Oncogene (2011) 30, 1108–1116 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE SUMO E3 ligase activity of TRIM proteins 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 pre- viously 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).