The prionlike domain of FUS is multiphosphorylated following DNA damage without altering nuclear localization.
The prionlike domain of FUS is multiphosphorylated following DNA damage without altering nuclear localization.
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DOI：
10.1091/mbc.e17-12-0735
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发表时间：
2018-08-01
影响因子：
3.3
通讯作者：
Shewmaker FP
中科院分区：
文献类型：
--
作者：
Rhoads SN;Monahan ZT;Yee DS;Leung AY;Newcombe CG;O'Meally RN;Cole RN;Shewmaker FP
FUS（融合在肉瘤中）是一种丰富的、主要参与RNA加工的核蛋白。在各种条件下，FUS与RNA和其他大分子功能性结合，形成独特的、可逆的相分离液体结构。相分离状态的持续和细胞质定位的增加都被假设为使FUS易发生不可逆聚集，这是肌萎缩侧索硬化和额颞叶痴呆亚型的病理标志。我们以前表明，FUS的朊病毒样结构域的磷酸化抑制相分离和毒性聚集，成比例地添加磷酸盐的数量。然而，FUS的朊病毒样结构域的磷酸化以前被报道促进其细胞质定位，可能有利于病理行为。在这里，我们使用质谱和人类细胞模型，以进一步确定FUS的朊病毒样结构域内的磷酸化位点，特别是在DNA损伤的压力。总共鉴定了28个推定位点，其中约一半是DNA依赖性蛋白激酶（DNA-PK）共有位点。开发定制抗体以确认这些位点中的两个（Ser-26和Ser-30）的磷酸化。这两个网站通常磷酸化的细胞FUS亚群后，各种DNA损伤的压力，但不一定相等或同时。重要的是，我们发现FUS的朊病毒样结构域的DNA-PK依赖性多磷酸化不会引起细胞质定位。
FUS (fused in sarcoma) is an abundant, predominantly nuclear protein involved in RNA processing. Under various conditions, FUS functionally associates with RNA and other macromolecules to form distinct, reversible phase-separated liquid structures. Persistence of the phase-separated state and increased cytoplasmic localization are both hypothesized to predispose FUS to irreversible aggregation, which is a pathological hallmark of subtypes of amyotrophic lateral sclerosis and frontotemporal dementia. We previously showed that phosphorylation of FUS’s prionlike domain suppressed phase separation and toxic aggregation, proportionally to the number of added phosphates. However, phosphorylation of FUS’s prionlike domain was previously reported to promote its cytoplasmic localization, potentially favoring pathological behavior. Here we used mass spectrometry and human cell models to further identify phosphorylation sites within FUS’s prionlike domain, specifically following DNA-damaging stress. In total, 28 putative sites have been identified, about half of which are DNA-dependent protein kinase (DNA-PK) consensus sites. Custom antibodies were developed to confirm the phosphorylation of two of these sites (Ser-26 and Ser-30). Both sites were usually phosphorylated in a subpopulation of cellular FUS following a variety of DNA-damaging stresses but not necessarily equally or simultaneously. Importantly, we found DNA-PK–dependent multiphosphorylation of FUS’s prionlike domain does not cause cytoplasmic localization.