Bidirectional interplay between SUMOylation and the tumor suppressor p14ARF

Tumor suppressor p14ARF is a lysine-less protein that suppresses tumorigenesis through different mechanisms, including enhancing the SUMOylation of its interactors. Although p14ARF is known to interact with the SUMO conjugating enzyme UBC9, the link between ARF and SUMOylation is poorly understood and the potential impact of SUMOylation on p14ARF is unknown. Herein, we show that p14ARF is modified by SUMO2 in vitro, both in transfected cells and under endogenous conditions. SUMO conjugates to the N-terminal part of the tumor suppressor protein increasing its stability. Degradation of p14ARF protein is induced by UBC9 downmodulation or the inhibition of SUMOylation, and is rescued by the NEDDylation inhibitor MLN4924. Treatment with MLN4924 also promotes p14ARF SUMOylation and the transcriptional transactivation of the SUMOylation machinery components SUMO1, SUMO2 and UBC9. This causes a global increase in SUMOylation that contributesto the upregulation of p14ARF levels. Importantly, p14ARF is required for the global increase in SUMOylation induced by MLN4924, and it plays an important role in the cytotoxic effect of the NEDDylation inhibitor on prostate cancer cells. Our results provide evidence that lysine-independent SUMOylation of p14ARF is a new post-translational mechanism regulating p14ARF stability and establishes a new link between inhibition of NEDDylation and SUMOylation. Our goal was to identify the genes involved in the response of PC3 cells to MLN4924, specifically those that depend on the expression of p14ARF. To achieve this, we generated PC3 cells with a knockout of the p14ARF gene (PC3-Sh-P14ARF KO). We then treated both the control PC3 cells and the P14 KO cells with either 1 mM MLN4924 or a vehicle for 3 days. Finally we compared the gene expression from RNA-seq data for PC3 cells and its derivatives (P14ARF-KO and/or MLN4924-treated).

肿瘤抑制因子p14ARF是一种无赖氨酸蛋白，可通过多种机制抑制肿瘤发生，包括增强其互作蛋白的类泛素化修饰（SUMOylation）。尽管已知p14ARF可与SUMO结合酶UBC9相互作用，但目前对ARF与SUMO化修饰之间的关联尚不清楚，且SUMO化修饰对p14ARF的潜在影响仍未明确。本研究证实，p14ARF在体外、转染细胞及内源性生理条件下均可被SUMO2修饰。SUMO结合于该肿瘤抑制蛋白的N端区域，可提升其蛋白稳定性。UBC9下调或SUMO化修饰抑制均可诱导p14ARF蛋白降解，而类泛素化修饰（NEDDylation）抑制剂MLN4924可挽救该降解过程。MLN4924处理还可促进p14ARF的SUMO化修饰，并介导SUMO化系统组分SUMO1、SUMO2及UBC9的转录激活，这会引发整体SUMO化修饰水平升高，进而推动p14ARF蛋白水平的上调。值得注意的是，p14ARF是MLN4924诱导整体SUMO化修饰升高的必需因子，且该蛋白在类泛素化修饰抑制剂对前列腺癌细胞的细胞毒性效应中发挥关键作用。本研究结果证实，p14ARF的赖氨酸非依赖型SUMO化修饰是调控其稳定性的全新翻译后机制，并搭建了类泛素化修饰抑制与SUMO化修饰之间的新关联。本研究的目标是筛选参与PC3细胞对MLN4924应答的基因，尤其是那些依赖p14ARF表达的基因。为此，我们构建了p14ARF基因敲除的PC3细胞（PC3-Sh-P14ARF KO）。随后分别用1 mM MLN4924或溶剂对照处理野生型PC3细胞及P14ARF基因敲除细胞，持续培养3天。最终通过RNA测序（RNA-seq）数据分析，对比PC3细胞及其衍生细胞（P14ARF基因敲除组和/或MLN4924处理组）的基因表达差异。