Oncogenic Activity of Glucocorticoid Receptor β Is Controlled by Ubiquitination-Dependent Interaction with USP49 in Glioblastoma Cells.
Oncogenic Activity of Glucocorticoid Receptor β Is Controlled by Ubiquitination-Dependent Interaction with USP49 in Glioblastoma Cells.
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糖皮质激素受体 β 的致癌活性由胶质母细胞瘤细胞中与 USP49 的泛素化依赖性相互作用控制
DOI：
10.1158/1541-7786.mcr-20-1068
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发表时间：
2022-01
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先前的研究表明，糖皮质激素受体β（GRβ）作为一种癌蛋白发挥作用，调节人胶质母细胞瘤（GBM）中的恶性表型和干细胞样细胞的维持。在糖皮质激素受体（GR）异构体中，GRβ和GRα高度同源，尽管这两种异构体在GBM中不同功能的潜在机制尚未阐明。在此，通过构建一个羧基末端（COOH - 末端）缺失突变体，我们确定GRβ可被泛素化。我们还发现其COOH末端对这种泛素化至关重要。第733位赖氨酸突变为精氨酸（K733R）阻断了GRβ的泛素化，表明K733是泛素化的一个关键位点。利用K733R构建非泛素化的GRβ，我们证明泛素化不仅调节GRβ的稳定性和核转位，而且是其在体外和体内致癌功能的重要机制。蛋白质相互作用分析进一步表明，泛素特异性蛋白酶49（USP49）是一种与GRβ结合的蛋白质，且这种相互作用依赖于GRβ的泛素化。USP49的敲低导致细胞增殖和侵袭减少，细胞凋亡增加。更重要的是，USP49敲低增加了泛素化并放大了GRβ的致癌作用，证实了泛素化对GRβ致癌性的决定性作用。综上所述，这些发现确定了泛素化是GRβ在GBM中执行致癌功能的一个关键过程，并且K733位点对GRβ的泛素化至关重要。
这项工作首次确定了由单个赖氨酸位点介导的泛素化和蛋白酶体降解对GRβ的激活，这决定了它在GBM中的致癌功能。
Previous studies have demonstrated that glucocorticoid receptor β (GRβ) functions as an oncoprotein, regulating the malignant phenotypes and stem-like cell maintaining in human glioblastoma (GBM). Of the glucocorticoid receptor (GR) isoforms, GRβ and GRα are highly homologous, though the mechanism underlying the distinct functions of these two isoforms in GBM has not been clarified. Here by establishing a carboxyl-terminal (COOH-terminal) deletion mutant, we determined that GRβ can be ubiquitinated. We also found that its COOH terminal is essential for this ubiquitination. The mutation of a lysine to arginine at residue 733 (K733R) blocked the ubiquitination of GRβ, indicating that K733 is a key site for ubiquitination. Using K733R to establish nonubiquitinated GRβ, we demonstrated that ubiquitination not only regulates the stability and nuclear translocation of GRβ, but is also a vital mechanism for its oncogenic functions in vitro and in vivo. Protein interaction assay further indicated that ubiquitin-specific protease 49 (USP49) is a GRβ-binding protein and the interaction depends on GRβ ubiquitination. USP49 knockdown resulted in a decrease of cell proliferation, invasion, and an increase of cell apoptosis. More importantly, USP49 knockdown increased ubiquitination and amplified the oncogenic effects of GRβ, confirming the decisive role of ubiquitination on GRβ carcinogenicity. Taken together, these findings established that ubiquitination is a vial process for GRβ the execution of oncogenic functions in GBM and that the K733 site is crucial for ubiquitination of GRβ. This work is the first identify of the activation GRβ by a single lysine point-mediated ubiquitination and proteasome degradation, which determines its oncogenic functions in GBM.