Fig 1: Phosphorylation of RRM2 at T33 protects GSS from proteasome degradation. a pRRM2 and RRM2 were measured by immunoblotting using anti-RRM2 antibodies following electrophoresis in gels containing Phos-tag™, while GSS was measured by immunoblotting using anti-GSS antibodies following electrophoresis in conventional gels. HepG2 and SMMC-7721 cells were cultured in the presence or absence of erastin (10 μM) for 24 h. Relative pRRM2/RRM2 ratios between groups were also calculated and graphed. b RRM2 was phosphorylated at T33. pRRM2 and RRM2 levels were measured by immunoblotting with anti-Myc antibodies following electrophoresis in Phos-tag™-containing gels of HepG2 cells ectopically expressing RRM2WT, RRM2T33A or RRM2T33E before and after treatment with erastin (10 μM) for 24 h. c RRM2 and GSS were measured by immunoblotting in RRM2−/− HepG2 and SMMC-7721 cells ectopically expressing RRM2WT, RRM2T33A or RRM2T33E before and after treatment with erastin (10 μM) for 24 h. d RRM2 and GSS were degraded by proteasomes. RRM2 and GSS were measured by immunoblotting in HepG2 cells with the indicated treatments. Erastin and MG132 were treated at concentrations of 10 μM and 8 μM, respectively, for 24 h. e Colocalization of GSS (upper) or RRM2 (lower) with PSMB5 in HepG2 cells cultured in the presence or absence of erastin (10 μM, 24 h). Scale bar, 20 μm. f Association of RRM2, GSS and PSMB5 in proteasomes isolated from HepG2 cells cultured in the presence or absence of erastin (10 μM, 24 h) in the presence of MG132 (8 μM, 24 h). Samples from affinity or control beads were analyzed in parallel. g Erastin (10 μM, 24 h) chase of GSS and RRM2 in RRM2−/− HepG2 and SMMC-7721 cells reconstituted with RRM2WT, RRM2T33A or RRM2T33E. The levels of GSS were also normalized to those of GAPDH, and the normalized level of GSS in the 0 h group was arbitrarily set to 100%. The data are shown as the mean ± SD from three biological replicates (including IB). *P < 0.05, **P < 0.01 indicates statistical significance. Data from a were analyzed using a one-way ANOVA test. Data from e were analyzed using Student’s t test
Fig 2: YAP ISGylation inhibits its ubiquitination and increases its stability.A Co-IP was performed in control, ISG15 overexpression or knockout A549 and H1299 cells using anti-YAP antibodies. The YAP level in each co-IP sample was adjusted to the same protein content. Indicated proteins were further analyzed by IB. B Co-IP was performed in control or UbCH8, HERC5, or βTrCP knockout A549 and H1299 cells using anti-YAP antibodies. The YAP level in each co-IP sample was adjusted to the same protein content. Indicated proteins were further analyzed by IB. C CHX (10 μg/ml) chase experiments were performed in control, ISG15 overexpression or knockout A549 and H1299 cells. The relative protein levels of YAP were shown as the ratios between YAP and GAPDH, and the “0 h” points were arbitrarily set to 100%. D Co-IP was performed in control, ISG15 overexpression or knockout A549 and H1299 cells using anti-YAP antibodies. The YAP level in each co-IP sample was adjusted to the same protein content. Indicated proteins were further analyzed by IB. E Co-localization of YAP and PSMB5 was analyzed in WT or ISG15−/− A549 cells before being photographed using a confocal microscope. Scale bar, 20 μm. F Association of YAP and PSMB5 analyzed by IB in proteasomes isolated from A549 cells with or without ISG15 overexpression or knockout in the presence of MG132 (8 μM, 24 h). Samples from affinity or control beads were analyzed in parallel. G YAP expression was analyzed by IB in WT or ISG15−/− A549 cells with 3-MA, CHQ, MG132, or Bort treatment or with ATG5 or PSMB5 knockout. H YAP mRNA level was analyzed in control, ISG15 overexpression or knockout A549, and H1299 cells. The data are shown as the mean ± SD from three biological replicates (including IB). Data in C were analyzed using a two-way ANOVA test. Data in H were analyzed using a one-way ANOVA test. NS nonsignificant.
Fig 3: TRIB2 and PCBP2 reduce K48-ubiquitination of GPX4.a CHX chase experiments of GPX4 in the control and Bel-7402 cells with TRIB2 knocked out, with or without the simultaneous overexpression of PCBP2. The relative protein levels of GPX4 were normalized to those of GAPDH, and the “0 h” point was arbitrarily set to 100%. b TRIB2 and PCBP2 regulated GPX4 ubiquitination. GPX4 or its K48-ubiquitination and total-ubiquitination level in the WCL of Bel-7402 and SMMC-7721 cells with or without TRIB2 knockout in the presence or absence of ectopic expression of PCBP2. The K48-Ub and total-Ub of GPX4 were normalized to that of GPX4 in the GPX4-IPs by ImageJ and indicated below the blots. c PCBP2 acts as the downstream of TRIB2 to prevent the association between GPX4 and proteasome in Bel-7402 and SMMC-7721 cells. The relative GPX4 levels were normalized to PSMB3 in proteasome and normalized to GAPDH at WCL. The relative GPX4 levels were also calculated as the ratio between the one in proteasome and the one at WCL. Besides, the PSMB5 activity was also parallel examined at WCL. Data were analyzed by one-way ANOVA and expressed as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. d, e The TRIB2 DQLVPD element and PCBP2 KH3 domain are critical to maintain GPX4 expression. GPX4 was measured by IB in the Bel-7402 cells transfected with the indicated plasmids. The relative GPX4 levels were normalized to that of GAPDH and indicated below the blots. f GPX4 in control and Bel-7402 cells with or without TRIB2 or PCBP2 knocked out in the presence or absence of overexpressed GPX4. The relative GPX4 levels were normalized to that of GAPDH and indicated below the blots. g Tumor growth in athymic mice inoculated with the same cells as shown in f. The weight and volume of a tumor are presented below the representative tumor image (n = 5/group); Scale bar = 3 mm. Data were analyzed by one-way ANOVA and expressed as mean ± SD. **P < 0.01; ****P < 0.0001. Images of all the immunoblots are representative of three independent experiments.
Fig 4: YAP ISGylation inhibits its interaction with βTrCP and promotes downstream transcription.A, B Co-IP was performed in control, ISG15 overexpression or knockout A549 and H1299 cells using anti-YAP (A) or anti-βTrCP (B) antibodies. The YAP (A) or βTrCP (B) level in each co-IP sample was adjusted to the same protein content. Indicated proteins were further analyzed by IB. C Co-localization of YAP and βTrCP was analyzed in WT or ISG15−/− A549 cells before being photographed using a confocal microscope. Scale bar, 20 μm. D Proximal protein ligation between YAP and βTrCP, as photographed by confocal microscope in WT or ISG15−/−A549 cells Scale bar, 50 μm. E YAP ubiquitination sites analyzed by PTMD and BDM-PUB databases. F Co-IP was performed in YAP−/− reconstituted A549 cells with YAPWT-HA, YAPK280R-HA, YAPK321R-HA, or YAPK497R-HA expressed. The YAP level in each co-IP sample was adjusted to the same protein content. G Association of YAP and PSMB5 analyzed by IB in proteasomes isolated from YAP−/− reconstituted A549 cells with ISG15, YAPWT-HA or YAPK497R-HA expressed in the presence of MG132 (8 μM, 24 h). Samples from affinity or control beads were analyzed in parallel. H Co-IP was performed using anti-YAP antibodies in YAP−/− reconstituted A549 cells with ISG15, YAPWT-HA or YAPK497R-HA expressed. The YAP level in each co-IP sample was adjusted to the same protein content. Indicated proteins were further analyzed by IB. I O-GlcNAcylation of YAP at T241 (YAPO241), phosphorylation of YAP at S127 (YAPP127) and S397 (YAPP397) was analyzed in YAP−/− reconstituted A549 cells with ISG15, YAPWT-HA or YAPK497R-HA expressed by IB. J, K Relative TEAD Luc (J), CTGF and ANKRD1 mRNA level (K) were measured using a pUAS-Luc/TEAD-Gal4 system in control, ISG15 overexpression or knockout A549 and H1299 cells. The data are shown as the mean ± SD from three biological replicates (including IB). Data in J, K were analyzed using a one-way ANOVA test. **P < 0.01, *P < 0.05.
Fig 5: PCBP2 is essential for TRIB2 to regulate PSMB5 activity.a Venn diagram showing three biologically independent MS results from the Bel-7402 cell immunoprecipitation with anti-TRIB2 antibodies. The candidates were further screened by UniProt online software and the literature to identify potential ubiquitination-related proteins. b Representative Coomassie blue stain image of the Bel-7402 cell immunoprecipitates pulled down by anti-TRIB2 or IgG antibodies (n = 3). c Ub in the control and Bel-7402 cells with MYPT1, GRP78, PARP1, or PCBP2 knocked down. d Ub in the control and Bel-7402 cells overexpressing PCBP1, PCBP2, or PCBP3 with or without TRIB2 knocked down. e Reciprocal co-IP results of purified TRIB2 and PCBP2 proteins in vitro. TRIB1, TRIB3, PCBP1, and PCBP3 were parallel examined to exclude non-specificity. f Positive correlation between PCBP2 and TRIB2 in the liver cancer specimens. TMA was performed by IHC using anti-PCBP2 and anti-TRIB2 antibodies (n = 208). The data were analyzed by Spearman’s rank correlation. g The effects of TRIB2 on Ub level were PCBP2-dependent. Ub in the control and Bel-7402 cells with or without TRIB2 knocked out or overexpressed, in the presence or absence of PCBP2 knocking out. h PCBP2 regulated Ub via PSMB5. Ub in Bel-7402 cells with PCBP2 and PSMB5 knocked out or overexpressed, as indicated. i TRIB2 regulated PSMB5 activity via PCBP2. A proteasome activity assay kit (AAT Bioquest) was used to evaluate PSMB5 activity in the Bel-7402 and SMMC-7721 cells under the same treatment as indicated in g (n = 3). Data were analyzed by one-way ANOVA and expressed as mean ± SD. ****P < 0.0001; NS non-significance. Images of all the immunoblots are representative of three independent experiments. The relative protein levels of the conj & poly Ub and mono Ub were normalized to those of GAPDH as calculated by ImageJ software and indicated just below the blots (c, d, g, h).
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