Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases characterized by the accumulation of cytoplasmic aggregates of RNA-binding proteins, including FUS. Under normal conditions, FUS predominantly localizes to the nucleus, where it participates in transcription regulation, RNA processing and DNA repair. Mutations in the FUS gene, particularly in the C-terminal nuclear localization signal (PY-NLS), are associated with familial forms of ALS, while cytoplasmic aggregates of wild-type FUS (FUSwt) are also observed in FTD. In this context, phosphorylation of tyrosine 526 (Y526) in the PY-NLS plays an important role, as it prevents binding to transportin-1 and thereby blocks nuclear import of FUS, leading to its cytoplasmic accumulation and aggregation. Phosphorylation of Y526 is catalyzed by Src family kinases (cAbl, cSrc and cFyn), which are active in different cellular compartments. We investigated the effect of the C-terminal mutations FUSR521C and FUSP525L on the nucleocytoplasmic distribution and aggregation of FUS in HEK293T cells, and how co-expression of FUS variants (FUSwt, FUSR521C and FUSP525L) with constitutively active kinases (cAbl, cSrc and cFyn) affects phosphorylation of Y526 and the number and morphology of cytoplasmic aggregates. Phosphorylation at Y526 (FUSp Y526) was detected with a specific antibody. We used immunocytochemistry, confocal microscopy and quantitative image analysis to evaluate the nucleocytoplasmic ratio of FUSp-Y526 and FUS. The results show that both mutated forms cause increased cytoplasmic accumulation of FUS, indicating severely reduced nuclear import. Co‑expression with cAbl increases cytoplasmic accumulation of FUSp‑Y526, while co‑expression with cSrc and cFyn confirms strong nuclear localization of FUSp‑Y526. Kinases also differentially affect the appearance or disappearance of aggregates of mutated FUS variants. Taken together, both mutations at the C‑terminus and in PY‑NLS, as well as phosphorylation of Y526, affect the distribution of FUS in the cell. The effect on intracellular distribution and aggregate formation of FUS variants depends on the type of kinase and the mutation. Our findings contribute to understanding the molecular mechanisms of FUS aggregation in ALS and open possibilities for further research into the role of tyrosine kinases in neurodegenerative diseases.
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