Regulation of FUS function by phosphorylation

Amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig’s disease) is a progressive and fatal neurodegenerative disease. The majority of ALS cases are sporadic, and approximately 10% are familial. Several ALS genes have been identified as their mutation can lead to familial ALS, including two genes encoding RNA processing proteins TDP-43 and fused in sarcoma (FUS, also named TLS). FUS is a ubiquitously expressed RNA-binding protein. In neurons and glial cells, FUS is almost exclusively localized to the nucleus but is also reported to transport mRNA for local translation in dendrites in neurons. In addition, FUS plays a role in a variety of processes including nucleocytoplasmic shuttling of mRNA, DNA repair, transcriptional regulation and mRNA splicing. However, little is known regarding how FUS function is regulated or how FUS mutations cause motor neuron degeneration and ALS. This application is based on our new results showing FUS is phosphorylated by protein kinase CK2. We have identified several potential phosphorylation sites within the RNA recognition motif (RRM). Our preliminary results showed co-expression of protein kinase CK2 mitigated the locomotive impairment of FUS transgenic flies, but not the CK2 phosphorylation-null mutant of FUS. This result suggests that phosphorylation of FUS by CK2 can directly attenuate the toxicity of FUS over-expression. Moreover, the CK2-dependent phosphorylation of FUS was also observed in mouse N2A and human primary fibroblast cells. These results led us to the hypothesis that phosphorylation of FUS by protein kinase CK2 plays a critical role in regulating FUS function. Three specific aims are designed to test the hypothesis. Aim 1 is to identify the exact CK2 phosphorylation site(s) in FUS using mass spectrometry. Aim 2 is to determine the effect of CK2 phosphorylation on RNA binding and subcellular localization of FUS. Aim 3 is to elucidate the contribution of individual CK2 sites to FUS-mediated neurotoxicity in cellular and Drosophila models. The proposed research will test a novel hypothesis and explore a new paradigm. We expect to produce novel insights into the mechanisms how FUS phosphorylation regulates the subcellular localization, RNA binding, and potential neurotoxicity of FUS. In addition, the expected results will also identify new molecular targets with translational values that will enable future drug development as potential ALS therapy.