Volume 21, Issue 9, Pages 2447-2457 (November 2017) TRIM48 Promotes ASK1 Activation and Cell Death through Ubiquitination-Dependent Degradation of the ASK1-Negative Regulator PRMT1  Yusuke Hirata, Kazumi Katagiri, Keita Nagaoka, Tohru Morishita, Yuki Kudoh, Tomohisa Hatta, Isao Naguro, Kuniyuki Kano, Tsuyoshi Udagawa, Tohru Natsume, Junken Aoki, Toshifumi Inada, Takuya Noguchi, Hidenori Ichijo, Atsushi Matsuzawa  Cell Reports  Volume 21, Issue 9, Pages 2447-2457 (November 2017) DOI: 10.1016/j.celrep.2017.11.007 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 21, 2447-2457DOI: (10.1016/j.celrep.2017.11.007) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 TRIM48 Promotes ASK1 Activation and K48-Linked Polyubiquitination (A) HEK293A cells were transfected with control siRNA or siRNAs targeting FBXO32, Roquin-2, RING1, or TRIM48 for 48 hr, and then the cells were treated with 0.5 mM H2O2 for the indicated time. The cell lysates were subjected to immunoblotting with the indicated antibodies. The arrowheads indicate bands corresponding to the indicated proteins, and the asterisk indicates a non-specific band. (B) HEK293A cells were transfected with Flag-ASK1-ΔGG for 24 hr and subsequently transfected with control siRNA or siRNA targeting TRIM48 for 48 hr. The cell lysates were subjected to immunoblotting with the indicated antibodies. The arrowheads indicate bands corresponding to the indicated proteins. Ctrl, Control. (C) HEK293A cells transfected with the indicated constructs were subjected to immunoblotting with the indicated antibodies. The arrowheads indicate bands corresponding to the indicated proteins. (D) HEK293A cells transfected with the indicated constructs were subjected to in vivo ubiquitination assays to assess Flag-ASK1 ubiquitination status. The arrowheads indicate bands corresponding to the indicated proteins. (E) In vivo ubiquitination assays were performed as in (D). The arrowheads indicate bands corresponding to the indicated proteins. (F) In vivo ubiquitination assays were performed as in (D). The arrowheads indicate bands corresponding to the indicated proteins. See also Figures S1, S2, and S3. Cell Reports 2017 21, 2447-2457DOI: (10.1016/j.celrep.2017.11.007) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 TRIM48 Interacts with PRMT1 and Promotes Ubiquitin-Dependent PRMT1 Degradation (A) HEK293A cells were transfected with an empty vector or a 6myc-TRIM48 plasmid for 24 hr and then treated with 5 μM MG132 for 4 hr. The cell lysates were immunoprecipitated with an anti-Myc antibody and subjected to immunoblotting with the indicated antibodies. The arrowheads indicate bands corresponding to the indicated proteins, and the asterisk indicates a non-specific band. (B) HEK293A cells transfected with the indicated constructs were subjected to in vivo ubiquitination assay to assess Flag-PRMT1 ubiquitination status. The arrowheads indicate bands corresponding to the indicated proteins. (C and D) In vitro ubiquitination assay using purified recombinant proteins from E. coli. (C) Recombinant proteins were subjected to Coomassie brilliant blue (CBB) staining and immunoblotting with the indicated antibodies. The arrowheads indicate bands corresponding to the indicated proteins, and the asterisk indicates a non-specific band. (D) In vitro ubiquitination reactions were performed using the recombinant proteins and subjected to immunoblotting with the indicated antibodies. The arrowheads indicate bands corresponding to the indicated proteins, and the asterisk indicates a non-specific band. (E and F) HEK293A cells were transfected with the indicated constructs for 24 hr and then treated with cycloheximide (CHX) for the indicated time. The cell lysates were subjected to immunoblotting with the indicated antibodies to evaluate the degradation rates of Flag-PRMT1 (E) and endogenous PRMT1 (F). The arrowheads indicate bands corresponding to the indicated proteins, and the asterisk indicates a non-specific band. See also Figures S4 and S5 and Table S1. Cell Reports 2017 21, 2447-2457DOI: (10.1016/j.celrep.2017.11.007) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 TRIM48-Dependent K48-Linked ASK1 Polyubiquitination Is Catalyzed by Roquin-2 (A) HEK293A cells were transfected with the indicated siRNAs 48 hr, subsequently transfected with the indicated constructs for 24 hr, and then subjected to in vivo ubiquitination assay. The arrowheads indicate bands corresponding to the indicated proteins. (B) HEK293A cells transfected with the indicated constructs were subjected to in vivo ubiquitination assays to assess Flag-ASK1ΔN ubiquitination status. The arrowheads indicate bands corresponding to the indicated proteins. (C) DNA sequences around the guide RNA (gRNA) target site in the exon 2 of the Roquin-2 gene in Roquin-2 WT and KO HEK293 cells (upper). A frameshift insertion (the underlined nucleotide) is introduced in the KO cells. Roquin-2 expression in these cells was assessed by immunoblot analysis (bottom). (D) Roquin-2 WT and KO HEK293 cells were transfected with the indicated constructs and subjected to in vivo ubiquitination assay to assess full-length Flag-ASK1 ubiquitination status. The arrowheads indicate bands corresponding to the indicated proteins, and the asterisk indicates a non-specific band. Cell Reports 2017 21, 2447-2457DOI: (10.1016/j.celrep.2017.11.007) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 TRIM48 Suppresses the Interactions between Trx and ASK1 and the Inhibitory Effects of Trx on ASK1 Activation (A) HEK293A cells were transfected with the indicated constructs for 24 hr. The cell lysates were immunoprecipitated with anti-mono and dimethyl arginine antibodies and subjected to immunoblotting with the indicated antibodies. The arrowheads indicate bands corresponding to the indicated proteins, and the asterisk indicates a non-specific band. (B) HEK293A cells were transfected with Flag-ASK1-ΔGG for 24 hr and subsequently transfected with control siRNA or siRNA targeting TRIM48 for 48 hr. The cell lysates were immunoprecipitated with an anti-Trx antibody and subjected to immunoblotting with the indicated antibodies. The arrowheads indicate bands corresponding to the indicated proteins. (C) HEK293A cells were transfected with control siRNA or siRNA targeting TRIM48 for 48 hr and then treated with the indicated concentrations of diamide for 15 min. The cell lysates were immunoprecipitated with an anti-rabbit immunoglobulin G (IgG) (negative control) or anti-Trx antibody and subjected to immunoblotting with the indicated antibodies. See also Figure S6A. Cell Reports 2017 21, 2447-2457DOI: (10.1016/j.celrep.2017.11.007) Copyright © 2017 The Author(s) Terms and Conditions

Figure 5 TRIM48 Promotes Oxidative Stress-Induced Cell Death by Negatively Regulating PRMT1 (A) HEK293A cells were transfected with the indicated siRNAs for 48 hr and then treated with 0.5 mM diamide for 2 hr before being subjected to cell death assay. The data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA followed by Dunnett’s test; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 (versus control). (B) Transfection and cell death assay were performed as in (A). The data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA followed by Dunnett’s test; ∗∗p < 0.01; ∗∗∗p < 0.001 (versus control). (C) HEK293A cells were transfected with single or combinations of siRNAs targeting the indicated genes for 48 hr and then treated with 0.5 mM diamide for 2 hr before being subjected to cell death assay. The data shown are the mean ± SEM (n = 3). Significant differences were determined by one-way ANOVA, followed by Dunnett’s test; ∗p < 0.05; ∗∗p < 0.01. See also Figures S6B, S6C, and S7. Cell Reports 2017 21, 2447-2457DOI: (10.1016/j.celrep.2017.11.007) Copyright © 2017 The Author(s) Terms and Conditions

Figure 6 TRIM48 Suppresses Tumor Growth in a Mouse Xenograft Model (A–E) A549 cells stably expressing empty vector, 6Myc-TRIM48 WT, or CS (A) were subcutaneously injected into nude mice, and the xenografts were analyzed as follows (the details are described in Experimental Procedures). (B) A representative image of tumor xenografts harvested at day 45. Bar, 10 mm. (C) Tumor volume was measured twice a week and is shown as the mean ± SEM (empty, TRIM48 WT: n = 6, TRIM48 CS: n = 5). Significant differences were determined by one-way ANOVA, followed by Dunnett’s test; ∗∗∗p < 0.001 (versus empty). (D and E) Paraffin sections of the harvested tumor xenografts were labeled by TUNEL, counterstained with DAPI, and observed by confocal fluorescence microscopy. (D) Representative images of the TUNEL-labeled sections. The arrowheads indicate TUNEL-positive nuclei. White bar, 50 μm. (E) The number of TUNEL-positive nuclei were counted in 3 fields for each section from 3 independent tumor xenografts and are shown as TUNEL-positive cells per square millimeter (mean ± SEM, n = 3). Significant differences were determined by one-way ANOVA, followed by Dunnett’s test; ∗∗∗p < 0.001 (versus empty). Cell Reports 2017 21, 2447-2457DOI: (10.1016/j.celrep.2017.11.007) Copyright © 2017 The Author(s) Terms and Conditions

Figure 7 A Proposed Model for TRIM48-Mediated Regulation of Oxidative Stress-Induced ASK1 Activation and Cell Death Methylation of ASK1 (78Arg and 80Arg) by PRMT1 increases the interactions between Trx and ASK1, resulting in suppression of ASK1 activation and cell survival under oxidative stress (Cho et al., 2012). TRIM48 promotes PRMT1 ubiquitination and degradation, leading to decreases in the ASK1-Trx interactions, which promotes oxidative-stress-induced ASK1 activation and cell death, and suppresses cancer development. TRIM48-dependent ASK1 activation is repressed by ubiquitin-dependent degradation through Roquin-2, an E3 ubiquitin ligase for ASK1 (Maruyama et al., 2014). Cell Reports 2017 21, 2447-2457DOI: (10.1016/j.celrep.2017.11.007) Copyright © 2017 The Author(s) Terms and Conditions