SUMO E3 ligase activity of TRIM proteins

Abstract

SUMOylation governs numerous cellular processes and is essential to most eukaryotic life. Despite increasing recognition of the importance of this process, an extremely limited number of small ubiquitin-like modifier (SUMO) protein ligases (E3s) have been identified. Here we show that at least some members of the functionally diverse tripartite motif (TRIM) superfamily are SUMO E3s. These TRIM proteins bind both the SUMO-conjugating enzyme Ubc9 and substrates and strongly enhance transfer of SUMOs from Ubc9 to these substrates. Among the substrates of TRIM SUMO E3s are the tumor suppressor p53 and its principal antagonist Mdm2. The E3 activity depends on the TRIM motif, suggesting it to be the first widespread SUMO E3 motif. Given the large number of TRIM proteins, our results may greatly expand the identified SUMO E3s. Furthermore, TRIM E3 activity may be an important contributor to SUMOylation specificity and the versatile functions of TRIM proteins.

Figure 1. PML promotes SUMOylation of p53 and Mdm2 in vivo

(a) Pml^−/− MEF cells were transfected with Flag-PML, p53, and SUMO1 as indicated. Cells were treated with proteasome inhibitor MG132 for 4 h (same below), and whole cell lysates (WCL) were analyzed by western blot. Molecular weight standards (in kDa) are shown on the left. (b) H1299 cells were transfected with Flag-PML, HA-p53, and His- or Flag-tagged SUMO1 as indicated. Cells were lysed in buffer containing guanidinium-HCl, and proteins conjugated to His-SUMO1 were pulled down by Ni²⁺-NTA beads (Ni beads). The bead-bound proteins and WCL were analyzed by western blot. (c) H1299 cells were transfected with PML isoform IV or VI, p53, SUMO1 and GFP as indicated. WCL were analyzed by western blot. (d) Pml^−/− cells were transfected with HA-PML, Flag-Mdm2, and His-SUMO1 as indicated. Cell lysates were incubated by Ni²⁺-NTA beads. WCL (left) and the bead-bound proteins (right) were analyzed by western blot.

Figure 2. Purified PML promotes SUMOylation of p53 and Mdm2 in vitro

(a) His-p53 purified from bacteria was incubated with SUMO E1(SAE1/SAE2), ATP, and either SUMO1 or SUMO2, in the presence or absence of Ubc9 and PML. The reaction mixes were analyzed by western blot using anti-p53 antibody. (b) In vitro SUMOylation of Mdm2 was performed with SUMO E1, His-SUMO1, and ATP, in the presence or absence of Ubc9 and PML. Proteins conjugated to His-SUMO1 were purified by Ni-NTA beads and analyzed by western blot using anti-Mdm2 antibody. (c) His-p53 was incubated with ATP and the indicated combinations of E1, Ubc9, and bacterial recombinant GST-PML or GST. The reaction mixes were analyzed as in (a).

Figure 3. The Ring and B boxes domains are required for SUMO E3 activity of PML

(a) Schematic representation of wild type PML isoform IV (called PML in this study), isoform VI, and various PML mutants. The RING domain (R) (aa 59–91), B1 box (aa 124–166), B2 box (aa 185–230), predicted coiled-coil region (CC) (aa 230–323), and SIM (aa 508–511) are indicated. PML mutations are: N (aa 1–233), C (aa 223–633), R^m (Cys57-to-Ser and Cys60-to-Ser, C57A/C60A), B1^m (C129A/C132A), B2^m (C189A/H194A), M4 (combination of R^m and B1^m), and M6 (combination of R^m, B1^m, and B2^m). Asterisks indicate the positions of the point mutations. (b) PML mutations affect Mdm2 SUMOylation. Pml^−/− cells were transfected with PML or each of the PML mutants, Mdm2, and His-SUMO1 as indicated. His-SUMO1-conjugated proteins pulled down by Ni²⁺-NTA beads and WCL were analyzed by western blot. (c) Pml^−/− MEF cells were transfected with p53, His-SUMO1, and Flag-tagged PML or each of the PML mutants. Cell lysates were analyzed by western blots. (d) In vitro SUMOylation of His-p53 (top) and Flag-Daxx (middle) by PML and PML point mutants. The relative levels of PML proteins are shown in the bottom. His-p53 was purified from bacteria and Flag-Daxx was purified from 293T cells.

Figure 4. Other TRIM proteins promote Mdm2 SUMOylation in vivo and in vitro

(a and b) 293T cells were transfected with His-SUMO1, HA-Mdm2, and EGFP unless indicated otherwise, together with the TRIM expression constructs or the corresponding vector controls (V). Proteins conjugated to His-SUMO1 and WCL were analyzed by western blot. (c) Summary of the effect of TRIM proteins on Mdm2 SUMOylation. The results are shown in (A) and (B), as well as in Figs. S3.

Figure 5. TRIM27 is associated with ubc9, p53 and Mdm2

(a) 293T cells were co-transfected with Flag-TRIM27 and/or HA-Ubc9. Cell lysates were incubated with anti-Flag M2 agarose. Immunoprecipitated proteins (IP) and whole cell lysates were analyzed by western blot. (b) 293T cells were co-transfected with Flag-TRIM27 and either HA-Mdm2 (left) or HA-p53 (right). Protein interactions were examined as in (a). *, a cleaved product of p53. (c) TRIM27 directly binds to p53. GST, GST-p53, and GST-Mdm2 immobilized on glutathione beads were incubated with Flag-TRIM27 purified from 293T cells. Top: The bound proteins and 10% of the input were analyzed by Western blotting with anti-Flag antibody. Bottom: GST proteins were analyzed by Coomassie staining. GST, full-length GST-p53 and GST-Mdm2 proteins are labeled by asterisks. The sizes of the molecular weight standards in the left lane are labeled (in kDa).

Figure 6. TRIM27 stabilizes Mdm2 through SUMOylation

(a) Pml^−/− MEF cells were transfected with His-SUMO1, HA-Mdm2, TRIM27, PML or PIASy as indicated. His-SUMO1 conjugates and WCL were analyzed by western blot. (b) Bacterially expressed p53 was incubated with SUMO E1, His-SUMO1/His-SUMO2, in the presence or absence of Ubc9 and Flag-TRIM27. Reaction mixes were analyzed by western blot using anti-p53 antibody. (c) In vitro Mdm2 SUMOylation reactions were performed with SUMO E1, ATP, His-SUMO1/SUMO2, in vitro-translated Mdm2, in the presence or absence of Ubc9 and Flag-TRIM27 purified from 293T cells. Proteins conjugated to His-SUMO were captured by Ni²⁺-NTA beads pull down and analyzed by western blot with anti-Mdm2 antibody. (d) His-p53 was incubated with ATP and Ubc9, E1, and bacterial recombinant GST or GST-TRIM27 as indicated. The reaction mixes were analyzed by western blot using anti-p53 antibody. (e and f) HA-Mdm2 were transfected into HeLa cells together with Flag-TRIM27 and/or His-SUMO1. Cells were treated with cycloheximide for the indicated durations. (e) Cell lysates were analyzed for the levels of Mdm2. (f) The same samples were analyzed by western blot with different exposure times for each blot to achieve comparable band intensity at time 0. (g) Ubc9 down-regulation reduces Mdm2 level. U2OS cells treated with Ubc9 siRNA control or control siRNA were transfected with HA-Mdm2, Flag-TRIM27, His-SUMO1 as indicated.