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. Overall design: 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).