Regulation of glioma migration and invasion via modification of Rap2a activity by the ubiquitin ligase Nedd4-1

Wang,Lei; Zhu,Bingxin; Wang,Shiquan; Wu,Yuxuan; Zhan,Wenjian; Xie,Shao; Shi,Hengliang; Yu,Rutong

doi:10.3892/or.2017.5572

Regulation of glioma migration and invasion via modification of Rap2a activity by the ubiquitin ligase Nedd4-1

Authors:
- Lei Wang
- Bingxin Zhu
- Shiquan Wang
- Yuxuan Wu
- Wenjian Zhan
- Shao Xie
- Hengliang Shi
- Rutong Yu
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Affiliations: Insitute of Nervous System Diseases, Xuzhou Medical University, Xuzhou, Jiangsu, P.R. China
Published online on: April 11, 2017 https://doi.org/10.3892/or.2017.5572
Pages: 2565-2574
Copyright: © Wang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Νeuronal precursor cell expressed and developmentally downregulated protein (Nedd4-1) is an E3 ubiquitin ligase with critical roles in the pathogenesis of cancer. Herein, we demonstrated that Nedd4-1 protein was upregulated in glioma tissues vs. that in non-cancerous tissues by western blotting and immunohistochemistry. Scratch migration and Transwell chamber assays indicated that downregulation of Nedd4-1 significantly reduced the migration and invasion of the glioma cell lines U251 and U87. Conversely, overexpression of Nedd4-1 obviously enhanced the migratory and invasive capacities in both cell lines. To investigate the role of Nedd4-1 and the intracellular pathways involved, we performed pull-down and co-immunoprecipitation assays, and recognized that Nedd4-1, TNIK and Rap2a formed a complex. Moreover, Nedd4-1 selectively ubiquitinated its specific substrates, the wild-type Rap2a (WT-Rap2a) and dominant-active Rap2a (DA-Rap2a) rather than the dominant-negative Rap2a (DN-Rap2a) in the U251 cells. Subsequently, we demonstrated that Rap2a was robustly ubiquitinated by Nedd4-1 along with the K63-linked, but not the K48-linked ubiquitin chain, which significantly inhibited GTP-Rap2a activity by GST-RalGDS pull-down assay. To further verify whether the ubiquitination of Rap2a by Nedd4-1 regulated the migration and invasion of glioma cells, Nedd4-1, HA-tagged ubiquitin and its mutants as well as WT-Rap2a were co-transfected in the U251 and U87 cell lines. The results confirmed that Nedd4-1 inhibited GTP-Rap2a activity, and promoted the migration and invasion of glioma cells. In brief, our findings demonstrated the important role of Nedd4-1 in regulating the migration and invasion of glioma cells via the Nedd4-1/Rap2a pathway, which may qualify Nedd4-1 as a viable therapeutic target for glioma.

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