Abl kinase-mediated FUS Tyr526 phosphorylation alters nucleocytoplasmic FUS localization in FTLD-FUS

Nuclear to cytoplasmic mislocalization and aggregation of multiple RNA-binding proteins (RBPs), including FUS, are the main neuropathological features of the majority of cases of amyotrophic lateral sclerosis (ALS) and frontotemporal lobular degeneration (FTLD). In ALS-FUS, these aggregates arise from disease-associated mutations in FUS, whereas in FTLD-FUS, the cytoplasmic inclusions do not contain mutant FUS, suggesting different molecular mechanisms of FUS pathogenesis in FTLD that remain to be investigated. We have previously shown that phosphorylation of the C-terminal Tyr526 of FUS results in increased cytoplasmic retention of FUS due to impaired binding to the nuclear import receptor TNPO1. Inspired by the above notions, in the current study we developed a novel antibody against the C-terminally phosphorylated Tyr526 FUS (FUSp-Y526) that is specifically capable of recognizing phosphorylated cytoplasmic FUS, which is poorly recognized by other commercially available FUS antibodies. Using this FUSp-Y526 antibody, we demonstrated a FUS phosphorylation-specific effect on the cytoplasmic distribution of soluble and insoluble FUSp-Y526 in various cells and confirmed the involvement of the Src kinase family in Tyr526 FUS phosphorylation. In addition, we found that FUSp-Y526 expression pattern correlates with active pSrc/pAbl kinases in specific brain regions of mice, indicating preferential involvement of cAbl in the cytoplasmic mislocalization of FUSp-Y526 in cortical neurons. Finally, the pattern of immunoreactivity of active cAbl kinase and FUSp-Y526 revealed altered cytoplasmic distribution of FUSp-Y526 in cortical neurons of post-mortem frontal cortex tissue from FTLD patients compared with controls. The overlap of FUSp-Y526 and FUS signals was found preferentially in small diffuse inclusions and was absent in mature aggregates, suggesting possible involvement of FUSp-Y526 in the formation of early toxic FUS aggregates in the cytoplasm that are largely undetected by commercially available FUS antibodies. Given the overlapping patterns of cAbl activity and FUSp-Y526 distribution in cortical neurons, and cAbl induced sequestration of FUSp-Y526 into G3BP1 positive granules in stressed cells, we propose that cAbl kinase is actively involved in mediating cytoplasmic mislocalization and promoting toxic aggregation of wild-type FUS in the brains of FTLD patients, as a novel putative underlying mechanism of FTLD-FUS pathophysiology and progression.