SUMO E3 Ligase Activity of TRIM Proteins

Home , SUMO protein, Tripartite motif family, UBA2

Y Chu1 and X Yang

Department of Cancer Biology and Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA, USA

SUMOylation governs numerous cellular processes and enzyme E1 (a heterodimer of Aos1/SAE1 and Uba2/ is essential to most eukaryotic life. Despite increasing SAE2), the SUMO-conjugating enzyme E2 (Ubc9) and recognition of the importance of this process, an one of the SUMO ligases or E3s. SUMO E3s confer extremely limited number of small ubiquitin-like modifier SUMOylation specificity and efficiency. However, in (SUMO) protein ligases (E3s) have been identified. Here contrast to the over 600 known ubiquitin E3s (Deshaies we show that at least some members of the functionally and Joazeiro, 2009), only a handful of SUMO E3s diverse tripartite motif (TRIM) superfamily are SUMO have been reported. These include protein inhibitor of E3s. These TRIM proteins bind both the SUMO- activated STAT proteins (PIAS proteins) (Hochstrasser, conjugating enzyme Ubc9 and substrates and strongly 2001; Johnson and Gupta, 2001; Kahyo et al., 2001), the enhance transfer of SUMOs from Ubc9 to these nuclear pore protein RanBP2 (Pichler et al., 2002) and substrates. Among the substrates of TRIM SUMO E3s the polycomb group protein Pc2 (Kagey et al., 2003). are the tumor suppressor p53 and its principal antagonist With the identification of numerous SUMOylation targets Mdm2. The E3 activity depends on the TRIM motif, (Golebiowski et al., 2009), it remains unclear whether a suggesting it to be the first widespread SUMO E3 motif. large number of SUMO E3s also exist and thus, how the Given the large number of TRIM proteins, our results specificity and efficiency of SUMOylation are achieved. may greatly expand the identified SUMO E3s. Further- Tripartite motif-containing (TRIM) proteins form more, TRIM E3 activity may be an important contributor a superfamily in metazoans with B20 members in to SUMOylation specificity and the versatile functions of Caenorhabditis elegans and B65 members in humans TRIM proteins. and mice (Ozato et al., 2008). These proteins are defined Oncogene (2011) 30, 1108–1116; doi:10.1038/onc.2010.462; by the TRIM/RBCC motif of a RING domain, one or published online 25 October 2010 two B-box domains (which, like the RING domain, are zinc-binding domains) and a predicted coiled-coil Keywords: TRIM proteins; SUMO E3 ligase; sumoylation; region. These domains are invariably arranged in the PML; Mdm2; p53 stated order and present at the N-terminal region of these proteins followed by a more variable C-terminal region. TRIM proteins have important roles in a wide range of processes including cell growth, tumor suppres- Introduction sion, DNA damage signaling, senescence, apoptosis, stem cell differentiation and immune responses against Covalent conjugation to small ubiquitin-like modifier viral, and particularly human immunodeficiency virus (SUMO) proteins is a common post-translational infection (Salomoni and Pandolfi, 2002; Dellaire and modification that alters various properties of the target Bazett-Jones, 2004; Nisole et al., 2005; Ozato et al., proteins, including stability, activity, cellular localiza- 2008; Schwamborn et al., 2009). For example, the tion and protein-protein interactions (Johnson, 2004; promyelocytic leukemia protein (PML), also known as Hay, 2005; Geiss-Friedlander and Melchior, 2007). TRIM19, a prototypical TRIM protein, is involved in a Mammalian cells expresses three SUMO proteins chromosomal translocation associated with the vast (SUMO 1–3). SUMO2 and SUMO3 are nearly identical majority of acute promyelocytic leukemia. PML is the in their sequence and function and share about B50% eponymous and main structural component of the PML sequence identity with SUMO1. Similar to the ubiqui- nuclear bodies, whose mechanism of action still remains tination pathway, the SUMO pathway consists of three elusive (Borden, 2002; Bernardi and Pandolfi, 2007). enzymatic steps performed by the SUMO-activating Likewise, TRIM27 (also known as Ret finger protein or RFP), acquires oncogenic activity when it is fused to the Ret receptor tyrosine kinase (Takahashi et al., 1985). Correspondence: Dr X Yang, Department of Cancer Biology and Abramson Family Cancer Research Institute, University of Pennsylvania Other examples include the potent anti-human immu- School of Medicine, 610 BRB II/III, 421 Curie Boulevard, Philadelphia, nodeficiency virus infection mediated by rhesus monkey PA 19096, USA. TRIM5-a (Stremlau et al., 2004), suppression of stem E-mail: [email protected] cell differentiation by TRIM32 (Schwamborn et al., 1Current address: Department of Pediatrics, Columbia University, New York, NY, USA. 2009) and the silencing of viral replication in stem cells Received 14 July 2010; revised 23 August 2010; accepted 25 August 2010; by TRIM28 (Wolf and Goff, 2007). However, despite an published online 25 October 2010 increasing awareness of TRIM proteins’ important SUMO E3 ligase activity Y Chu and X Yang 1109 cellular functions, the biochemical basis for these may be a SUMO E3. We first examined whether PML functions is poorly understood. Certain TRIM proteins stimulates substrate SUMOylation in mammalian cells. exhibit ubiquitin E3 activity, which is attributed to The tumor suppressor p53 is a known SUMOylation the RING domain (Meroni and Diez-Roux, 2005). The substrate (Rodriguez et al., 1999). We expressed p53 and properties of the other motifs with the characteristic the glutathione-S-transferase (GST) fusion of SUMO1 TRIM region remain unknown. In the current study, in PmlÀ/À MEF cells in the presence or absence of PML we show that TRIM proteins are a new class of isoform IV (called PML in this study). This isoform was SUMO E3s. Unlike the other SUMO E3s, the activity of chosen because it can directly bind to p53 (Rodriguez TRIM proteins requires intact RING and B-box et al., 1999). In the presence but not absence of PML, domains. Our results provide a biochemical framework a slower migrating form of p53 was detected with a for understanding SUMOylation and the function of size expected for its conjugation to GST-SUMO1 TRIM proteins. (Figure 1a). In a similar experiment where Flag-tagged SUMO1 was used, p53 conjugation to Flag-SUMO1 was also strongly increased by the coexpressed PML Results (Figure 1a). Conjugation occurred mainly at a previously determined p53 SUMOylation site, Lys386 PML stimulates substrate SUMOylation in mammalian (Rodriguez et al., 1999), as change of this site to Arg cells abolished the SUMO conjugation (Figure 1a). PML is heavily modified by SUMO, and overexpression To confirm that PML stimulates SUMOylation of PML in yeast cells enhanced overall SUMOylation of p53, we expressed p53 and 6xHis-tagged SUMO1 (Kamitani et al., 1998; Muller et al., 2000; Quimby et al., (His-SUMO1), alone or together with PML, in the 2006). These observations led us to reason that PML p53-deficient human lung cancer H1299 cells. The

Flag-p53 WT KR Flag-PML -+-+-+-+ Flag-PML(VI) ----+++ GST-SUMO1 ++------Flag-PML(IV) - +++++- - Flag-SUMO1 --++--++ p53-GST His-SUMO1 - ++-++ -SUMO1 75 p53- 100 SUMO1 p53-Flag 75 p53 -SUMO1 50 p53 50 250 PML- SUMO1 250 PML- 150 100 PML(IV) 150 SUMO1 75 PML(VI) 100 PML GFP GFP 123456 1 2 3 4 5 6 7 8

HA-p53 + ++ - ++ WCL Ni beads Flag-PML - ++++ + + HA-PML - + ++ + His-SUMO1 + +++ - - -++ His-SUMO1 +++- Flag-SUMO1 -----+ +++ Flag-Mdm2 ++++ +++ p53-His 75 -SUMO1 150 Mdm2- 150 SUMO1

Ni beads 50 100 100 p53- 75 Mdm2 75 SUMO1 75 p53 50 250 PML- 250 PML- 150 SUMO1 150

WCL SUMO1 100 100 PML PML 100 75 75 567 Actin GFP GFP 123456 1234 Figure 1 PML promotes SUMOylation of p53 and Mdm2 in vivo.(a) PmlÀ/À 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 þ -NTA beads (Ni beads). The bead-bound proteins and WCL were analyzed by western blots. (c) H1299 cells were transfected with PML isoform IV or VI, p53, SUMO1 and GFP as indicated. WCL were analyzed by western blot. (d) PmlÀ/À cells were transfected with HA-PML, Flag-Mdm2 and His-SUMO1 as indicated. Cell lysates were incubated by Ni2 þ -NTA beads. WCL (left) and the bead-bound proteins (right) were analyzed by western blots.

Oncogene SUMO E3 ligase activity Y Chu and X Yang 1110 His-SUMO1-conjugated p53 protein was then captured SUMO1 SUMO2 SUMO1 by Ni-NTA beads (nickel-charged affinity resins) under PML +-+ + + - PML +-+ denaturing conditions and analyzed by western blots. Ubc9 - + + - + + Ubc9 -++ 75 150 PML enhanced conjugation of p53 to His-SUMO1 in a 100 p53- SUMO SUMO1 75 dose-dependent manner (Figure 1b, top panel). The -Mdm2 specificity of the Ni-NTA capture was verified by the 100 p53 lack of p53 binding to the beads when His-SUMO1 was 50 50 123 replaced by Flag-SUMO1 (Figure 1b). Furthermore, a 123 456 shorter PML isoform (isoform VI, Figure 3a) that cannot interact with p53, failed to stimulate p53 SUMOylation SUMO1 SUMO2 GST -+- -+- ---+ (Figure 1c). In these experiments, PML was also GST-PML + + - + + - + + ++ - conjugated to SUMO1, as expected (Figures 1a–d). Ubc9 +++ -++ -+++ To examine the generality of PML’s ability to E1 - + + + + + +++ + 75 p53- enhance SUMOylation in vivo, we tested two other SUMO SUMOylation substrates: the ubiquitin ligase Mdm2 p53 and the transcriptional factor c-Jun (Muller et al., 2000), 50 both of which interact with PML (Bernardi et al., 1 2 3 45678910 2004; Salomoni et al., 2005). PML strongly increased Figure 2 Purified PML promotes SUMOylation of p53 and Mdm2 in vitro.(a) His-p53 purified from bacteria was incubated conjugation of both SUMO1 and SUMO2 to Mdm2 with SUMO E1 (SAE1/SAE2), ATP and either SUMO1 or (Figures 1d and Supplementary Figure 1). Likewise, SUMO2, in the presence or absence of Ubc9 and PML. The PML promoted conjugation of SUMO1 to c-Jun reaction mixes were analyzed by western blot using anti-p53 (Supplementary Figures 1b, c). In contrast, PML did antibody. (b) In vitro SUMOylation of Mdm2 was performed with not enhance modification of IkBa, a SUMOylation SUMO E1, His-SUMO1 and ATP, in the presence or absence of Ubc9 and PML. Proteins conjugated to His-SUMO1 were purified substrate that resides mainly in the cytosol (Supplemen- by Ni-NTA beads and analyzed by western blot using anti-Mdm2 tary Figure 1c). Therefore, PML enhances SUMOyla- antibody. (c) His-p53 was incubated with ATP and the indicated tion of multiple nuclear proteins. combinations of E1, Ubc9 and bacterial recombinant GST-PML or GST. The reaction mixes were analyzed as in (a).

SUMO E3 activity of PML and its structural determinants also showed reduced SUMO E3 activity in vitro towards To determine whether PML itself possesses SUMO E3 both p53 and Daxx, another SUMOylation substrate activity, we performed in vitro SUMOylation assays (Figure 3d). Therefore, the RING and B-box domains using purified recombinant proteins. Flag-PML protein are likely required for the E3 activity of PML. was expressed in 293T cells and His-p53 in bacteria, both of which were affinity-purified (Supplementary Figures 2a and b). When incubated in a SUMOylation SUMO E3 activity of other TRIM proteins reaction mix, the addition of Flag-PML strongly To determine whether SUMO E3 activity is a shared enhanced His-p53 modification by both SUMO1 and feature of TRIM proteins, we screened a total of 14 SUMO2 (Figure 2a). Flag-PML also enhanced Mdm2 other TRIM proteins from various subgroups that are modification by SUMO1 (Figure 2b). To rule out the distinguished by their C-terminal regions (Ozato et al., possibility that the SUMO E3 activity was originated 2008). SUMOylation of Mdm2 was noticeably enhanced from trace amounts of contaminating protein from 293T by half of these TRIM proteins representing all the cells, a GST fusion of PML was expressed in bacteria, major subgroups (Figures 4a and b, and Supplementary which lack the SUMOylation system, and purified with Figures 3a and b). Of note, a few TRIM family glutathione beads (Supplementary Figure 2c). GST- proteins—including TRIM27 and TRIM32—exhibited PML strongly enhanced conjugation of p53 to SUMO1 much higher activity in stimulating Mdm2 SUMOyla- and SUMO2. This activity depended on both SUMO E1 tion compared with PML. and Ubc9 (Figure 2c). To investigate the structural determinant of PMLs’ TRIM27 is associated with Ubc9, p53 and Mdm2 SUMO E3 activity, we generated PML deletion mutants To verify the SUMO E3 activity of these TRIM that contain either the N-terminal or the C-terminal proteins, we chose TRIM27 for further analysis as it region (Figure 3a). The N-terminal region, but not the strongly stimulated Mdm2 SUMOylation in vivo.An C-terminal region, stimulated Mdm2 SUMOylation in E3, in addition to promoting the transfer of ubiquitin or vivo (Figure 3b). To assess the role of individual zinc- a ubiquitin-like protein from the E2 to the substrate, is binding domains within the characteristic N-terminal characterized by its binding to both the E2 and TRIM region, we mutated two key zinc-chelating substrates, either directly or indirectly (Hershko and residues (Cys or His) within each domain to either Ser Ciechanover, 1998; Hochstrasser, 2001). PML fulfills or Ala (Figure 3a). Mutations in each of these domains this binding requirement as it interacts with Ubc9 as well (Rm,B1m and B2m), as well as two combined mutations as with p53, Mdm2 and c-Jun (Duprez et al., 1999; (M4 and M6), reduced PML’s SUMO E3 activity Guo et al., 2000; Wei et al., 2003). To examine towards p53 in vivo (Figure 3c). The purified mutants whether TRIM27 interacts with Ubc9, we expressed

Oncogene SUMO E3 ligase activity Y Chu and X Yang 1111

1 RCCB1 B2 630 Flag-PML -WTWTWT N C M6 PML His-SUMO1 ++ -++++ PML(VI) 560 Flag-Mdm2 +++++-+ 150 PML-N Mdm2- Ni

PML-C beads SUMO1 100 m PML-R ** 150 Mdm2- PML-B1m ** SUMO1 100 Mdm2 PML-B2m ** 75 PML-M4 ** ** 250 PML- PML-M6 **** ** 150 SUMO1 100 PML

m m 75 C

PML WT WTWT R M4 M6 - B2 WCL ATP +- ++++++ 50 UbcH9 -+++++++ p53- N SUMO1 37

p53 Actin Daxx- GFP SUMO1 172 3 4 5 6 Daxx

1 2345678 PML - WTRm B1m M4B2m M6

WT B2Rm M4 M6 m p53- 250 75 SUMO1 150 100 p53 75 PML 50 250 PML- 150 SUMO1 50 Cleaved 100 PML PML 37 GFP 1 2 3 4 5 6 7 8 9 101112 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), Rm (Cys57-to-Ser and Cys60-to-Ser, C57A/C60A), B1m (C129A/C132A), B2m (C189A/H194A), M4 (combination of Rm and B1m) and M6 (combination of Rm,B1m and B2m). Asterisks indicate the positions of the point mutations. (b) PML mutations affect Mdm2 SUMOylation. PmlÀ/À 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 þ -NTA beads and WCL were analyzed by western blot. (c) PmlÀ/À 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.

Flag-TRIM27 and HA-Ubc9 in 293T cells. A coimmu- SUMOylation in PmlÀ/À MEFs, and their combination noprecipitation assay showed their specific interaction generated an additive effect (Figure 6a). Thus, TRIM27 (Figure 5a). A similar assay also revealed the interaction acts on Mdm2 independently of PML. We also compared of TRIM27 with Mdm2 and p53 (Figure 5b). The the E3 activities of TRIM27 and PML with that of a well- interaction of TRIM27 with p53, but not with Mdm2, established SUMO E3, PIASy. The activities of TRIM27 was likely direct as shown by an in vitro pull-down assay and PML appeared to be weaker than that of PIASy with recombinant proteins (Figure 5c). Collectively, (Figure 6a). these results indicate that TRIM27 binds to both Ubc9 To confirm that TRIM27 functions as a SUMO E3 and substrates. ligase toward Mdm2, we performed in vitro SUMOyla- tion assays. TRIM27 purified from 293T cells (Supple- mentary Figure 2b) enhanced conjugation of both TRIM27 enhances Mdm2 stability through SUMO1 and SUMO2 to Mdm2 and p53 in an Ubc9- SUMOylation dependent manner (Figures 6b and c). Furthermore, TRIM27 directly interacts with PML and partially GST-TRIM27 purified from bacteria, but not GST resides in the PML nuclear bodies (Cao et al., 1998). alone (Supplementary Figure 2c), enhanced conjugation To test whether PML influences the ability of TRIM27 of both SUMO1 and SUMO2 to p53. This activity relied to stimulate Mdm2 SUMOylation, we expressed TRIM27 on SUMO E1 and Ubc9 (Figure 6d). and PML individually or in combination, together with SUMOylation enhances Mdm2 stability by inhibiting Mdm2. TRIM27 and PML alone enhanced Mdm2 its ubiquitination (Lee et al., 2006). When coexpressed

Oncogene SUMO E3 ligase activity Y Chu and X Yang 1112

V PML Trim5 Trim27 Trim32 + ++ + ++ ++ + ++ +++++ + subfamily gene Mdm2 SUMOylation His-SUMO1 +++ + + - + + + + + 150 C-I TRIM1 ++ Mdm2- TRIM18 - 100 SUMO1 Ni beads TRIM9 -/+ 150 TRIM36 ++ 100 Mdm2

WCL TRIM46 - Actin α GFP C-IV TRIM5 * - TRIM11 - TRIM22 ++ V V Trim46 Trim36 Trim28 Trim9 Trim27 TRIM25 - +++ ++ + ++ + ++ + ++ +++ TRIM27 ++++ 150 Mdm2- TRIM39 + SUMO1

Ni beads 100 C-V TRIM19 + 100 (PML) Mdm2 C-Vl TRIM24 -

WCL Actin TRIM28 + GFP C-Vll TRIM32 ++

Figure 4 Other TRIM proteins promote Mdm2 SUMOylation in vivo and in vitro.(a, 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, b), as well as in Supplemental Figure 3c.

with Mdm2 in mammalian cells, the TRIM proteins that the TRIM motif and their biochemical activity has increased Mdm2 SUMOylation almost always elevated remained elusive. To our knowledge, the present study the steady-state levels of Mdm2 (Figures 4a and b, and provides the first activity linked to these domains. The Supplementary Figures 3a and b). A cycloheximide SUMO E3 motif of TRIM proteins is thus, distinct from chase experiment showed that the half-life of Mdm2 was the other known SUMO E3 motifs including those markedly extended by TRIM27 (Figures 6e and f). present in RanBP2 and Pc2, although the E3 activity of Mdm2 was also stabilized by expression of exogenous the five mammalian protein inhibitor of activated STAT SUMO1 alone and more drastically by the combined proteins and their yeast homologs, Siz1 and Siz2, expression of SUMO1 and TRIM27 (Figures 6e and f). is associated with a different RING-like domain, This effect of TRIM27 was diminished when Ubc9 was SP-RING. However, like the other known E3s, TRIM knocked down by small interfering RNA (siRNA) proteins stimulate the conjugation of both SUMO1 and (Figure 6g). These results suggest that TRIM27 stabi- SUMO2/3 to targets. Selective attachment of different lizes Mdm2 through SUMOylation. SUMO variants to substrates in vivo by TRIM proteins may be in part controlled by isopeptidases as suggested by a recent study for the SUMOylation of RanGAP1 (Zhu et al., 2009). Discussion The SUMO E3 activity appears to be prevalent among TRIM proteins. A screen of 14 TRIM proteins The present study suggests that TRIM proteins are a from various subgroups on a single substrate (Mdm2) new class of SUMO E3. This finding may expand the suggested that more than half of them have SUMO E3 existing number of SUMO E3s many folds and offer a activity. This is likely an underestimate of the actual basis for understanding the regulation of SUMOylation. number of SUMO E3s within the TRIM family as other Furthermore, SUMO E3 activity may also explain the TRIM proteins may have different substrates. A SUMO diversity of functional roles ascribed to TRIM proteins. E3 is expected to bind to both Ubc9 and its substrates. The SUMO E3 activity of TRIM proteins relies on PML binds to Ubc9 by its RING domain and to its the RING domain, extending the well-described ubiqui- substrates p53 and Mdm2 by its C-terminal region. tin E3 role of this domain. It also requires intact B1- and Other TRIM proteins may act similarly. The conse- B2-boxes. These two zinc-binding domains fold into quence for SUMOylation varies among different target similar ternary structures to the RING domain, proteins. The effect of SUMOylation on p53 function is suggesting that all three domains share an evolutionarily still a matter of debate (Hoeller et al., 2006). Although ancestral motif. B-boxes almost always exist as part of Mdm2 SUMOylation has been proposed to enhance

Oncogene SUMO E3 ligase activity Y Chu and X Yang 1113 could be a TRIM protein. Identification of endogenous Flag-TRIM27+ - + SUMO E3 for Mdm2 should help define the functional HA-Ubc9 - ++ consequence of Mdm2 SUMOylation. Ubc9 The TRIM family is notable not only because of its IP TRIM27 large size but also because of their roles in a wide range of Ubc9 processes, including those that protect against cancer, viral TRIM27 WCL infection and neurological disorders. We propose that, the Actin SUMO E3 activity is a unifying biochemical mechanism 1 23 for these functions. For example, TRIM proteins are often identified as part of subcellular proteinaceous bodies (Reymond et al., 2001). The best-studied bodies, the PML HA-Mdm2 - ++ HA-p53 - ++ nuclear bodies, contain a large number of stable and Flag-TRIM27 + -+ Flag-TRIM27 + -+ transient components and the function of these transient components is often altered when they flux through the Mdm2 p53 IP IP bodies. The function of these proteins may be altered TRIM27 TRIM27 either transiently or permanently by PML-mediated p53 SUMOylation. To date, the function of the majority of Mdm2 TRIM proteins still remains elusive, but the identification WCL * WCL TRIM27 TRIM27 of TRIM proteins as SUMO E3s should be instrumental Actin Actin in understanding these proteins. 1 2 3 4 5 6 Some TRIM proteins are also ubiquitin E3 ligases (Meroni and Diez-Roux, 2005; Gillot et al., 2009). Consistent with a recent report on the ubiquitin E3 activity of TRIM27 (Meroni and Diez-Roux, 2005; Gillot et al., 2009), we found that TRIM27 can also promote p53 ubiquitination (Supplementary Figure 3c). Thus, a single

GST GST-p53 GST-Mdm2 Input TRIM protein can have dual E3 activities. A continuum TRIM27 may exist among TRIM proteins in the relative strength of the ubiquitin and SUMO E3 activity; some TRIM 250 proteins may modulate their target protein primarily 150 through ubiquitination while others through SUMOyla- * 100 tion. A TRIM protein may also attach ubiquitin and SUMO onto the same target simultaneously or sequen- 75 * tially to achieve versatile outcomes. It is now of great interest to determine the regulation of the TRIM proteins’ 50 SUMO E3 activity, its physiological targets and its dynamic interplay with the ubiquitin E3 activity. 37

25 * Materials and methods M41 2 3 Figure 5 TRIM27 is associated with ubc9, p53 and Mdm2. Reagents (a) 293T cells were cotransfected with Flag-TRIM27 and/or HA- Antibodies against PML (PG-M3, sc-966, H-238 and sc-5621), Ubc9. Cell lysates were incubated with anti-Flag M2 agarose. p53 (DO-1, sc-126), Ubc9 (sc-10759) and Mdm2 (SMP14, Immunoprecipitated proteins (IP) and whole-cell lysates were sc-965), and horseradish peroxidase-conjugated anti-rabbit, anti- analyzed by western blot. (b) 293T cells were cotransfected with mouse and anti-goat IgGs were purchased from Santa Cruz Flag-TRIM27 and either HA-Mdm2 (left) or HA-p53 (right). Biotechnology (Santa Cruz, CA, USA). Antibodies against p53 Protein interactions were examined as in (a). *a cleaved product of (Ab-6) and Mdm2 (Ab-1) were purchased from EMD Chemicals p53. (c) TRIM27 directly binds to p53. GST, GST-p53 and GST- (Gibbstown, NJ, USA), TRIM27 antibody (18791) from Mdm2 immobilized on glutathione beads were incubated with Flag-TRIM27 puriﬁed from 293T cells. Top: The bound proteins Immuno-Biological Lab (Minneapolis, MN, USA), and Flag and 10% of the input were analyzed by western blotting with anti- peptides and anti-Flag antibody (M2) from Sigma (St Louis, Flag antibody. Bottom: GST proteins were analyzed by Coomassie MO, USA). Glutathione Sepharose 4B (17-0756-01) (GE staining. GST, full-length GST-p53 and GST-Mdm2 proteins are healthcare, Waukesha, WI, USA); SUMO E1, Ubc9, SUMO1, labeled by asterisks. The sizes of the molecular weight standards in SUMO2 and ATP (Boston Biochem, Boston, MA, USA) and the left lane are labeled (in kDa). MG132 (BML-PI102) (Enzo life science, Plymouth Meeting, PA, USA) were purchased from the indicated sources.

Plasmids Mdm2 stability (Lee et al., 2006), it is also stimulated by Plasmids for expressing PML (isoform IV), TRIM27, the tumor suppressor Arf (Tago et al., 2005), which TRIM32, TRIM5d, Mdm2, c-Jun, IkBa, SUMO1 and inhibits the function of Mdm2 (Sherr, 2006). The SUMO2 in mammalian cells and/or in the in vitro translation SUMO E3 for Mdm2 has not been identiﬁed, but it system were constructed in pRK5 with an N-terminally fused

Oncogene SUMO E3 ligase activity Y Chu and X Yang 1114 TRIM27 - + +-+-- SUMO1 SUMO2 SUMO1 SUMO2 PML4 ---++-+ TRIM27 +-+ +-+ TRIM27 +-++-+++-++ PIASy -----++ Ubc9- + + - + + SUMO1 ++-++++ Ubc9 - + + -++-++ 75 100 150 150 SUMO Mdm2- 75 -p53 SUMO Ni SUMO1 -Mdm2 beads 100 p53 100 150 50 50 123 456 123 456789 100 Mdm2 PML- 150 SUMO1 SUMO2 SUMO1 100 WCL GST --+ --+ --+ 75 PML PIASy GST-TRIM27 ++- ++- + + - Ubc9 -++ + ++ - ++ 50 TRIM27 Actin E1 +++ -++ + + + 1 2 3 4 5 6 7 p53-SUMO p53 Flag-TRIM27 -+-+ 123 456 456 His-SUMO1 --++ CHX (min) 04080040800408004080 siRNA Ctrl Ubc9 Mdm2 Flag-TRIM27 -++ ++--- Actin His-SUMO1 - --++++- Mdm2 Flag-TRIM27 -+-+ TRIM27 His-SUMO1 --++ Ubc9 CHX (min) 0 40 80 120 0 40 80 120 0 40 80 120 0 40 80 120 GFP Mdm2 Actin 12345678 Figure 6 TRIM27 stabilizes Mdm2 through SUMOylation. (a) PmlÀ/À MEF cells were transfected with His-SUMO1, HA-Mdm2, TRIM27, PML or protein inhibitor of activated STATy 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 þ -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, 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 downregulation reduces Mdm2 level. U2OS cells treated with Ubc9 siRNA or control siRNA were transfected with HA-Mdm2, Flag-TRIM27 and His-SUMO1 as indicated.

Flag, HA, or 6xHis (His) tag, or GST as indicated. Other 10 mM b-mercaptoethanol) and C (same as B except pH ¼ 6.3). TRIM plasmids were kindly provided by Drs V Yu, A-M Beads with bound proteins were then boiled in SDS sample Herr, F Rauscher III and TC Cox. TRIM25 was purchased buffer, the proteins were fractioned by SDS–PAGE and were from Addgene (Cambridge, MA, USA). For expressing analyzed by western blot for the presence of conjugated proteins in Escherichia coli, full-length PML and TRIM27 Mdm2, p53 or PML. were fused to GST in pGEX-1ZT, a derivative of pGEX-1lT. p53 was fused to the 6xHis tag at the C-terminus and the Flag Protein generation and purification tag at the N-terminus in pET28a. Point mutations were made Flag-tagged PML, its mutants and TRIM27 were expressed in by overlap PCR. All plasmids generated for this study were PmlÀ/À MEFs or 293T cells through transient transfection and confirmed by sequencing. purified by anti-Flag M2 beads as described (Tang et al., 2004, 2006). Whole-cell lysates were made in IP-lysis buffer (50 mM In vivo SUMOylation assays Tris–HCl at pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.5% In vivo SUMOylation assays were performed as described NP-40, 1 mM phenylmethanesulfonylfluoride (PMSF), 1 mM previously with minor changes (Chen and Chen, 2003). Cells dithiothreitol (DTT) and EDTA-free ‘complete’ protease cultured in 6-cm plates were transfected with His-SUMO1 inhibitors). The lysates were immunoprecipitated with the or His-SUMO2 expression plasmids, MDM2, GFP and TRIM anti-Flag M2 beads for 4 h to overnight. After extensive wash, protein expression plasmids. At 24 h after transfection, cells the proteins were eluted from beads with 100 mg/ml 3xFlag were treated with MG132 (20 nM) for 4 h. Cells from each plate peptide in elution buffer (50 mM Tris–HCl at pH 7.5, 150 mM were collected into two aliquots. One aliquot was lysed in lysis NaCl, 5 mM DTT, 25 mM ZnCl2 and 10% glycerol). buffer and analyzed by western blot to examine the expression GST-fusion proteins and His-p53/pET28a were induced and of transfected proteins. The second aliquot was lysed in buffer purified in E. coli BL21(DE3). E. coli BL21(DE3) expressing A(6M guanidinium-HCl, 0.1 M Na2HPO4/NaH2PO4,10mM the appropriate GST-fusion proteins was cultured in Terrific Tris–Cl pH 8.0, 5 mM imidazole and 10 mM b-mercaptoetha- Broth medium with 50 mM ZnCl2. The proteins were induced nol) and incubated with Ni2 þ -NTA beads (Qiagen, Valencia, for 4 h with 0.2 mM isopropyl-b-D-thiogalactoside, followed CA, USA) for 4 h at room temperature or overnight at 4 1C. by centrifugation and re-suspended in lysis buffer (50 mM The beads were washed sequentially with buffers A, B (8 M Tris–HCl at pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM urea, 0.1 M Na2PO4/NaH2PO4,10mM Tris–HCl pH 8.0 and PMSF, 5 mM DTT, 25 mM ZnCl2 and EDTA-free ‘complete’

Oncogene SUMO E3 ligase activity Y Chu and X Yang 1115 protease inhibitors). Cells were sonicated and then centrifuged In vitro GST pull-down assay for 10 min at 4 1C. Extracts were incubated with 30 ml GST pull down was performed as described previously glutathione Sepharose 4B beads (50% slurry) for 4 h at 4 1C. (Townson et al., 2006) with some modifications. GST only After extensive washing, the beads with the GST-fusion or GST-fusion proteins were induced in E. coli BL21. Cells proteins were used for in vitro SUMOylation assay. BL21 were lysed in lysis buffer (50 mM Tris–HCl at pH 7.5, 150 mM (DE3) containing His-p53/pET28a was cultured in Terrific NaCl, 1% Triton X-100, 1 mM EDTA, 0.1% SDS, 1 mM PMSF Broth medium without ZnCl2. His-p53 was captured with and complete protease cocktail) and sonicated. GST of 400 mg TALON His-Tag purification resins (Clontech, Mountain or 1 mg of GST-fusion protein bacterial crude extracts View, CA, USA). After extensive washing, His-p53 was was incubated with 30 ml glutathione Sepharose 4B beads eluted from the TALON beads by 1x phosphate-buffered (50% slurry). Flag-TRIM27 was purified from 293T cells as saline containing 300 mM imidazole. His-p53 was then passed described above. For the binding assay, the beads were through a PD-10 desalting column (GE Healthcare) pre- incubated in lysis buffer and Flag-TRIM27 elute was added equilibrated with a buffer containing 20 mM Tris-HCl, pH8.0, and incubated at 4 1C. The beads were washed three times with 150 mM NaCl, 0.1 mM EDTA and 5% glycerol. lysis buffer. Bound proteins were eluted in SDS sample buffer, resolved by SDS–PAGE, and analyzed by western blot. In vitro translation In vitro-translated proteins were made using TNT Coupled Protein half-life analysis Transcription/Translation System with S6 RNA polymerase HeLa cells transfected with appropriate vectors were cultured (Promega, Madison, WI, USA). for 18 h. Cells were divided into four fractions and further cultured on 60-mm dishes overnight. The cells were treated with 50 mg/ml cycloheximide and collected at different time In vitro SUMOylation assays points. Cells were lysed in RIPA buffer (50 mM Tris–HCl at pH In vitro SUMOylation reactions were performed at 37 1C for 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 0.1% SDS, 1 h in 20 ml volume containing purified His-p53 (B100 ng) 0.1% sodium deoxycholate, 1 mM PMSF and complete or in vitro translated HA-Mdm2 (4 ml), mammalian or bacterial protease cocktail). Proteins were separated by SDS–PAGE cell-expressed PML or TRIM27 protein (5–10 ng), SAE1/ and analyzed by western blots. SAE2 (125 nM), Ubc9 (50 nM) and His-SUMO1 or His-SUMO2 (32 mM). The reaction buffer contained 50 mM Tris–HCl pH 7.5, 2.5 mM Mg2 þ -ATP and 2.5 mM DTT. siRNA treatment Mdm2-containing reaction mixes were incubated with Ni2 þ - Cells were seeded in 6-cm plates and transfected with 80 pmol NTA beads. After washing in urea buffer, the conjugated siRNA per well using Lipofetamine 2000 according to the Mdm2 was detected by anti-Mdm2 antibody (SMP14, Santa manufacturer’s instruction. Ubc9 siRNA was ordered from Cruz Biotechnology). p53-containing mix was directly fractio- Thermo Scientific Dharmacon (Lafayette, CO, USA; Cat# nated by SDS–PAGE (8%) and analyzed by western blot using M-004910-00). Control siRNA was ordered from Qiagen anti-p53 antibody (DO-1). (Cat# 1027281).

Immunofluorescence microscopy In vitro ubiquitination assay In vitro ubiquitination reactions were performed at 37 1C for Cellsculturedoncoverslipswerefixedwith4%paraformaldehyde B for 15 min at room temperature, permeabilized with 0.2% Triton 2 h in 20 ml volume containing purified His-p53 ( 100 ng) or X-100, blocked with 1% bovine serum albumin and incubated bacterial-expressed TRIM27 protein (5–10 ng), E1 (156 nM), Ubc5a (50 nM) and Ubiquitin (147 mM). The reaction buffer with anti-TRIM27, anti-p53, anti-Mdm2 and anti-PML antibodies 2 þ as indicated, followed by Texas-red conjugated anti-Rabbit IgG contained 50 mM Tris–HCl pH 7.5, 2.5 mM Mg -ATP and (Vector Laboratories, Burlingame, CA, USA) and FITC-con- 2.5 mM DTT. p53 ubiquitination was analyzed by western blot jugated anti-mouse IgG antibody (Zymed Laboratories, San using anti-p53 antibody (DO-1). Francisco, CA, USA). The cells were mounted with 40-6- diamidino-2-phenylindole-containing medium (Vector Labora- tories) and the images were acquired with a confocal microscope. Conflict of interest

Immunoprecipitation The authors declare no conflict of interest. Whole-cell lysates were made in lysis buffer (50 mM Tris–HCl at pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 0.5% NP-40, 1 mM PMSF, 1 mM EDTA, 1 mM DTT and EDTA- Acknowledgements free ‘complete’ protease inhibitors (Roche, Indianapolis, IN, USA: 04693132001). The lysates were then immunoprecipi- We thank Dr PP Pandolfi for providing PmlÀ/À MEF cells and tated with the anti-Flag M2 beads for 4 h to overnight. Beads Drs V Yu, A-M Herr, F Rauscher III and TC Cox for TRIM- were washed multiple times and boiled in SDS-containing expressing plasmids. We also thank E Fischer and S Slattery loading buffer. Protein samples were resolved by SDS–PAGE for technical assistance and A Stonestrom for help with and transferred onto polyvinylidene fluoride membrane and manuscript preparation. Supported by NIH (CA088868 and probed with the indicated antibodies. GM060911) to XY.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Oncogene