Role of SUMO in RNF4-mediated Promyelocytic Leukemia Protein (PML) Degradation: Sumoylation of pml and phospho-switch control of its sumo binding domain dissected in living cells*

Promyelocytic leukemia protein (PML) is a tumor suppressor acting as the organizer of subnuclear structures called PML nuclear bodies (NBs). Both covalent modification of PML by the small ubiquitin-like modifier (SUMO) and non-covalent binding of SUMO to the PML SUMO binding domain (SBD) are necessary for PML NB formation and maturation. PML sumoylation and proteasome-dependent degradation induced by the E3 ubiquitin ligase, RNF4, are enhanced by the acute promyelocytic leukemia therapeutic agent, arsenic trioxide (As₂O₃). Here, we established a novel bioluminescence resonance energy transfer (BRET) assay to dissect and monitor PML/SUMO interactions dynamically in living cells upon addition of therapeutic agents. Using this sensitive and quantitative SUMO BRET assay that distinguishes PML sumoylation from SBD-mediated PML/SUMO non-covalent interactions, we probed the respective roles of covalent and non-covalent PML/SUMO interactions in PML degradation and interaction with RNF4. We found that, although dispensable for As₂O₃-enhanced PML sumoylation and RNF4 interaction, PML SBD core sequence was required for As₂O₃- and RNF4-induced PML degradation. As confirmed with a phosphomimetic mutant, phosphorylation of a stretch of serine residues, contained within PML SBD was needed for PML interaction with SUMO-modified protein partners and thus for NB maturation. However, mutation of these serine residues did not impair As₂O₃- and RNF4-induced PML degradation, contrasting with the known role of these phosphoserine residues for casein kinase 2-promoted PML degradation. Altogether, these data suggest a model whereby sumoylation- and SBD-dependent PML oligomerization within NBs is sufficient for RNF4-mediated PML degradation and does not require the phosphorylation-dependent association of PML with other sumoylated partners.Promyelocytic leukemia protein (PML)⁵ is a tumor suppressor (1) whose gene is translocated in cases of acute promyelocytic leukemia (2). PML functions as the organizer of PML NBs, which are dynamic structures harboring numerous transiently and permanently localized proteins (3). The importance of PML NB structural integrity was first revealed in acute promyelocytic leukemia because, in this malignancy, the abnormal fusion protein PML/RARα leads to NB disruption. Patient treatment with As₂O₃ induces the reversion of the acute promyelocytic leukemia phenotype as well as PML/RARα degradation and PML NB reformation (4).PML is a target for the post-translational modification by SUMO, an ubiquitin-like protein that is covalently coupled to PML lysine residues 65, 160, and 490 via a process called sumoylation (5, 6). Among the four human SUMO paralogs identified, SUMO1, -2, and -3 were found to be conjugated to target proteins. It involves an enzymatic cascade for the transfer of the mature SUMO and the formation of an isopeptide bond between the COOH-terminal glycine of SUMO and a lysine from the target protein. Sumoylation is a reversible process due to the existence of several deconjugating enzymes.PML NB formation requires both the covalent linkage (sumoylation) (reviewed in Ref. 7) and the non-covalent interactions of SUMO with PML through a SUMO binding domain (SBD also named SIM for SUMO interacting motif) (8). Interestingly, PML SBD contains specific serines, acting as substrates for the caseine kinase-2 (CK2), which are implicated in PML ubiquitination and degradation (9) and which phosphorylation status could regulate the function of the SBD.Because sumoylation of proteins is dynamic and reversible, this post-translational modification is difficult to follow in vivo and its detection mainly relies on the identification of sumoylated protein species by Western blot following cell lysis. Recently, we used bioluminescence resonance energy transfer (BRET) to detect covalent linkage of ubiquitin (ubiquitination) in living mammalian cells and in real time (10). In brief, BRET monitors the interaction between a protein fused to a luciferase and a protein fused to yellow or green fluorescent protein (YFP or GFP), upon addition of a luciferase substrate; it is a proximity-based assay that requires that the donor of energy (luciferase fusion) and the acceptor (YFP or GFP fusions) are within 50 to 100 Å for an efficient energy transfer (11–13). However, a demonstration that BRET may provide a method of choice to follow the dynamics of protein sumoylation in living cells is lacking. Here, we developed a sensitive and quantitative SUMO BRET assay for the detection of PML interactions with SUMO in living cells. We proved that BRET can be used to detect both SUMO covalent and non-covalent interactions with PML (model, Fig. 1H). For this purpose, we used the PMLIII isoform in which sumoylation is induced by As₂O₃ and triggers a proteasome-dependent PML degradation (14); the degradation process involves the ubiquitination of poly-SUMO covalently coupled to PML by the poly-SUMO-specific E3 ubiquitin ligase RNF4 (15–17). Altogether, our BRET results indicate that, As₂O₃ and/or RNF4-induced PML degradation are dependent on the integrity of both PML sumoylation target sites and the PML SBD core sequence but not on the CK2 serine phosphorylation sites within the SBD. However, phosphorylation of these serines is required for most PML SBD-dependent non-covalent interactions. This phospho-regulation of PML SBD (“SBD phospho-switch”) establishes another link between the phosphorylation and SUMO, different from the phospho-sumoyl switch (18).Open in a separate windowFIGURE 1.BRET reveals both covalent and non-covalent PML/SUMO1 interactions as well as As₂O₃-induced PML sumoylation in living cells. A and B, detection of PML/SUMO1 interactions by BRET¹ (A) or BRET² (B) titration assays using HEK293T cells transfected for expression of increasing amounts of YFP-SUMO1 (BRET¹) or GFP-SUMO1 (BRET²) and a fixed amount of Luc fusion. Negative controls: BRET pairs including PML_C57,60A-Luc (a non-sumoylatable mutant with Cys⁵⁷ and Cys⁶⁰ mutated to Ala) or YFP-SUMO1G (a SUMO1 that cannot be processed) (dotted line) (A) and Luc fused to a NLS (B). C and D, detection of covalent and non-covalent PML/SUMO1 interactions by BRET¹ (C) or BRET² (D) titration assays in the presence (dotted lines, empty symbols) or absence (solid lines and symbols) of As₂O₃ in HEK293T cells transfected for expression of PML_WT-Luc or its sumoylation deficient mutant PML_3K-Luc in pairs with either YFP-SUMO1 (BRET¹) or GFP-SUMO1 (BRET²). Negative control: PML_WT-Luc in pairs with YFP-SUMO1G. E, kinetics of As₂O₃-induced PML_WT-Luc sumoylation revealed by BRET¹ (assay on attached cells) and BRET² (cells in suspension). F, dose-response curve to As₂O₃ treatment for PML_WT-Luc or PML_3KR-Luc/YFP-SUMO1 BRET¹ pairs. Negative control: PML_C57,60A-Luc/YFP-SUMO1. G, comparison of As₂O₃-induced sumoylation of PML_WT, PML_3KR, and its single lysine mutants at an identical YFP acceptor/Luc donor expression ratio as derived from titration curves. As₂O₃ treatment (C–G): 5 μm, 4 h exposure for BRET¹ and Western blot or 10 μm, 70-min exposure for BRET². H, model for the covalent (sumoylation) and non-covalent interactions between a tested protein fused to Luc and SUMO fused to a fluorescent protein (YFP) that generates a BRET signal. The black arrows indicate the bioluminescent transfer of energy (or BRET) that occurs between Luc and GFP fusion upon exposure to the cell-permeable luciferase substrate.