The role of phosphorylation in nuclear-cytoplasmic localization of Fused in Sarcoma (FUS)

Identification: Johnson, Michelle


Description

The role of phosphorylation in nuclear-cytoplasmic localization of Fused in Sarcoma (FUS)  
 
Johnson, MA1,2, Holler, CJ1, Taylor, G1, Tansey, MG.2 and Kukar, TL1
1Department of Pharmacology and 2Neurology, 3Department of Physiology, Emory University School of Medicine
 
Frontal temporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) share many genetic and neuropathological markers of diseases. One such neuropathological marker that occurs in about 5-10% of cases is the abnormal cytoplasmic aggregation of Fused in Sarcoma (FUS) protein. FUS is a RNA/DNA binding protein involved in gene transcription, mRNA splicing, DNA-repair pathways, and mRNA transport. To accomplish its many roles in the cell, FUS shuttles between the nucleus and cytoplasm carrying mRNA transcripts to distinct cellular locations. However, in FTD and ALS with FUS pathology, nuclear/cytoplasmic shuttling of FUS is disrupted resulting in its mislocalization into insoluble inclusions in the cytoplasm. Recently, studies from our group demonstrated that DNA-PK mediated phosphorylation of FUS (p-FUS) at N-terminal residues triggers cytoplasmic accumulation of p-FUS. Nonetheless, it remains unclear what mechanism mediates p-FUS mislocalization to the cytoplasm. Results from past studies suggest nuclear export of FUS may be partially mediated through CRM1, the major protein receptor for the exportin-1 receptor complex. Therefore, two approaches were used to determine whether active nuclear export of FUS in a CRM1-dependent manner causes cytoplasmic accumulation of p-FUS. SH-SY5Y human neuroblastoma cells were either transfected with expression plasmids encoding mutant FUS missing either the PY-NLS (Proline-Tyrosine-nuclear localization sequence) or the NES (nuclear export sequence), the sequences responsible for its shuttling behavior; or cells were treated with leptomycin B to block CRM1-mediated nuclear export. Next, cells were treated with calicheamicin 1�� (CLM) to activate the DNA repair response that triggers DNA-PK to phosphorylate FUS. Cell lysates were fractionated into nuclear and cytoplasmic fractions and then subjected to SDS-PAGE and western blot analyses to determine whether p-FUS cytoplasmic accumulation was dependent on CRM1 or the FUS NES. Completion of these studies will not only inform the extent to which phosphorylation plays a role in FUS pathology, but will also help validate p-FUS as a therapeutic target in FTD and ALS with FUS pathology.

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