| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 5845934 | Toxicology and Applied Pharmacology | 2015 | 11 Pages |
•As3 + is a carcinogen and also a therapeutic agent for leukemia.•PML becomes insoluble in RIPA and SUMOylated by As3 +.•Sb3 + modifies PML similar to As3 +.•Functional RING motif is necessary for As3 +-induced PML modification.
Promyelocytic leukemia (PML), which is a tumor suppressor protein that nevertheless plays an important role in the maintenance of leukemia initiating cells, is known to be biochemically modified by As3 +. We recently developed a simple method to evaluate the modification of PML by As3 + resulting in a change in solubility and the covalent binding of small ubiquitin-like modifier (SUMO). Here we semi-quantitatively investigated the SUMOylation of PML using HEK293 cells which were stably transfected with PML-VI (HEK-PML). Western blot analyses indicated that PML became insoluble in cold RadioImmunoPrecipitation Assay (RIPA) lysis buffer and was SUMOylated by both SUMO2/3 and SUMO1 by As3 +. Surprisingly SUMO1 monomers were completely utilized for the SUMOylation of PML. Antimony (Sb3 +) but not bismuth (Bi3 +), Cu2 +, or Cd2 + biochemically modified PML similarly. SUMOylated PML decreased after removal of As3 + from the culture medium. However, unSUMOylated PML was still recovered in the RIPA-insoluble fraction, suggesting that SUMOylation is not requisite for changing the RIPA-soluble PML into the RIPA-insoluble form. Immunofluorescence staining of As3 +-exposed cells indicated that SUMO2/3 was co-localized with PML in the nuclear bodies. However, some PML protein was present in peri-nuclear regions without SUMO2/3. Functional Really Interesting New Gene (RING)-deleted mutant PML neither formed PML nuclear bodies nor was biochemically modified by As3 +. Conjugation with intracellular glutathione may explain the accessibility of As3 + and Sb3 + to PML in the nuclear region evading chelation and entrapping by cytoplasmic proteins such as metallothioneins.
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