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Ubiquitination of E3 ubiquitin ligase TRIM5a and its potential role Keiko Yamauchi, Keiji Wada, Kunikazu Tanji, Makoto Tanaka and Tetsu Kamitani Department of Cardiology, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA Host cell barriers to the early phase of immunodefi- ciency virus replication explain the current distribution of these viruses among humans and nonhuman primate species [1,2]. HIV-1, the cause of AIDS in humans, can efficiently enter the cells of Old World monkeys but encounters a block before reverse tran- scription. Recently, this species-specific restriction at the postentry stage was shown to be mediated mainly by TRIM5a, a member of the tripartite motif (TRIM) family [3,4]. However, the precise mechanism of this is still unknown, because the molecular function of TRIM5a has not been defined. TRIM proteins contain RING, B-box and coiled-coil domains [5]. In addition, some TRIM proteins, including TRIM5a and Ro52 (also called TRIM21), possess a B30.2 (SPRY) domain at their C-terminus. Although the domain structure of the TRIM family is known, the functions of most TRIM proteins have not been determined. Recently, however, we defined the function of Ro52, showing that it is an enzyme for the ligation of ubiquitin [6–10]. Ubiquitin, a 76 amino acid polypeptide, is highly conserved in evolution, with only three amino acid dif- ferences between the human and yeast homologs [11]. The C-terminus of ubiquitin contains a conserved Gly residue, which is activated to form a thiol–ester linkage with the Cys residue of the E1 ubiquitin-activating enzyme. Activated ubiquitin is then transferred to the E2 ubiquitin-conjugating enzyme to form another thiol–ester linkage. Subsequently, with the aid of E3 Keywords ligase; Ro52; TRIM5; ubiquitin; YopJ Correspondence T. Kamitani, Department of Cardiology, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Unit 1101, Houston, TX 77030, USA Fax: +1 713 563 0424 Tel: +1 713 563 0413 E-mail: tkamitani@mdanderson.org (Received 7 December 2007, revised 24 January 2008, accepted 30 January 2008) doi:10.1111/j.1742-4658.2008.06313.x HIV-1 efficiently infects susceptible cells and causes AIDS in humans. Although HIV can also enter the cells of Old World monkeys, it encoun- ters a block before reverse transcription. Data have shown that this species-specific restriction is mediated by tripartite motif (TRIM)5a, whose molecular function is still undefined. Here, we show that TRIM5a func- tions as a RING-finger-type E3 ubiquitin ligase both in vitro and in vivo and ubiquitinates itself in cooperation with the E2 ubiquitin-conjugating enzyme UbcH5B. In addition to the self-ubiquitination, we show that TRIM5a is ubiquitinated by another E3 ubiquitin ligase, Ro52, and deubiquitinated by YopJ, one of the pathogenic proteins derived from Yersinia species. Thus, the ubiquitination of TRIM5a is catalyzed by itself and Ro52 and downregulated by YopJ. Unexpectedly, although TRIM5a is ubiquitinated, our results have revealed that the proteasome inhibitors MG115 and MG132 do not stabilize it in HeLa cells, suggesting that the ubiquitination of TRIM5a does not lead to proteasomal degradation. Importantly, TRIM5a is clearly conjugated by a single ubiquitin molecule (monoubiquitination). Our monoubiquitin-fusion assay suggests that mono- ubiquitination is a signal for TRIM5a to translocate from cytoplasmic bodies to the cytoplasm. Abbreviations DAPI, 4¢,6-diamidino-2-phenylindole; EGFP, enhanced green fluorescent protein; HA, hemagglutinin; HEK, human embryonic kidney; HIF, hypoxia-inducible factor; MBP, maltose-binding protein; RH, RGS-poly-His; TRIM, tripartite motif; UbG, truncated ubiquitin. 1540 FEBS Journal 275 (2008) 1540–1555 ª 2008 The Authors Journal compilation ª 2008 FEBS ubiquitin ligase, ubiquitin becomes covalently attached to the Lys residues of target proteins through the formation of isopeptide bonds [11]. The internal Lys residue at position 48 of ubiquitin can also form an isopeptide bond with the C-terminal Gly residue of another ubiquitin molecule to create a polyubiquitin chain in some cases. This chain serves as a pro- teasome-targeting signal [11]. In the proteasome, polyubiquitinated proteins are degraded in an ATP- dependent manner [11]. By targeting polyubiquitinated proteins to the proteasome for degradation, ubiquitina- tion plays a critical role in many biological events [11]. Ubiquitination is negatively regulated by deubiquiti- nating enzymes, which remove ubiquitin from target proteins [12]. As described above, Ro52 is a RING-finger protein that belongs to a TRIM family [4]. Previous results from several laboratories indicated that the RING- finger proteins recruit E2 ubiquitin-conjugating enzymes and act as E3 ubiquitin ligases [13,14]. Recently, we showed that Ro52 functions as an E3 ubiquitin ligase in a RING-finger-dependent manner as well as other RING-type E3 ligases and that Ro52 is ubiquitinated by itself (self-ubiquitination) through its ligase activity [6–9]. Furthermore, we showed that the self-ubiquitinated Ro52 is selectively deubiquitinated by UnpEL (also known as Usp4) [8,10], which is a deubiquitinating enzyme. Because of the structural sim- ilarity between Ro52 and TRIM5a, we hypothesized that TRIM5a also has E3 ligase activity, which enables it to conjugate ubiquitin to itself (self-ubiquitination) and Ro52 (cross-ubiquitination), and that the ubiquiti- nated TRIM5a is selectively deubiquitinated by UnpEL. Indeed, TRIM5d, an isoform generated by alternative splicing, was previously shown to have E3 ubiquitin ligase activity in vitro [15]. Although TRIM5d lacks the C-terminal B30.2 domain, it possesses other domains found in TRIM5a, suggesting that TRIM5a has E3 ubiquitin ligase activity. In this study, we tested the hypotheses described above to characterize the molecular function of TRIM5a and its regulator. Results TRIM5a and Ro52 are phylogenetically and structurally similar On human chromosome 11p15, the trim5 gene is located with a cluster of other trim genes, including ro52, trim68, trim6, trim34, trim22 and Trim3.Itisof particular interest that trim6, trim34, trim5 and trim22 are assembled at adjacent loci [16] (Fig. 1A). This chromosomal localization suggests that these trim genes were generated by amplification from a single gene on chromosome 11p15. To investigate the respec- tive molecular evolution of these gene products, a phy- logenetic study was performed (Fig. 1B). As expected, TRIM5a, TRIM6, TRIM34, and TRIM22, the genes for which are clustered at the chromosomal loci, are also clustered in the phylogenetic tree. Importantly, A B C Fig. 1. Relationship between Ro52 and TRIM5. (A) Loci of trim genes on human chromosome 11p15. (B) Phylogenetic tree of TRIM family members encoded by genes on human chromo- some 11p15. Amino acid sequences of TRIM family members were aligned using CLUSTAL W. The alignment was then used to build trees in MEGA3.1, using the neighbor-joining method. The scale bar represents evolutionary distance in substitutions ⁄ amino acid residues. (C) Schematic representation of domain structure of human Ro52 and TRIM5a. K. Yamauchi et al. Ubiquitination of TRIM5a and its role FEBS Journal 275 (2008) 1540–1555 ª 2008 The Authors Journal compilation ª 2008 FEBS 1541 the evolutionary distance of TRIM5a is very close to that of Ro52, TRIM6, and TRIM34, suggesting that TRIM proteins such as Ro52, TRIM5a, TRIM6 and TRIM34 have a similar function. Next, we investigated the domain structure of TRIM5a and Ro52. As shown in Fig. 1C, both proteins possess RING-finger and B-box domains in the N-terminal region. In the central region, Ro52 contains two separated coiled-coil domains, whereas TRIM5a contains a fused coiled-coil domain. In the C-terminal region, both proteins con- tain a B30.2 domain. Thus, the domain structure of TRIM5a is almost identical to that of Ro52, implying that the two proteins have similar functions. TRIM5a is ubiquitinated in the presence of UbcH5B in vitro Because TRIM5a was phylogenetically and structurally similar to Ro52, which is a RING-type E3 ubiquitin ligase, we hypothesized that TRIM5a also functions as an E3 enzyme. However, this hypothesis raised the question as to what the substrate of the TRIM5a-med- iated ubiquitination is. Previously, we found that Ro52 acts as an E3 enzyme and ubiquitinates itself (self- ubiquitination) [6–8], suggesting that TRIM5a likewise acts as an E3 enzyme and ubiquitinates itself. We therefore performed an in vitro ubiquitination assay to test this possibility. In the assay, maltose-binding protein (MBP)-fused TRIM5a was expressed in bacteria and purified using amylose resin beads. MBP–TRIM5a immobilized on the beads was then incubated with recombinant E1 enzyme and different recombinant E2 enzymes (UbcH2, UbcH5B, UbcH7, UbcH10, and hCDC34, which were produced in bacteria) in the presence of RGS-poly-His (RH)-tagged ubiquitin. In this in vitro system, MBP–TRIM5a served as both a potential sub- strate and a potential E3 enzyme for its self-ubiquitina- tion. After the incubation, MBP–TRIM5a was solubilized and analyzed by western blotting, using antibodies to RH and to MBP. As shown in Fig. 2A, the incubation of MBP–TRIM5a in the reaction mix- ture containing UbcH2, UbcH7, UbcH10 or hCDC34 did not result in the ubiquitination of MBP–TRIM5a, whereas the incubation of MBP–TRIM5a in the reac- tion mixture containing UbcH5B resulted in both the monoubiquitination and polyubiquitination of MBP–TRIM5a. These results indicate that TRIM5a is ubiquitinated in vitro and that this ubiquitination is catalyzed by UbcH5B but not by other E2 enzymes. Interestingly, Ro52, which is phylogenetically and structurally close to TRIM5a, also catalyzes ubiquiti- nation in cooperation with UbcH5B [7]. TRIM5a functions as an E3 enzyme and ubiquitinates itself in vitro In general, ubiquitin conjugates to the substrate in the presence of E1, E2 and E3 enzymes. These proteins are the minimum ones required for ubiquitination to occur. To confirm whether these proteins are also essential for the ubiquitination shown in Fig. 2A, we performed another in vitro ubiquitination assay (Fig. 2B). As a positive control, amylose resin beads coated with MBP–TRIM5a were incubated in the complete reaction mixture containing RH–ubiquitin, recombinant E1 enzyme, and recombinant UbcH5B (E2 enzyme) (Fig. 2B, lane 5). As a negative control, amylose resin beads alone (i.e. not coated with MBP–TRIM5a) were incubated in the complete reaction mixture (Fig. 2B, lane 1). In the other reactions, amylose resin beads coated with MBP–TRIM5a were incubated in an incomplete reaction mixture lacking one of these com- ponents (Fig. 2B, lanes 2–4). After the incubation, MBP–TRIM5a was solubilized and analyzed by western blotting using antibody to RH and antibody to MBP. As shown in Fig. 2B, incubation of MBP–TRIM5a in the complete reaction mixture resulted in the ubiquitina- tion of MBP–TRIM5a (lane 5), whereas incubation of MBP–TRIM5a in the incomplete reaction mixture lack- ing one component did not lead to the ubiquitination of MBP–TRIM5a (lanes 2–4). These results indicate that ubiquitin, E1 enzyme and UbcH5B (E2 enzyme) are the minimum requirement for the in vitro ubiquitination of TRIM5a. Because the reaction mixtures used in this assay did not contain any E3 enzymes other than TRIM5a, these results also indicate that TRIM5a functions as an E3 enzyme and ubiquitinates itself. In vitro self-ubiquitination of TRIM5a is mediated by its RING-finger domain TRIM5a possesses a RING-consensus sequence (Cys-X 2 -Cys-X 9–39 -Cys-X 1–3 -His-X 2–3 -Cys-X 2 -Cys-X 4–48 - Cys-X 2 -Cys) between amino acids 15 and 58 [3,17] (Fig. 3A). This sequence coordinates two zinc ions in a ‘cross-braced’ fashion [17,18]. Recent results from sev- eral laboratories have indicated that the RING-finger proteins recruit E2 enzymes through their RING domain and act as an E3 enzyme [13]. This E3 activity of RING-finger proteins has been shown to be abol- ished by a mutation of the conserved Cys or His resi- due described above [7,19,20]. To determine whether the E3 activity of TRIM5a is dependent on its RING- finger domain, we substituted Ala for the conserved Cys15 in the RING-finger domain to generate a TRIM5a mutant (C15A) (Fig. 3A). Then, we tested Ubiquitination of TRIM5a and its role K. Yamauchi et al. 1542 FEBS Journal 275 (2008) 1540–1555 ª 2008 The Authors Journal compilation ª 2008 FEBS whether this mutation abolishes the E3 activity of TRIM5a, using an in vitro ubiquitination assay. In the assay, MBP-fused wild-type TRIM5a or its C15A mutant was expressed in bacteria and purified using amylose resin beads. MBP–TRIM5a immobilized on the beads was then incubated with RH–ubiquitin, recombinant E1 enzyme, and recombinant UbcH5B (E2 enzyme). After the incubation, MBP–TRIM5a was solubilized and analyzed by western blotting, using antibody to RH and antibody to MBP. As shown in Fig. 3B, the wild-type TRIM5a ubiquitinated itself (lanes 1 and 3), whereas the C15A mutant did not ubiquitinate itself at all (lanes 2 and 4). These results indicate that the in vitro self-ubiquitination of TRIM5a is dependent on its RING-finger domain. Thus, we confirmed that TRIM5a is a RING-motif-dependent E3 enzyme. TRIM5a is self-ubiquitinated in human embryonic kidney (HEK) 293T cells The E3 activity of TRIM5a was determined by in vitro assays, as described in the preceding sections. There- fore, this raised the question of whether TRIM5a func- tions as an E3 enzyme in human cells. To determine this, we performed an in vivo ubiquitination assay using the wild-type TRIM5a and its RING-finger mutant (C15A). In brief, RH-tagged wild-type TRIM5a or its C15A mutant was expressed with or without hemagglutinin epitope (HA)-tagged ubiquitin in HEK293T cells. The cells were then harvested and lysed under denaturing conditions. Afterwards, TRIM5a–RH (wild-type or C15A) in the lysate was precipitated by TALON beads, solubilized, and ana- lyzed by western blotting, using antibody to HA to detect ubiquitinated TRIM5a–RH, and antibody to RH to detect both nonubiquitinated and ubiquitinated TRIM5a–RH. As shown in Fig. 4, the wild-type TRIM5a was monoubiquitinated and also polyubiqui- Fig. 2. In vitro self-ubiquitination of TRIM5a. (A) UbcH5B-depen- dent self-ubiquitination of TRIM5a. MBP-fused TRIM5a was puri- fied using amylose resin beads and incubated with the reaction mixture containing RH-tagged ubiquitin, recombinant E1 enzyme, and various poly-His-tagged recombinant E2 enzymes (UbcH2, UbcH5B, UbcH7, UbcH10, and hCDC34). After this reaction, MBP– TRIM5a immobilized on the beads was washed to remove the reaction mixture and solubilized in SDS treatment solution. MBP– TRIM5a was then analyzed by western blotting, using antibody to RH to detect ubiquitinated MBP–TRIM5a (upper panel), and anti- body to MBP to detect both nonubiquitinated and ubiquitinated MBP–TRIM5a (lower panel). Molecular size markers are shown on the left in kilodaltons (kDa). (B) Minimum requirements for the in vitro self-ubiquitination of TRIM5a. In the in vitro ubiquitination assay, the complete reaction mixture contained RH–ubiquitin, E1 enzyme, and UbcH5B as an E2 enzyme. To determine the mini- mum requirements for the self-ubiquitination of TRIM5a, MBP– TRIM5a immobilized on amylose resin beads was incubated in the incomplete reaction mixture lacking one of these components (lanes 2–4). As a positive control, MBP–TRIM5a immobilized on amylose resin beads was incubated in the complete mixture (lane 5). As a negative control, amylose resin beads alone without immobilization of MBP–TRIM5a were incubated in the complete mixture (lane 1). After the reaction, the beads were treated in SDS- containing solution to solubilize MBP–TRIM5a. Then, MBP–TRIM5a was analyzed by western blotting, using antibody to RH to detect ubiquitinated MBP–TRIM5a (upper panel), and antibody to MBP to detect both nonubiquitinated and ubiquitinated MBP–TRIM5 a (lower panel). The incomplete reaction mixture shown in lanes 2, 3 and 4 lacked RH–ubiquitin, E1 enzyme, and UbcH5B, respectively. A B K. Yamauchi et al. Ubiquitination of TRIM5a and its role FEBS Journal 275 (2008) 1540–1555 ª 2008 The Authors Journal compilation ª 2008 FEBS 1543 tinated (or multimonoubiquitinated) when overexpres- sed with HA–ubiquitin in HEK293T cells (lanes 3 and 7). In contrast, the ubiquitination of the C15A mutant was extremely weak, even when overexpressed with HA–ubiquitin in HEK293T cells (Fig. 4, lanes 4 and 8). This faint ubiquitination of TRIM5a(C15A) might have been catalyzed by the wild-type TRIM5a or other E3 ubiquitin ligases that are endogenously expressed in HEK293T cells. These results indicate that TRIM5a ubiquitinates itself through the function of its RING- finger domain in HEK293T cells. Ro52 strongly ubiquitinates itself and TRIM5a in HEK293T cells TRIM5a functions as an E3 ubiquitin ligase, because it ubiquitinates itself both in vitro (Figs 2 and 3) and in vivo (Fig. 4), as does Ro52 [7]. Because TRIM5a is structurally similar to Ro52 (Fig. 1C), we wondered whether TRIM5a and Ro52 cross-ubiquitinate (or trans-ubiquitinate) each other in addition to undergo- ing self-ubiquitination. In other words, we wondered whether Ro52 ubiquitinates TRIM5a and whether TRIM5a ubiquitinates Ro52. To test the first possibil- ity, we performed the in vivo ubiquitination assay, using a wild-type Ro52 as an E3 ubiquitin ligase. As a substrate, we used a RING mutant of Ro52 (positive control) or of TRIM5a to avoid the self- ubiquitination. Specifically, RH-tagged Ro52(C16A) or TRIM5a(C15A) was expressed with HA-tagged ubiqu- itin and FLAG-tagged Ro52 (wild-type or its mutant C16A) in HEK293T cells. The cells were then harvested and lysed under denaturing conditions. Ro52(C16A)–RH or TRIM5a(C15A)–RH in the lysate was precipitated with cobalt-coated TALON beads, solubilized in SDS solution, and then analyzed by wes- tern blotting, using antibody to RH to detect both nonubiquitinated and ubiquitinated forms, and anti- body to HA to detect ubiquitinated forms. As shown in Fig. 5A, both Ro52(C16A)–RH (upper panel) and TRIM5a(C15A)–RH (lower panel) were strongly Fig. 4. E3 activity of wild-type TRIM5a and its RING mutant C15A in HEK293T cells. RH-tagged wild-type TRIM5a or its RING mutant C15A was expressed with or without HA-tagged ubiquitin in HEK293T cells by plasmid transfection. Twenty hours after trans- fection, the cells were harvested and lysed under denaturing conditions. TRIM5a–RH (wild-type or C15A) in the lysate was pre- cipitated with cobalt-coated TALON beads and solubilized in 2% SDS solution. The solubilized TRIM5a–RH was then analyzed by western blotting, using antibody to RH to detect both nonubiquiti- nated and ubiquitinated TRIM5a–RH (lanes 1–4), and antibody to HA to detect ubiquitinated TRIM5a–RH (lanes 5–8). AB Fig. 3. E3 activity of wild-type TRIM5a and its RING mutant in vitro. (A) Schematic representation of the RING-finger domain of TRIM5a. The amino acid sequence and structure of the RING-finger domain are shown. Asterisks indicate conserved Cys and His residues in the RING-finger domain. Arrows indicate Cys15, which was replaced by Ala to generate the TRIM5a(C15A). (B) In vitro ubiquitination assay using wild-type TRIM5a and its RING mutant C15A. MBP–TRIM5a (wild-type) or MBP–TRIM5a(C15A) was purified with amylose resin beads from bacterial lysate and incubated with the reaction mixture containing RH–ubiquitin, E1 enzyme, and UbcH5B. After the reaction, MBP–TRIM5a immobilized on the beads was solubilized and analyzed by western blotting, using antibody to MBP to detect both nonubiquitinated and ubi- quitinated MBP–TRIM5a (lanes 1 and 2), and antibody to RH to detect ubiquitinated MBP–TRIM5a (lanes 3 and 4). Ubiquitination of TRIM5a and its role K. Yamauchi et al. 1544 FEBS Journal 275 (2008) 1540–1555 ª 2008 The Authors Journal compilation ª 2008 FEBS monoubiquitinated and polyubiquitinated when coex- pressed with wild-type FLAG–Ro52 in HEK293T cells (lanes 4 and 9). In contrast, the ubiquitination of Ro52(C16A)–RH and TRIM5a(C15A)–RH was extre- mely weak when wild-type FLAG–Ro52 was not coex- pressed in HEK293T cells (Fig. 5A, lanes 3 and 8) and when FLAG-tagged inactive Ro52(C16A) was coex- pressed in HEK293T cells (Fig. 5A, lanes 5 and 10). This faint ubiquitination of Ro52(C16A) and TRIM5a(C15A) might have been catalyzed by the wild-type Ro52, TRIM5a, or other E3 ubiquitin ligases that are endogenously expressed in HEK293T cells. These results indicate that Ro52 ubiquitinates both itself and TRIM5a in HEK293T cells. TRIM5a ubiquitinates itself, but not Ro52, in HEK293T cells Next, we examined whether TRIM5a ubiquitinates Ro52 in HEK293T cells, using a wild-type TRIM5a as an E3 ubiquitin ligase. As a substrate, we used a RING mutant of Ro52 or of TRIM5a (positive con- trol) to avoid self-ubiquitination. Specifically, RH- tagged Ro52(C16A) or TRIM5a(C15A) was expressed with HA–ubiquitin and FLAG–TRIM5a (wild-type or its mutant C15A) in HEK293T cells. The cells were then harvested and lysed under denaturing conditions. Ro52(C16A)–RH or TRIM5a(C15A)–RH in the lysate was precipitated with cobalt-coated TALON beads, solubilized in SDS solution, and then analyzed by wes- tern blotting, using antibody to RH and antibody to HA. As shown in the upper panel of Fig. 5B, the Ro52(C16A)–RH was weakly monoubiquitinated and polyubiquitinated in HEK293T cells when wild-type FLAG–TRIM5a was not coexpressed (lanes 3 and 8) A B C Fig. 5. In vivo assay of self-ubiquitination and cross-ubiquitination between Ro52 and TRIM5a. (A) In vivo ubiquitination by Ro52 E3 ubiquitin ligase. To examine the ubiquitination of RH-tagged Ro52(C16A) and TRIM5a(C15A) by FLAG–Ro52, Ro52(C16A)–RH or TRIM5a(C15A)–RH was expressed with HA–ubiquitin and FLAG– Ro52 (wild-type or C16A) in HEK293T cells by plasmid transfection. Twenty hours after transfection, the cells were harvested and lysed under denaturing conditions. Ro52(C16A)–RH or TRIM5a(C15A)–RH in the lysate was precipitated with TALON beads and solubilized in 2% SDS solution. The solubilized Ro52(C16A)–RH (upper panel) or TRIM5a(C15A)–RH (lower panel) was then analyzed by western blotting, using antibody to RH to detect both nonubiquitinated and ubiquitinated forms (lanes 1–5), and antibody to HA to detect the ubiquitinated form (lanes 6–10). (B) In vivo ubiquitination by TRIM5a E3 ubiquitin ligase. To examine the ubiquitination of RH-tagged Ro52(C16A) or TRIM5a(C15A) by FLAG–TRIM5a, Ro52(C16A)–RH or TRIM5a(C15A)–RH was expressed with HA–ubiquitin and FLAG– TRIM5a (wild-type or C15A) in HEK293T cells by plasmid transfec- tion. Twenty hours after transfection, the cells were harvested and lysed under denaturing conditions. Ro52(C16A)–RH or TRIM5a(C15A)–RH in the lysate was precipitated with cobalt- coated TALON beads and solubilized in 2% SDS solution. The solu- bilized Ro52(C16A)–RH (upper panel) or TRIM5a(C15A)–RH (lower panel) was then analyzed by western blotting, using antibody to RH to detect both nonubiquitinated and ubiquitinated forms (lanes 1–5), and antibody to HA to detect the ubiquitinated form (lanes 6–10). A nonspecific band is indicated by an asterisk. (C) Schematic sum- mary of self-ubiquitination and cross-ubiquitination between Ro52 and TRIM5a. K. Yamauchi et al. Ubiquitination of TRIM5a and its role FEBS Journal 275 (2008) 1540–1555 ª 2008 The Authors Journal compilation ª 2008 FEBS 1545 and when FLAG-tagged inactive TRIM5a(C15A) was coexpressed (lanes 5 and 10). Importantly, the level of the ubiquitination of Ro52(C16A) was not changed when wild-type FLAG–TRIM5a was coexpressed (Fig. 5B, lanes 4 and 9), suggesting that Ro52 is not ubiquitinated by TRIM5a in HEK293T cells. The faint ubiquitination of Ro52(C16A) seen in Fig. 5B (lanes 3–5 and 8–10) seemed to be catalyzed by the wild-type Ro52 or other E3 ubiquitin ligases that were endogenously expressed in HEK293T cells. In contrast, TRIM5a(C15A)–RH was more strongly ubiquitinated by wild-type FLAG–TRIM5a (Fig. 5B, lower panel, lanes 4 and 9). TRIM5a is ubiquitinated by Ro52 more strongly than TRIM5a in HEK293T cells As summarized in Fig. 5C, we showed two things using the in vivo ubiquitination assay. First, TRIM5a is ubiq- uitinated by itself and Ro52. Second, Ro52 is ubiquiti- nated by itself, but not by TRIM5a. These results raised a question: which E3 ligase predominantly ubiq- uitinates TRIM5a? In other words, is TRIM5a ubiqui- tinated more strongly by itself or by Ro52? To address this question, we performed an in vivo ubiquitination assay (Fig. 6). FLAG-tagged wild-type TRIM5a and wild-type Ro52 were used as E3 ubiquitin ligases, and TRIM5a(C15A)–RH was used as a substrate. In brief, FLAG-tagged wild-type TRIM5a and Ro52 were expressed with HA–ubiquitin and TRIM5a(C15A)–RH in HEK293T cells, by plasmid transfection. The cells were then harvested. Some of the cells were lysed in the SDS treatment solution, and FLAG-tagged proteins were analyzed by western blotting, using antibody to FLAG. As shown in the upper panel of Fig. 6, the expression levels of FLAG–TRIM5a and Ro52 were almost equal (lane 3 versus lane 4). The rest of the cells were also lysed under denaturing conditions to precipi- tate TRIM5a(C15A)–RH with TALON beads. TRIM5a(C15A)–RH was then solubilized in 2% SDS solution and analyzed by western blotting, using anti- body to HA and antibody to RH. As shown in the middle panel of Fig. 6, TRIM5a(C15A)–RH is ubiqui- tinated by FLAG–Ro52 (wild-type) more strongly than by FLAG–TRIM5a (wild-type) in HEK293T cells (lane 4 versus lane 3), suggesting that Ro52 has higher E3 ligase activity for this ubiquitination. UnpEL ⁄ Usp4 deubiquitinates Ro52, but not TRIM5a, in HEK293T cells Recently, we showed that UnpEL is an isopeptidase used to deubiquitinate Ro52 [10]. Because TRIM5a and Ro52 are phylogenetically and structurally similar, as described above, we hypothesized that TRIM5a is also deubiquitinated by UnpEL. To test this hypothe- sis, we performed an in vivo deubiquitination assay using UnpEL as described previously [10,21] (Fig. 7). As a control, we used a deubiquitinating enzyme, YopJ (Fig. 8) (see below). Specifically, TRIM5a and ubiquitin were expressed in HEK293T cells along with empty vector, wild-type UnpEL, or UnpEL(C311A), Fig. 6. Ubiquitination of TRIM5a by TRIM5a and Ro52: a compara- tive study. To compare the ligase activities of TRIM5a and Ro52 for the ubiquitination of TRIM5a,anin vivo ubiquitination assay was performed. FLAG-tagged TRIM5a (wild-type) and Ro52 (wild type) were expressed with HA–ubiquitin and TRIM5a(C15A)–RH as a substrate in HEK293T cells. Twenty hours after transfection, the cells were harvested. Some of the cells were lysed and analyzed by western blotting, using antibody to FLAG to show the expres- sion level of FLAG–TRIM5a (wild-type) and Ro52 (wild-type) (upper panel). The rest of the cells were also lysed under denaturing conditions to precipitate TRIM5a(C15A)–RH with TALON beads. TRIM5a(C15A)–RH was then solubilized in 2% SDS solution and analyzed by western blotting, using antibody to HA (middle panel) and antibody to RH (lower panel). Ubiquitination of TRIM5a and its role K. Yamauchi et al. 1546 FEBS Journal 275 (2008) 1540–1555 ª 2008 The Authors Journal compilation ª 2008 FEBS in which Ala was substituted for the active site Cys311. TRIM5 a was then precipitated using TALON beads, after which it was solubilized and then analyzed by western blotting to detect ubiquitinated TRIM5a (Fig. 7, lanes 1–3). To demonstrate the isopeptidase activity of UnpEL [10], Ro52 was also used as a posi- tive control for the substrate (Fig. 7, lanes 4–6). As shown in the upper and lower panels of Fig. 7, there was strong ubiquitination of Ro52 when Ro52 and ubiquitin were coexpressed with empty vector (lane 4). Importantly, however, their coexpression with wild- type UnpEL greatly reduced the level of ubiquitinated Ro52, because of UnpEL’s isopeptidase activity (Fig. 7, lane 5). In contrast, the coexpression of Ro52 and ubiquitin with UnpEL(C311A) did not affect the ubiquitination of Ro52 (Fig. 7, lane 6), because of the substitution of Ala for the active site Cys311 in UnpEL(C311A). Thus, we clearly detected the isopep- tidase activity of UnpEL when Ro52 was the substrate but not when TRIM5a was the substrate (Fig. 7, lanes 1–3). Specifically, we detected ubiquitination of TRIM5a when TRIM5a and ubiquitin were coex- pressed with empty vector (Fig. 7, lane 1). Unexpect- edly, however, their coexpression with wild-type UnpEL did not reduce the level of ubiquitinated TRIM5a (Fig. 7, lane 2), indicating that UnpEL does not deubiquitinate TRIM5a in HEK293T cells. YopJ deubiquitinates both Ro52 and TRIM5a in HEK293T cells YopJ is one of the Yersinia outer proteins encoded by pathogenic Yersinia species. In particular, YopJ is a cysteine protease that is thought to remove ubiquitin or a ubiquitin-like modification from target proteins in host cells [22]. As described above, we chose YopJ as a control against UnpEL because we initially expected that UnpEL would deubiquitinate both Ro52 and TRIM5a, but YopJ would not. To test the possibility that YopJ would not deubiquitinate either Ro52 or TRIM5a, we performed an in vivo deubiquitination assay. First, we used Ro52 as a substrate. Specifically, Ro52 and ubiquitin were expressed in HEK293T cells along with empty vector, wild-type YopJ, or YopJ(C172S), in which Ser was substituted for the active site Cys172. Ro52 was then precipitated, solubi- lized, and analyzed by western blotting to detect ubi- quitinated Ro52 (Fig. 8A). As shown in the upper and lower panels of Fig. 8A, we detected strong ubiquitina- tion of Ro52 when Ro52 and ubiquitin were co- expressed with empty vector (lane 2). Surprisingly, in contrast, their coexpression with wild-type YopJ greatly reduced the level of ubiquitinated Ro52, due to its isopeptidase activity (Fig. 8A, lane 4). The coex- pression of Ro52 and ubiquitin with YopJ (C172S), however, did not affect the ubiquitination of Ro52 (Fig. 8A, lane 6), because of substitution of Ser for the active site Cys172 in this mutant YopJ. Thus, the detection of isopeptidase activity of YopJ when Ro52 was used as a substrate was unexpected. Because TRIM5a and Ro52 are phylogenetically and structurally similar, as described above, we then hypothesized that TRIM5a is also deubiquitinated by YopJ. To test this hypothesis, we performed the same in vivo deubiquitination assay as described above. As shown in Fig. 8B, we strongly detected the ubiquitina- Fig. 7. In vivo deubiquitination by isopeptidase activity of human UnpEL. HA-tagged ubiquitin was coexpressed with RH-tagged TRIM5a (lanes 1–3) or Ro52 (lanes 4–6) in HEK293T cells. In addi- tion, empty vector (lanes 1 and 4), FLAG–UnpEL (wild-type) (lanes 2 and 5) or FLAG-tagged UnpEL mutant with a single substitution (C311A) (lanes 3 and 6) was also coexpressed. The cells were lysed in 6 M guanidine hydrochloride. TRIM5a–RH or Ro52–RH in the lysate was then precipitated with cobalt-coated TALON beads and analyzed by western blotting, using antibody to HA to detect ubiquitinated TRIM5a–RH or Ro52–RH (upper panel), and antibody to RH to detect all derivatives of TRIM5a–RH or Ro52–RH (lower panel). K. Yamauchi et al. Ubiquitination of TRIM5a and its role FEBS Journal 275 (2008) 1540–1555 ª 2008 The Authors Journal compilation ª 2008 FEBS 1547 tion of TRIM5a when TRIM5a and ubiquitin were co- expressed with empty vector (lane 2). In contrast, their coexpression with wild-type YopJ greatly reduced the level of ubiquitinated TRIM5a, because of its isopepti- dase activity (Fig. 8B, lane 4). The coexpression of TRIM5a and ubiquitin with YopJ(C172S), however, did not affect the ubiquitination of TRIM5a (Fig. 8B, lane 6). This is because an active site Cys172 was replaced by Ser in YopJ(C172S). Thus, we found by chance that YopJ deubiquitinates both Ro52 and TRIM5a in HEK293T cells. Finally, we examined the enzymatic specificity of YopJ, using hypoxia-inducible factor (HIF)1a(DC) as a negative control for the substrate. HIF1a(DC), an N-terminal fragment (amino acids 1–330) of HIF1a, was previously shown to be polyubiquitinated [7,23]. To confirm that YopJ does not deubiquitinate HIF1a(DC), HIF1a(DC) and ubiquitin were expressed with empty vector, wild-type YopJ or YopJ(C172S) in HEK293T cells. HIF1a(DC) was then precipitated, solubilized, and analyzed by western blotting to detect ubiquitinated HIF1a(DC) (Fig. 8C). As shown in the upper panel of Fig. 8C, we detected strong ubiquitina- tion of HIF1a(DC) when HIF1a(DC) and ubiquitin were coexpressed with empty vector (lane 2) or YopJ(C172S) (lane 6). As expected, the wild-type YopJ did not affect this ubiquitination (Fig. 8C, lane 4), indicating that YopJ does not deubiquitinate HIF1a(DC). Ubiquitinated Ro52 and TRIM5a are not stabilized by proteasome inhibitors In the sections above, we demonstrated the ubiquitina- tion of TRIM5a in vitro and in vivo. Because we previ- ously showed that self-ubiquitination of Ro52 does not target it to the proteasome for degradation [7], we hypothesized that ubiquitination of TRIM5a does not lead to proteasomal degradation either. To test this hypothesis, we performed an in vivo ubiquitination assay (Fig. 9). Specifically, using the proteasome inhib- itor MG115 (Fig. 9A) or MG132 (Fig. 9B), we inhib- ited the proteasomal degradation in HeLa cells to determine whether the ubiquitinated TRIM5a was accumulated. Briefly, TRIM5a–RH was coexpressed with HA–ubiquitin in HeLa cells in the presence or absence of proteasome inhibitor MG115 or MG132. The cells were then harvested and lysed under denatur- ing conditions. Afterwards, TRIM5a–RH in the lysate was precipitated with TALON beads, solubilized, and then analyzed by western blotting using antibody to HA to detect ubiquitinated TRIM5a–RH, and AB C Fig. 8. In vivo deubiquitination by isopeptidase activity of YopJ. (A) In vivo deubiquitination assay of YopJ using Ro52 as a substrate. (B) In vivo deubiquitination assay of YopJ using TRIM5a as a substrate. (C) In vivo deubiquitination assay of YopJ using HIF1a N-terminal fragment (DC) as a substrate. RH-tagged Ro52, TRIM5a or HIF1a(DC) was expressed without HA–ubiquitin (lanes 1, 3, and 5) or with HA–ubiquitin (lanes 2, 4, and 6) in HEK293T cells. In addition, empty vector (lanes 1 and 2), FLAG–YopJ (wild-type) (lanes 3 and 4) or FLAG-tagged YopJ mutant with a single substitution (C172S) (lanes 5 and 6) was also coexpressed. The cells were lysed in 6 M guanidine hydrochloride. RH-tagged substrate, such as Ro52, TRIM5a, or HIF1a(DC), in the lysate was then precipitated with cobalt-coated TALON beads and analyzed by western blotting, using antibody to HA to detect ubiquitinated substrate (upper panel), and antibody to RH to detect all derivatives of the substrate (lower panel). A nonspecific band is indicated by an asterisk. Ubiquitination of TRIM5a and its role K. Yamauchi et al. 1548 FEBS Journal 275 (2008) 1540–1555 ª 2008 The Authors Journal compilation ª 2008 FEBS antibody to RH to detect both nonubiquitinated and ubiquitinated TRIM5a–RH. As a positive control for the effect of proteasome inhibitor on proteasomal degradation of ubiquitinated proteins, we used HIF1a(DC), because we had previously detected a clear effect of MG132 on the proteasomal degradation of HIF1a(DC) [7,23]. As a negative control for the effect of proteasome inhibitor, we used Ro52 [7]. As shown in the lower panel of Fig. 9, the treatment with proteasome inhibitors (MG115 and MG132) did not increase the expression of either ubiquitinated TRIM5a–RH (lane 9 versus lane 12) or unubiquitinat- ed TRIM5a–RH (lane 3 versus lane 6). These results suggested that the ubiquitination of TRIM5a does not lead to its proteasomal degradation in HeLa cells. As expected, a negative control Ro52 was not stabilized by the treatment with MG115 and MG132 either (Fig. 9, middle panel). In contrast, a positive control HIF1a(DC) was stabilized by the treatment with MG115 and MG132. As shown in the upper panel of Fig. 9, the treatment with MG115 and MG132 increased the expression of HIF1 a(DC)–RH (lane 3 versus lane 6). Furthermore, the treatment increased the amount of ubiquitinated HIF1a(DC)–RH (Fig. 9, lane 9 versus lane 12), because MG115 and MG132 inhibited the proteasomal degradation of the ubiquiti- nated HIF1a(DC)–RH, resulting in its accumulation. These results suggested that the ubiquitination of HIF1a(DC) targets it to proteasomal degradation. Monoubiquitin-fusion of Ro52 and TRIM5a causes their translocation from cytoplasmic bodies to cytoplasm in human cells As described above, the ubiquitination of Ro52 and TRIM5a did not cause their proteasomal degradation. This raises the following question: what is the biologi- cal relevance of the ubiquitination of Ro52 and TRIM5a? Because monoubiquitination appeared to be dominant in their ubiquitination (see Discussion), we investigated the biological relevance of their mono- ubiquitination, using monoubiquitin-fused Ro52 and TRIM5a. Specifically, we examined whether the direct fusion of a monoubiquitin to Ro52 or TRIM5 a causes its translocation. In the molecule of monoubiquitin- fused protein, however, the monoubiquitin links to the N-terminal Met residue of the protein with an a-pep- tide bond. This linkage is artificial, not being found naturally in cells. In the molecule of naturally mono- ubiquitinated protein in cells, the monoubiquitin links to the target Lys residue of the protein with an isopep- tide bond. Thus, the monoubiquitin-fusion product is different from the natural monoubiquitination product, A B Fig. 9. Effects of proteasome inhibitors on the expression of HIF1a, Ro52 and TRIM5a in HeLa cells. (A) Expression of RH- tagged HIF1a(DC), Ro52 and TRIM5a in MG115-treated cells. (B) Expression of RH-tagged HIF1a(DC), Ro52 and TRIM5a in MG132- treated cells. HIF1a(DC)–RH, Ro52–RH or TRIM5a –RH was coex- pressed in HeLa cells with empty vector (lanes 2, 5, 8, and 11) or HA-tagged ubiquitin (lanes 3, 6, 9, and 12). The HeLa cells were cultured for 6 h in the absence (lanes 1–3 and 7–9) or presence (lanes 4–6 and 10–12) of a proteasome inhibitor, either MG115 or MG132. After incubation, the cells were harvested and lysed under denaturing conditions. RH-tagged proteins in the lysate were pre- cipitated with cobalt-coated TALON beads and solubilized in 2% SDS solution. The solubilized RH-tagged proteins were then ana- lyzed by western blotting in which both nonubiquitinated and ubi- quitinated forms were detected by antibody to RH (lanes 1–6), and the ubiquitinated form was detected by antibody to HA (lanes 7–12). K. Yamauchi et al. Ubiquitination of TRIM5a and its role FEBS Journal 275 (2008) 1540–1555 ª 2008 The Authors Journal compilation ª 2008 FEBS 1549 [...]... is strongly ubiquitinated by Ro52 Importantly, the ubiquitination of TRIM5a by Ro52 is stronger than its self -ubiquitination, suggesting that Ro52 regulates TRIM5a, but TRIM5a does not regulate Ro52, by ubiquitination in cells In addition to investigating the ubiquitination of TRIM5a, we investigated its deubiquitination We found that the ubiquitination of TRIM5a is downregulated by a deubiquitinating... Given this, we then asked, what is the biological relevance of ubiquitination of TRIM5a? Importantly, when we performed the in vivo ubiquitination assay of TRIM5a, we clearly detected the monoubiquitination of TRIM5a in addition to its polyubiquitination Strangely, the monoubiquitination was even stronger than the polyubiquitination when TRIM5a was detected by western blotting using the antibody to.. .Ubiquitination of TRIM5a and its role K Yamauchi et al but they are structurally similar Therefore, using the direct fusion of a monoubiquitin to target proteins, we are able to investigate their monoubiquitination [24] To investigate the subcellular translocation of monoubiquitin-fused Ro52 and TRIM5a, the cDNA of monoubiquitin (UbG) was first fused to that of Ro52–enhanced green... self -ubiquitination play in the function of TRIM5a, and what are the substrates, other than the TRIM5a itself, in the TRIM5a- mediated ubiquitination? Recently, Diaz-Griffero et al reported that TRIM5a is polyubiquitinated, resulting in its rapid degradation by the 26S proteasome [28] This observation seems to partially answer our first question, because the selfubiquitination of TRIM5a might lead to its. .. only with a single RH-epitope of the polyubiquitinated TRIM5a RH On the basis of this explanation of our findings, the monoubiquitinated form clearly exists in the actual population of ubiquiti- nated TRIM5a Because monoubiquitination plays roles in proteasome-unrelated events, such as protein trafficking and interaction with other proteins [30–33], the monoubiquitination of TRIM5a might be involved FEBS... enzymes and act as E3 ubiquitin ligases [13,14] Furthermore, some groups recently reported that TRIM family members, such as ARD1 [26], TRIM37 [27], and Ro52 [7], function as E3 ubiquitin ligases In addition, TRIM5d, an alternative splicing product of the trim5 gene, was shown to have E3 ubiquitin ligase activity in vitro [15] On the basis of these reports, we hypothesized that TRIM5a is also an E3 ubiquitin. .. degradation In other words, the activity of TRIM5a might be negatively regulated by its feedback mechanism Indeed, an E3 ubiquitin ligase Nrdp1 is regulated by a similar mechanism Namely, Nrdp1 polyubiquitinates itself, resulting in its proteasomal degradation [29] On the basis of these previous observations, we initially thought that the self -ubiquitination of TRIM5a leads to its proteasomal degradation To... The Authors Journal compilation ª 2008 FEBS 1551 Ubiquitination of TRIM5a and its role K Yamauchi et al in these events To test this hypothesis, we directly fused monoubiquitin to the N-terminal Met residue of TRIM5a and expressed it in HEK293 cells The monoubiquitin-fused TRIM5a diffusely located to the cytoplasm in the cells, whereas TRIM5a without monoubiquitin fusion mainly localized to cytoplasmic... ubiquitin ligase In the study presented here, we tested this hypothesis using both in vitro and in vivo ubiquitination assays As expected, we observed that TRIM5a ubiquitinated itself in the presence of E1 enzyme and 1550 E2 enzyme UbcH5B, indicating that TRIM5a functions as an E3 enzyme for its self -ubiquitination, thus proving our hypothesis These findings, however, raised two questions: what role does... 2008 FEBS K Yamauchi et al Ubiquitination of TRIM5a and its role A B EGFP EGFP C D Ro52-EGFP Ro52-EGFP E F UbG-Ro52-EGFP UbG-Ro52-EGFP G H TRIM5α-EGFP TRIM5α-EGFP I J UbG-TRIM5α-EGFP UbG-TRIM5α-EGFP + DAPI Fig 10 Subcellular location of Ro52 and TRIM5a and their monoubiquitin-fused forms in HEK293 cells EGFP alone, Ro52–EGFP, UbG-fused Ro52–EGFP, TRIM5a EGFP or UbG-fused TRIM5a EGFP was expressed in . 6. Ubiquitination of TRIM5a by TRIM5a and Ro52: a compara- tive study. To compare the ligase activities of TRIM5a and Ro52 for the ubiquitination of TRIM5a, anin. sum- mary of self -ubiquitination and cross -ubiquitination between Ro52 and TRIM5a. K. Yamauchi et al. Ubiquitination of TRIM5a and its role FEBS Journal 275 (2008)