Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are both rapidly progressive neurodegenerative diseases. They are both characterized by progressing neuron death. Mutations in different genes can contribute to development of these diseases, as well as post translational modification of RNA-binding proteins. One of genes, where mutation can occur, is FUS gene, encoding FUS protein (FUsed in Sarcoma). It is an RNA/DNA-binding protein, which is primarily located in the cell nucleus and has roles in many processes like gene transcription, RNA metabolism, DNA damage response and cell stress response. Because it functions in both cell nucleus and cytoplasm, it must shuttle between them to execute normal functions. A post translational modification like phosphorylation of FUS last C-terminal tyrosine residue by Src family kinases could be involved in that. We wanted to evaluate the effect of phosphorylation of FUS at its last tyrosine (52) on intracellular distribution of normal FUS and FUS without C-terminal domain (NLS) in differentiated SH-SY5Y FlpIn cells.
We used previously generated SH-SY5Y FlpIn cell lines, SH-SY5Y-mSc, SH-SY5Y-mScFUS and SH-SY5Y-mScFUSdNLS. Upon induction they all express red fluorescent proteins mScarlet (mSc), FUS protein tagged with mScarlet (mScFUS) or FUS protein with deleted C-terminal end, tagged with mSarlet (mScFUSdNLS). We confirmed successful expression of mSc, mScFUS and mScFUSdNLS in cells with fluorescent confocal microscopy and western blot analyses. First we adapted a differentiation protocol based on literature and our empircal work. We differentiated the cells of all three FlpIn cell lines to neuron-like cells by adding retinoic acid and neurotrophic factor BDNF. We confirmed differentiated phenotype of the cells and their expression of neuron-specific proteins with fluorescent confocal microscopy and western blot analyses. We transfected differentiated SH-SY5Y FlpIn cells of all three cell lines with plasmids encoding active c-Src, c-Fyn and c-Abl kinases. For comparison we also transfected undifferentiated SH-SY5Y FlpIn cells with them. Prior transfection we induced the cells with doxycycline to enable expression of mSc, mScFUS and mScFUSdNLS. We evaluated the effect of kinase mediated phosphorylation of FUS on its intracellular distribution by using specific anti-FUSp-526Y antibodies respectively. We demonstrated that in cells expressing active c-Src and c-Fyn kinase, phosphorylated FUS p-526Y is mostly found in the nucleus, whereas in cells expressing active c-Abl kinase, FUSp-526Y is mostly found in the cytoplasm. Phosphorylation of the FUS protein last tyrosine residue inhibits its import into the nucleus and disrupts normal shuttling of FUS between nucleus and cytoplasm. This way it potentially contributes to aggregation of FUS protein in the cytoplasm and formation of toxic FUS aggregates.
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