Technical CommentsPosttranslational Modifications

Comment on “SUMO deconjugation is required for arsenic-triggered ubiquitylation of PML”

See allHide authors and affiliations

Science Signaling  09 Aug 2016:
Vol. 9, Issue 440, pp. tc1
DOI: 10.1126/stke.9.440.tc1

Figures

  • Fig. 1

    Arsenic induces an efficient Lys65-independent degradation of PML or PML/RARA. (A) Arsenic trioxide was prepared as previously described (1) and intraperitoneally injected into mice (129sv background) at 5 μg/g for the indicated times. Total cell extracts were obtained from mouse livers ground in liquid nitrogen using Tissue Lyser II (Thermo Fisher, Life Technologies) and immediately lysed in Laemmli buffer. Immunoblots using mouse monoclonal anti-mouse PML antibody, clone 36.1-104 (Merck Millipore, MAB3738), and rabbit anti-actin (Sigma, A2066) antibodies, and horseradish peroxidase–conjugated secondary antibodies (1:20,000; The Jackson Laboratory) were performed as described previously (4, 7). Note the rapid SUMOylation and degradation of endogenous PML isoform in hepatocytes after in vivo treatment, as previously described (4, 16). Pml−/− mice (129sv background) demonstrate the specificity of PML detection (right). Two representative experiments, each using two mice, of n = 4 independent experiments are depicted. (B) Hematopoietic progenitors were purified and transduced using pMSCV (mouse stem cell virus)–IRES-EGFP (MIE) retroviral vectors expressing wild-type (WT) PML/RARA (P/R) as in (1). Time course of PML/RARA SUMOylation and degradation in transformed primary hematopoietic progenitors treated with 10−7 M arsenic trioxide (Fluka), directly lysed in Laemmli buffer, and detected by immunoblotting with rabbit polyclonal anti-RARA antibody, are shown. n = 2 independent experiments. (C) APL leukemic mice were treated with arsenic for 1 hour. The mice were obtained from serial transplantation of PML/RARA transgenic leukemic bone marrow cells into FVB (Friend leukemia virus B) strain, as previously described (1). PML/RARA SUMOylation and degradation as detected by rabbit polyclonal anti-RARA antibody from two independent experiments are shown. (D) Degradation of PML (left) or its SUMOylation mutants (right) stably expressed in CHO cells (4, 7) (detected by chicken anti-human PML antibody) after treatment with 10−6 M arsenic trioxide and direct lysis in Laemmli buffer. Retinoid X receptor α (RXRA) was detected with rabbit polyclonal anti-RXRA antibody (Santa Cruz Biotechnology). (E) Degradation of PML/RARA or its K65AR mutant (detected by rabbit polyclonal anti-RARA) in primary hematopoietic progenitors treated with 10−7 M arsenic after transduction. Note the different SUMO conjugates in untreated cells. All experiments in cell lines in (D) and (E) were repeated at least three times.

  • Fig. 2

    Lys65 is dispensable for PML and PML/RARA SUMOylation. (A) Western blot analyses of PML/RARA hyperSUMOylation (detected with rabbit polyclonal anti-RARA antibody) 1 hour after arsenic trioxide injections in PML/RARA or PML/RARA K65/490R-driven APL mice. Two arsenic-treated mice are shown for the PML/RARA K65/490R mutant. Data are representative of two independent experiments. (B) Arsenic treatment and SUMO1 knockdown [sequence and use as described in (7)] in CHO cells stably expressing PML or PML K65/490R. Immunoblotting was performed with chicken anti-human PML antibody and rabbit polyclonal anti-RXRA antibody. Results in (B) were independently obtained at least three times.

Navigate This Article