplasmic localization.
FIGURE 6:  Phosphorylation of the FUS prionlike domain is not linked to cytoplasmic localization. (A) H4 cells following calicheamicin treatment, with and without DNA-PK inhibitor (NU7441), were fixed for immunocytochemical analysis using commercial FUS and anti-FUS(pSer30) antibodies; n = 3. (B) Quantification of nuclear and cytoplasmic fluorescence signals was done to determine the percentage of cytoplasmic localization; error bars represent 95% confidence interval. (C) Lysates were analyzed by Western blotting; n = 3. (D) H4 cells expressing ectopic PM FUS (6 or 12 glutamate substitutions in the prionlike domain) were visualized by immunofluorescence microscopy; n = 3. (E) Quantification of nuclear and cytoplasmic fluorescence signal was done to determine the percentage of cytoplasmic localization of FUS-wt and FUS-12E from C; error bars represent 95% confidence intervals. (F) U-2 OS cells expressing GFP-fused ectopic phosphomimetic FUS were visualized by live-cell imaging; n = 2.   Because the entire FUS PTM repertoire that follows calicheamicin treatment is unknown and is not necessarily limited to PrLD modifications, it is possible that PrLD phosphorylation favors cytoplasmic localization but is masked by other PTMs. We used H4 cells and a dual-expression plasmid system that expresses GFP and FUS phosphomimetic variants as separate proteins. The FUS variants were specifically modified at DNA-PK sites within the PrLD to determine their effects on FUS subcellular localization (phosphomimetics discussed in Figure 1C and Supplemental Figure S8; see Materials and Methods for sites). GFP fluorescence was used to determine which transfected cells expressed ectopic proteins at similar levels. The localization of ectopic FUS, FUS-6E, or FUS-12E was evaluated by immunofluorescence microscopy. We found that FUS, FUS-6E, and FUS-12E were all mostly nuclear (Figure 6D). Quantification yielded no significant differences in localization (Figure 6E). A similar experiment was performed in live U-2 OS cells expressing ectopic GFP-FUS fusion proteins. Again, no difference in localization was observed by introducing phosphomimetic substitutions (Figure 6F).