FUS is primarily a nuclear protein that regulates transcription, processing, and transport of mRNA molecules in the cell. In stress conditions, it is sequestered into stress granules, temporarily halting the energy-intensive process of synthesizing less important proteins. Due to unknown mechanisms, these stress granules can form insoluble aggregates that are harmful to the cell. Toxic FUS protein aggregates are found in the brains of patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), although the mechanism of their formation differs between the two diseases. In ALS, the cytoplasmic aggregates contain the mutated FUS protein, whereas in FTD they contain the wild-type FUS protein. One possible cause of the abnormal aggregation of the FUS protein in FTD is post-translational modifications, including phosphorylation of Y526 in the nuclear localization signal (NLS), which impedes its binding to the nuclear receptor transportin-1 and transport into the nucleus. Tyrosines in the FUS protein are phosphorylated by Src family kinases, which are activated upon oxidative stress. To investigate how c-Src kinase affects the intracellular distribution of FUS protein, we cultured three SH-SY5Y Flp-In cell lines inducibly expressing mScarlet, mScarlet fused to FUS protein, and mScarlet fused to FUSΔNLS protein, lacking the nuclear localization signal. We observed that oxidative stress induced by the addition of arsenite triggers the formation of FUS-positive stress granules in the cytoplasm, with the highest number present in SH-SY5Y-mSc-FUSΔNLS cells. These cells express the most aggregation-prone FUSΔNLS protein. After transfection with constitutively active c-Src kinase, SH-SY5Y-mSc-FUS cells contained the most FUS-positive granules, indicating that phosphorylation of the FUS protein accelerates their formation. Since the commercial antibody against FUS does not recognize all phosphorylated FUS molecules, the FUS-positive granules could represent FUS that has already been dephosphorylated during the maturation of stress granules. We also found that the c-Src kinase can influence the stability of stress granules during stress. Due to the impact of the FUSp-Y526 protein on the dynamics of stress granules, Src kinases could become important therapeutic targets for treating neurodegenerative diseases in the future.
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