title: Regulation of glioma migration and invasion via modification of Rap2a activity by the ubiquitin ligase Nedd4-1.
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.
authors.affiliation: Insitute of Nervous System Diseases, Xuzhou Medical University, Xuzhou, Jiangsu, P.R. China.
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.affiliation: Insitute of Nervous System Diseases, Xuzhou Medical University, Xuzhou, Jiangsu, P.R. China.
authors.forename: Bingxin
authors.initials: B
authors.lastname: Zhu
authors.affiliation: Insitute of Nervous System Diseases, Xuzhou Medical University, Xuzhou, Jiangsu, P.R. China.
authors.forename: Shiquan
authors.initials: S
authors.lastname: Wang
authors.affiliation: Insitute of Nervous System Diseases, Xuzhou Medical University, Xuzhou, Jiangsu, P.R. China.
authors.forename: Yuxuan
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Insitute of Nervous System Diseases, Xuzhou Medical University, Xuzhou, Jiangsu, P.R. China.
authors.forename: Wenjian
authors.initials: W
authors.lastname: Zhan
authors.affiliation: Insitute of Nervous System Diseases, Xuzhou Medical University, Xuzhou, Jiangsu, P.R. China.
authors.forename: Shao
authors.initials: S
authors.lastname: Xie
authors.affiliation: Insitute of Nervous System Diseases, Xuzhou Medical University, Xuzhou, Jiangsu, P.R. China.
authors.forename: Hengliang
authors.initials: H
authors.lastname: Shi
authors.affiliation: Insitute of Nervous System Diseases, Xuzhou Medical University, Xuzhou, Jiangsu, P.R. China.
authors.forename: Rutong
authors.initials: R
authors.lastname: Yu
mesh_terms: D001932:Brain Neoplasms / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D002465:Cell Movement; D056827:Endosomal Sorting Complexes Required for Transport / Q000235:genetics / Q000378:metabolism*; D005910:Glioma / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D000075702:Nedd4 Ubiquitin Protein Ligases; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination; D020668:rap GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D056827:Endosomal Sorting Complexes Required for Transport; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; D044767:Ubiquitin-Protein Ligases; C116756:RAP2A protein, human; D020668:rap GTP-Binding Proteins