CNRS, FRE 3235, Université Paris Descartes, Paris, France.
PLoS One. 2012;7(9):e44949. doi: 10.1371/journal.pone.0044949. Epub 2012 Sep 18.
PML, the organizer of nuclear bodies (NBs), is expressed in several isoforms designated PMLI to VII which differ in their C-terminal region due to alternative splicing of a single gene. This variability is important for the function of the different PML isoforms. PML NB formation requires the covalent linkage of SUMO to PML. Arsenic trioxide (As₂O₃) enhances PML SUMOylation leading to an increase in PML NB size and promotes its interaction with RNF4, a poly-SUMO-dependent ubiquitin E3 ligase responsible for proteasome-mediated PML degradation. Furthermore, the presence of a bona fide SUMO Interacting Motif (SIM) within the C-terminal region of PML seems to be required for recruitment of other SUMOylated proteins within PML NBs. This motif is present in all PML isoforms, except in the nuclear PMLVI and in the cytoplasmic PMLVII. Using a bioluminescence resonance energy transfer (BRET) assay in living cells, we found that As₂O₃ enhanced the SUMOylation and interaction with RNF4 of nuclear PML isoforms (I to VI). In addition, among the nuclear PML isoforms, only the one lacking the SIM sequence, PMLVI, was resistant to As₂O₃-induced PML degradation. Similarly, mutation of the SIM in PMLIII abrogated its sensitivity to As₂O₃-induced degradation. PMLVI and PMLIII-SIM mutant still interacted with RNF4. However, their resistance to the degradation process was due to their inability to be polyubiquitinated and to recruit efficiently the 20S core and the β regulatory subunit of the 11S complex of the proteasome in PML NBs. Such resistance of PMLVI to As₂O₃-induced degradation was alleviated by overexpression of RNF4. Our results demonstrate that the SIM of PML is dispensable for PML SUMOylation and interaction with RNF4 but is required for efficient PML ubiquitination, recruitment of proteasome components within NBs and proteasome-dependent degradation of PML in response to As₂O₃.
PML 是核体 (NBs) 的组织者，以 PMLI 至 VII 的几种同工型表达，这些同工型在其 C 末端区域因单个基因的选择性剪接而不同。这种可变性对于不同 PML 同工型的功能很重要。PML NB 的形成需要 SUMO 与 PML 的共价连接。三氧化二砷 (As₂O₃) 增强了 PML 的 SUMO 化，导致 PML NB 大小增加，并促进其与 RNF4 的相互作用，RNF4 是一种多 SUMO 依赖性泛素 E3 连接酶，负责蛋白酶体介导的 PML 降解。此外，PML C 末端区域内存在真正的 SUMO 相互作用基序 (SIM)，似乎是募集 PML NB 内其他 SUMO 化蛋白所必需的。该基序存在于所有 PML 同工型中，除了核 PMLVI 和细胞质 PMLVII。我们在活细胞中使用生物发光共振能量转移 (BRET) 测定法发现，As₂O₃ 增强了核 PML 同工型 (I 至 VI) 的 SUMO 化和与 RNF4 的相互作用。此外，在核 PML 同工型中，只有缺乏 SIM 序列的 PMLVI 对 As₂O₃ 诱导的 PML 降解具有抗性。同样，PMLIII 中的 SIM 突变使其对 As₂O₃ 诱导的降解失去敏感性。PMLVI 和 PMLIII-SIM 突变体仍然与 RNF4 相互作用。然而，它们对降解过程的抗性是由于它们不能被多泛素化，并且不能有效地募集蛋白酶体 20S 核心和 11S 复合物的β调节亚基在 PML NBs 中。PMLVI 对 As₂O₃ 诱导的降解的这种抗性可通过 RNF4 的过表达减轻。我们的结果表明，PML 的 SIM 对于 PML SUMO 化和与 RNF4 的相互作用不是必需的，但对于 PML 的有效泛素化、NB 内蛋白酶体成分的募集以及蛋白酶体依赖性的 PML 降解是必需的，以响应 As₂O₃。
