TY - JOUR
T1 - The prionlike domain of FUS is multiphosphorylated following DNA damage without altering nuclear localization
AU - Rhoads, Shannon N.
AU - Monahan, Zachary T.
AU - Yee, Debra S.
AU - Leung, Andrew Y.
AU - Newcombe, Cameron G.
AU - O’Meally, Robert N.
AU - Cole, Robert N.
AU - Shewmaker, Frank P.
N1 - Funding Information:
We thank Michael Panagos and Sarah Walsh for assistance in the laboratory, Nick Fawzi for experimental suggestions, Jesse Guidry for mass spectrometry advice, Robert Kortum for sharing immuno-blotting equipment and expertise, Dennis McDaniel and Rachel Cox for help with fluorescence microscopy, Jeremy Smyth and Darya Karabasheva for sharing microscopy equipment and expertise, Jeff Harmon for reviewing the manuscript, Rok Tkavc for help with the GammaCell irradiator, and Molly Jia for her assistance in creating the custom polyclonal antibodies. Support from the National Institute of General Medical Sciences of the National Institutes of Health under Award Numbers R35GM119790 and R01GM118530 made these studies possible.
Publisher Copyright:
© 2018 Rhoads et al.
PY - 2018/8/1
Y1 - 2018/8/1
N2 - 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.
AB - 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.
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U2 - 10.1091/mbc.E17-12-0735
DO - 10.1091/mbc.E17-12-0735
M3 - Article
C2 - 29897835
AN - SCOPUS:85052874155
VL - 29
SP - 1786
EP - 1797
JO - Molecular Biology of the Cell
JF - Molecular Biology of the Cell
SN - 1059-1524
IS - 15
ER -