Ubiquitination Destabilizes Protein Sphingosine Kinase 2 to Regulate Glioma Malignancy.

Hongliang Wang,Yuyang Wang,Gang Zong,Chunchun Ma,Jinghai Wan,Bing Zhao,Jie He,Erbao Bian

doi:10.3389/fncel.2021.660354

Abstract

Gliomas are the most common and lethal malignant tumor in the central nervous system. The tumor oncogene sphingosine kinase 2 (SphK2) was previously found to be upregulated in glioma tissues and enhance glioma cell epithelial-to-mesenchymal transition through the AKT/β-catenin pathway. Nevertheless, ubiquitination of SphK2 protein has yet to be well elucidated. In this study, mass spectrometry analysis was performed to identify proteins that interacted with SphK2 protein. Co-immunoprecipitation (co-IP) and immunoblotting (IB) were used to prove the specific interaction between SphK2 protein and the neural precursor cell-expressed developmentally downregulated 4-like (NEDD4L) protein. Fluorescence microscopy was used for detecting the distribution of related proteins. Ubiquitylation assay was utilized to characterize that SphK2 was ubiquitylated by NEDD4L. Cell viability assay, flow cytometry assay, and transwell invasion assay were performed to illustrate the roles of NEDD4L-mediated SphK2 ubiquitination in glioma viability, apoptosis, and invasion, respectively. We found that NEDD4L directly interacted with SphK2 and ubiquinated it for degradation. Ubiquitination of SphK2 mediated by NEDD4L overexpression suppressed glioma cell viability and invasion but promoted glioma apoptosis. Knockdown of NEDD4L presented opposite results. Moreover, further results suggested that ubiquitination of SphK2 regulated glioma malignancy via the AKT/β-catenin pathway. in vivo assay also supported the above findings. This study reveals that NEDD4L mediates SphK2 ubiquitination to regulate glioma malignancy and may provide some meaningful suggestions for glioma treatment.

Highlights

Gliomas are the most lethal intracranial tumor with poor prognosis, and there is still no effective therapy up to now (Ouyang et al, 2015)
We found that neural precursor cell-expressed developmentally downregulated 4-like (NEDD4L) interacted with sphingosine kinase 2 (SphK2) and mediated its degradation in a ubiquitinationproteasome-dependent manner
SphK2 was immunoprecipitated from the cultured HEK293T cells by using Flag beads and HA-tagged NEDD4L was detected by immunoblotting

Summary

Introduction

Gliomas are the most lethal intracranial tumor with poor prognosis, and there is still no effective therapy up to now (Ouyang et al, 2015). SphKs could promote cell growth and tumor progression via converting the backbone of sphingolipids and sphingosine into S1P (Neubauer and Pitson, 2013; Lee et al, 2015; Neubauer et al, 2016). In gliomas, both SphK1 and SphK2 were abnormally upregulated in tumor tissues and cell lines, and a higher expression of SphKs means lower survival times of glioma patients (Li et al, 2008; Liu et al, 2018). This raises our interests to explore the ubiquitination of SphK2 in gliomas

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Results

Conclusion