title: Solubility shift and SUMOylaltion of promyelocytic leukemia (PML) protein in response to arsenic(III) and fate of the SUMOylated PML.
abstract: 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 As(3+). We recently developed a simple method to evaluate the modification of PML by As(3+) 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 As(3+). Surprisingly SUMO1 monomers were completely utilized for the SUMOylation of PML. Antimony (Sb(3+)) but not bismuth (Bi(3+)), Cu(2+), or Cd(2+) biochemically modified PML similarly. SUMOylated PML decreased after removal of As(3+) 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 As(3+)-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 As(3+). Conjugation with intracellular glutathione may explain the accessibility of As(3+) and Sb(3+) to PML in the nuclear region evading chelation and entrapping by cytoplasmic proteins such as metallothioneins.
authors.affiliation: Center for Environmental Risk Research, National Institute for Environmental Studies, Japan; Graduate School of Pharmaceutical Sciences, Chiba University, Japan. Electronic address: seishiro@nies.go.jp.
authors.forename: Seishiro
authors.initials: S
authors.lastname: Hirano
authors.affiliation: Center for Environmental Risk Research, National Institute for Environmental Studies, Japan.
authors.forename: Mihoko
authors.initials: M
authors.lastname: Tadano
authors.affiliation: Center for Environmental Health Sciences, National Institute for Environmental Studies, Japan; Graduate School of Pharmaceutical Sciences, Chiba University, Japan.
authors.forename: Yayoi
authors.initials: Y
authors.lastname: Kobayashi
authors.affiliation: Center for Environmental Risk Research, National Institute for Environmental Studies, Japan.
authors.forename: Osamu
authors.initials: O
authors.lastname: Udagawa
authors.affiliation: Graduate School of Pharmaceutical Sciences, Chiba University, Japan.
authors.forename: Ayaka
authors.initials: A
authors.lastname: Kato
mesh_terms: D000965:Antimony / Q000633:toxicity; D018053:Arsenites / Q000633:toxicity*; D002712:Chlorides / Q000633:toxicity; D005978:Glutathione / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D000071156:Promyelocytic Leukemia Protein; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D025842:SUMO-1 Protein / Q000378:metabolism; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism; D017670:Sodium Compounds / Q000633:toxicity*; D012995:Solubility; D058207:Sumoylation; D013997:Time Factors; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000737:chemistry / Q000378:metabolism*; D014452:Ubiquitins / Q000378:metabolism
chemical_list: D018053:Arsenites; D002712:Chlorides; D009687:Nuclear Proteins; D000071156:Promyelocytic Leukemia Protein; D025842:SUMO-1 Protein; C495501:SUMO1 protein, human; C117909:SUMO2 protein, human; C492189:SUMO3 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; D017670:Sodium Compounds; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D014452:Ubiquitins; C070576:PML protein, human; C017947:sodium arsenite; D000965:Antimony; D005978:Glutathione; C054672:antimony trichloride
keywords: Arsenic; Immunofluorescence; Metalloid; Promyelocytic leukemia; SUMO