PMC:6085830 / 6901-11178 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/6085830","sourcedb":"PMC","sourceid":"6085830","source_url":"https://www.ncbi.nlm.nih.gov/pmc/6085830","text":"The FUS prionlike domain is phosphorylated at numerous serines and threonines following calicheamicin treatment\nDeng and coworkers found several pharmacological agents caused a characteristic increase in the apparent molecular weight of the FUS protein band in Western blots (Deng et al., 2014): staurosporine (kinase inhibitor), calicheamicin (DNA damaging agent), and calyculin-A (phosphatase inhibitor). When they treated H4 neuroglioma cells with staurosporine, it yielded a high-molecular-weight FUS species that on phosphatase treatment returned to the untreated size, suggesting that the gel shift was due to phosphorylation. We reproduced their results showing an increase in FUS’s apparent molecular weight in multiple human cell lines treated with staurosporine, calicheamicin, and calyculin-A (Figure 1A and Supplemental Figure S1A). Likewise, when we subjected immunoprecipitated FUS from calicheamicin and calyculin-A–treated cells to phosphatase before Western blotting, the FUS bands returned to their original, untreated position (Figure 1B). Additionally, we expressed FUS in H4 cells with various numbers of phosphomimetic substitutions (replacing S or T with E; see Materials and Methods for specific sites) to simulate varying amounts of phosphorylation in the PrLD. The mimetic FUS Western blot bands migrated at increasingly higher apparent molecular weights (Figure 1C) with the number of phosphomimetic substitutions correlated to the increase in band shift. A stepwise change became prominent above the unmodified FUS band with ∼4 substitutions. The phosphomimetic with 12 substitutions (12E) migrated most similarly to FUS from staurosporine-, calicheamicin-, or calyculin-A–treated cells. From this, we concluded the large increase in apparent molecular weight in Western blots was consistent with multiple phosphorylation of FUS’s PrLD.\nFIGURE 1: The prionlike domain of cellular FUS contains numerous phosphorylation sites. (A) Lysates from multiple human cell lines treated with calicheamicin or calyculin-A showed FUS migrating with a larger apparent molecular weight in Western blot; HEK293T, n = 5; H4, n = 4; U-2 OS, n = 2. (B) Treatment of immunoprecipitated FUS with phosphatase caused FUS to return to its normal apparent size; *Heavy chain of the immunoglobulin G used for immunoprecipitation; n = 2. (C) Substituting glutamate at potential serine or threonine phosphorylation sites caused ectopic FUS in H4 cells to migrate similarly to endogenous FUS from cells treated with calicheamicin or calyculin-A; see Materials and Methods for exact substitution sites; n = 2. (D) Twenty-eight serines and threonines have been identified in this and other studies as putative sites of phosphorylation (bold and underlined; DNA-PK consensus sites are shown in red). Here sites were identified by mass spectrometry following immunoprecipitation of FUS from lysates of human cell lines treated with the DNA-damaging agents calicheamicin or camptothecin, or the phosphatase-inhibitor calyculin-A. Our previous work with recombinant DNA-PK indicated that all 12 S/TQ DNA-PK consensus motifs in the PrLD could be phosphorylated in vitro (Monahan et al., 2017). We preliminarily mapped three of these sites using mass spectrometry analysis of immunoprecipitated FUS from HEK293T cells treated with calyculin-A: Ser-26, Ser-30, and Ser-61. Here, using refinements of our previous proteomic approach to identify PTMs, we focused our attention on the DNA-damaging agents calicheamicin and camptothecin, due to the previously implicated link between DNA damage and FUS phosphorylation. Numerous unambiguous S and T sites were identified (Supplemental Figure S2A). Several ion fragments appeared to contain two phosphates, albeit with less certainty (Supplemental Figure S2B). Site combinations within individual peptides were as follows: Ser-26/Ser-30, Ser-30/Ser-42, Thr-109/Ser-115, and Ser-115/Ser-117. In total, we found seven DNA-PK consensus sites in the PrLD to be phosphorylated following DNA damage with Ser-26 and Ser-30 being the most prominent across multiple experiments (Figure 1D). In general, the PrLD appears to be highly susceptible to multiphosphorylation in cells with as many as 28 candidate sites found by us and others (Figure 1D).","divisions":[{"label":"Title","span":{"begin":0,"end":111}},{"label":"Figure caption","span":{"begin":1865,"end":3026}}],"tracks":[{"project":"2_test","denotations":[{"id":"29897835-24899704-70504538","span":{"begin":289,"end":293},"obj":"24899704"},{"id":"29897835-28790177-70504539","span":{"begin":3182,"end":3186},"obj":"28790177"}],"attributes":[{"subj":"29897835-24899704-70504538","pred":"source","obj":"2_test"},{"subj":"29897835-28790177-70504539","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#93eca2","default":true}]}]}}