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Chromosomal localization of Ewing sarcoma EWSR1/FLI1 protein promotes the induction of aneuploidy
Park, Hyewon Kim, Haeyoung Hassebroek, Victoria Azuma, Yoshiaki Slawson, Chad Azuma, Mizuki
Park, Hyewon
Kim, Haeyoung
Hassebroek, Victoria
Azuma, Yoshiaki
Slawson, Chad
Azuma, Mizuki
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Abstract
Ewing sarcoma is a pediatric bone cancer that expresses the chimeric protein EWSR1/FLI1. We previously demonstrated that EWSR1/FLI1 impairs the localization of Aurora B kinase to the midzone (the midline structure located between segregating chromosomes) during anaphase. While localization of Aurora B is essential for faithful cell division, it is unknown whether interference with midzone organization by EWSR1/FLI1 induces aneuploidy. To address this, we generated stable Tet-on inducible cell lines with EWSR1/FLI1, using CRISPR/Cas9 technology to integrate the transgene at the safe-harbor AAVS1 locus in DLD-1 cells. Induced cells expressing EWSR1/FLI1 displayed an increased incidence of aberrant localization of Aurora B, and greater levels of aneuploidy, compared with noninduced cells. Furthermore, the expression of EWSR1/FLI1-T79A, containing a threonine (Thr) to alanine (Ala) substitution at amino acid 79, failed to induce these phenotypes, indicating that Thr 79 is critical for EWSR1/FLI1 interference with mitosis. In contrast, the phosphomimetic mutant EWSR1/FLI1-T79D (Thr to aspartic acid (Asp)) retained the high activity as wild-type EWSR1/FLI1. Together, these findings suggest that phosphorylation of EWSR1/FLI1 at Thr 79 promotes the colocalization of EWSR1/FLI1 and Aurora B on the chromosomes during prophase and metaphase and, in addition, impairs the localization of Aurora B during anaphase, leading to induction of aneuploidy. This is the first demonstration of the mechanism for EWSR1/FLI1-dependent induction of aneuploidy associated with mitotic dysfunction and the identification of the phosphorylation of the Thr 79 of EWSR1/FLI1 as a critical residue required for this induction.
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Date
2020-12-10
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Elsevier
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Keywords
Ewing sarcoma, EWSR1/FLI1, Aurora B, Aneuploidy, Mitosis
Citation
Park, H., Kim, H., Hassebroek, V., Azuma, Y., Slawson, C., & Azuma, M. (2021). Chromosomal localization of Ewing sarcoma EWSR1/FLI1 protein promotes the induction of aneuploidy. The Journal of biological chemistry, 296, 100164. https://doi.org/10.1074/jbc.RA120.014328