Function and subcellular location of Ro52β

BBRC Biochemical and Biophysical Research Communications 340 (2006) 872–878 www.elsevier.com/locate/ybbrc

Function and subcellular location of Ro52b Keiji Wada, Kunikazu Tanji, Tetsu Kamitani

*

Department of Cardiology, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA Received 28 November 2005 Available online 27 December 2005

Abstract Autoantigen Ro52a was recently identified as an E3 ubiquitin ligase. Its splicing variant Ro52b, which lacks a leucine zipper, has not been characterized yet. We therefore characterized Ro52b in contrast to Ro52a. Our biochemical assays revealed that both Ro52a and Ro52b function as E3 ubiquitin ligases and self-ubiquitinate in cooperation with UbcH5B in vitro. In addition, both Ro52a and Ro52b are ubiquitinated when overexpressed with ubiquitin in HEK293T cells, suggesting that both function as E3 ligases and self-ubiquitinate in vivo. However, cytological studies revealed that Ro52a mainly localizes to the cytoplasmic rod-like structures, whereas Ro52b diffusely localizes to both the cytoplasm and the nucleus. Since the leucine zipper plays a role in the homodimerization and heterodimerization of Ro52a, the dimerization might be required for the localization of Ro52a to the rod-like structures. On the basis of these results, Ro52a and Ro52b appear to ubiquitinate their particular substrates at different locations.
2005 Elsevier Inc. All rights reserved. Keywords: Ro52; Sjo¨gren’s syndrome; Ubiquitin; TRIM; RING finger; RBCC; Ubiquitin ligase; E3; UnpEL; Usp4

Anti-Ro/SSA antibodies are autoantibodies that are most commonly found in patients with Sjo¨gren’s syndrome. They play a pathogenic role in a variety of clinical manifestations, including skin lesions and congenital heart block. Ro52 is one of the autoantigens recognized by the anti-Ro/SSA autoantibodies. So far, a and b isoforms of Ro52 have been reported. Ro52a, a comparatively large isoform (52 kDa), is a RING-finger protein that belongs to an RBCC (RING-finger/B-box/coiledcoil) family (also known as the TRIM family) [1]. Ro52b, a comparatively small isoform, is a product of mRNA derived from the alternative splicing of exon 3 to exon 5, skipping exon 4, which results in a smaller protein with a predicted molecular weight of 45 kDa [2]. Recently, we reported that Ro52a functions as an E3 ubiquitin ligase and ubiquitinates itself [3]. We also reported that Ro52a colocalizes with UnpEL to the cytoplasmic rod-like structures and is selectively deubiquitinated by UnpEL [4]. Thus, we have characterized the *

Corresponding author. Fax: +1 713 563 0424. E-mail address: [email protected] (T. Kamitani).

0006-291X/$ - see front matter
2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.12.084

molecular function and subcellular location of Ro52a and identified its interacting protein UnpEL as a potential regulator. However, Ro52b has not yet been characterized to this extent. Although Ro52b is a minor splicing variant in comparison with Ro52a [5], the expression of Ro52b seems to be clinically important. This was indicated by the finding that fetal congenital heart block caused by maternal anti-Ro/SSA autoantibodies might be triggered by the expression of Ro52b in the fetal heart. Since the expression of Ro52b is maximal at the time of cardiac ontogeny when maternal antibodies gain access to the fetal circulation, just prior to the clinical detection of brady-arrhythmia, Ro52b has been implicated in the development of congenital heart block [5]. It is therefore important to learn more about Ro52b in order to shed light on the pathogenesis of congenital heart block and perhaps other disorders in which anti-Ro/SSA antibodies play a pathogenic role. In the study described here, we investigated Ro52b using the same assay methods that we previously used to characterize Ro52a and compared Ro52b with Ro52a.

K. Wada et al. / Biochemical and Biophysical Research Communications 340 (2006) 872–878

Results and discussion Ro52b structurally lacks the leucine zipper To compare human Ro52a and Ro52b, we first summarized their domain structure, which was previously reported [2]. As shown in Fig. 1, Ro52a possesses a RING finger

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Cell culture. Human embryonic kidney HEK293 cells and HEK293T cells (American Type Culture Collection, Manassas, VA) were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum and antibiotics. Antibodies. Mouse anti-FLAG antibody (M5) was purchased from Sigma Chemical Company (St. Louis, MO). Mouse anti-RH antibody (specific for the amino acid sequences RGSHHHH and GGSHHHH) was purchased from QIAGEN (Santa Clara, CA). Mouse anti-HA antibody (16B12) was purchased from Covance (Richmond, CA). Rabbit anti-MBP antibody was purchased from NewEngland Biolabs (Beverly, MA). Preparation of Ro52b cDNA. The cDNA of Ro52b [2] was generated by a recombinant polymerase chain reaction [6] using the cDNA of Ro52a [7] as a template. Plasmid construction and transfection. To express Ro52b tagged with RH-epitope (RGSHHHHHH) at the C-terminus in HEK293T cells, the cDNA of Ro52b was subcloned into pcDNA3/RH-C [8], as we previously did for the expression of Ro52a [7]. To express ubiquitin tagged with HA at the N-terminus, pcDNA3/HA-N was used [9]. The plasmids were transfected into HEK293T cells using FuGENE6 (Roche Applied Science, Indianapolis, IN) and 20 h later, the transfected cells were harvested for TALON-bead precipitation. In vitro ubiquitination assay. For the in vitro ubiquitination assay, we first expressed several recombinant proteins in bacteria using the eukaryotic expression vectors pMAL-c2 (New England BioLabs) and pTrcHisB (Invitrogen, Carlsbad, CA). These proteins include maltosebinding protein (MBP)-fused Ro52a (MBP-Ro52a) [3], MBP-Ro52b, and MBP-Ro52a (C16A) [3], RH-tagged ubiquitin (RH-Ub) [3], and poly-Histagged E2 ubiquitin-conjugating enzyme UbcH5B [3]. Next, bead-immobilized MBP-Ro52 was incubated with RH-Ub, E1 ubiquitin-activating enzyme (Boston Biochem, Cambridge, MA), and poly-His-tagged UbcH5B in reaction buffer (50 mM Tris–HCl [pH 7.5], 2 mM ATP, 4 mM MgCl2, and 2 mM DTT) for 30 min at 37 C. After the reaction, the beads were washed with washing buffer (25 mM Tris–HCl [pH 7.5], 100 mM NaCl, and 0.5% NP-40) and treated for 1 h at 50 C in sample treating solution containing 2% SDS and 5% b-mercaptoethanol. Finally, the solubilized MBP-Ro52 was analyzed by Western blotting using anti-RH antibody to detect ubiquitinated Ro52 and anti-MBP antibody to detect both unubiquitinated and ubiquitinated Ro52. Treatment with proteasome inhibitor. Cells were treated with the proteasome inhibitor MG132 (Calbiochem, San Diego, CA), as described previously [10]. In brief, 1 · 106 HEK293T cells were transfected by FuGENE 6. After an overnight culture, the culture medium was replaced with fresh medium containing MG132 (20 lM). The cells were further cultured in the medium at 37 C for 6 h. Then the cells were harvested, and the total cell lysates were prepared for TALON-bead precipitation. TALON-bead precipitation of RH-tagged Ro52. To investigate the in vivo ubiquitination of Ro52a and Ro52b, HA-tagged ubiquitin was coexpressed with Ro52a-RH or Ro52b-RH in HEK293T cells by the cotransfection method. Since the sequence of the RH tag is RGSHHHHHH, RH-tagged proteins can be purified by cobalt-immobilized resin beads (TALON beads; Clontech, Palo Alto, CA) [3,4]. The total cell lysate of the transfectants expressing RH-tagged Ro52 (a or b isoform) and HA-ubiquitin was prepared in lysis buffer (20 mM Tris–HCl [pH 8.0], 6 M guanidine–HCl, and 100 mM NaCl). DNA in the sample was sheared with a 22-gauge needle, and then the lysate was centrifuged at 100,000g for 30 min at 15 C. The supernatant was incubated with TALON beads for 1 h at room temperature. The beads were washed once with lysis buffer and then washed twice with washing buffer (20 mM Tris–HCl [pH 7.0], 15 mM imidazole, 8 M urea, and 100 mM NaCl). Finally, the beads were washed twice with phosphate-buffered saline (PBS) and treated for 1 h at 50 C in sample treating solution containing 2% sodium dodecyl sulfate (SDS) and 5% b-mercaptoethanol, followed by SDS–polyacrylamide gel electrophoresis (SDS–PAGE). In vivo deubiquitination assay. To investigate the isopeptidase activity of UnpEL against ubiquitinated Ro52a and Ro52b, we performed an

in vivo deubiquitination assay, as described previously [4]. Briefly, FLAGtagged UnpEL was expressed in HEK293T cells with HA-ubiquitin and an RH-tagged substrate (Ro52a-RH or Ro52b-RH) using plasmid cotransfection method. Twenty hours after transfection, cells were lysed in the lysis buffer containing 6 M guanidine–HCl (see above description). The RH-tagged substrate in the lysate was then precipitated with cobalt-coated TALON beads, washed, and solubilized in sample treating solution containing 2% SDS and 5% b-mercaptoethanol (see above). Finally, the RHtagged substrate was analyzed by Western blotting using anti-RH antibody to detect all derivatives of the substrate and anti-HA antibody to detect the substrate conjugated with HA-ubiquitin. Western blotting. Protein samples were treated at 50 C for 1 h in 2% SDS treating solution containing 5% b-mercaptoethanol. After SDS– PAGE, Western blotting was performed using the protocol provided with the ECL detection system (Amersham Pharmacia Biotech). Horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody or anti-rabbit IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was used as a secondary antibody. Immunocytochemistry. To investigate the subcellular location of Ro52a, Ro52b, and UnpEL in cultured cells, we performed immunocytochemical studies, as described previously [4]. Briefly, HEK293 cells were cultured on a coverslip in a 3.5-cm dish, and 1 lg of pEGFP-N1 [4], pEGFP-N1/Ro52a [4], or pEGFP-N1/Ro52b (pEGFP-N1 inserted with the Ro52b cDNA) was transfected with or without 1 lg pcDNA3/FLAGUnpEL [4]. After 24 h, the cells were fixed with a 4% paraformaldehyde solution [pH 7.5] for 30 min and permeabilized with 0.1% Triton X-100 for 15 min at room temperature. The cells cotransfected with pcDNA3/ FLAG-UnpEL were first labeled with mouse anti-FLAG antibody. After washing, the cells were labeled with Alexa Fluor 594-conjugated antimouse IgG antibody (Molecular Probes, Eugene, OR). Finally, the cells were counterstained with 4 0 ,6-diamidino-2-phenylindole (DAPI; 5 lg/ml PBS) for 5 min and analyzed under a fluorescence microscope. The localization of enhanced green fluorescent protein (EGFP) alone, Ro52aEGFP, and Ro52b-EGFP was shown by the green fluorescence of EGFP, and the localization of FLAG-UnpEL was shown by the red fluorescence of Alexa Fluor 594. Their colocalization was shown by the merging of both fluorescences.

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Fig. 1. The protein domains of human Ro52a and Ro52b. The two isoforms are products of alternative splicing of a single gene. Ro52a (52 kDa) possesses a RING finger and a B box at the N-terminal region, two coiled coils in the central domain with the second as a leucine zipper encoded in exon 4, and a B30.2 domain at the C-terminus. Ro52b (45 kDa) is identical to Ro52a but lacks 77 amino acids (amino acid residues 169–245) inclusive of the second coiled-coil domain with the leucine zipper.

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and a B box at the N-terminal region. This RING-finger motif is required for the activity of E3 ubiquitin ligase [3]. In the central domain, Ro52a possesses two coiled coils, the second of which is a leucine zipper encoded in exon 4. Recently, Ro52a was shown to form homodimers through its leucine zipper [11]. At the C-terminus, Ro52a possesses a B30.2 domain (also known as a rfp-like domain). Ro52b, however, is a product of mRNA derived from the alternative splicing of exon 3 to exon 5, skipping exon 4, which results in the deletion of 77 amino acids, including the second coiled-coil domain with the leucine zipper [2]. Other than that, Ro52b is identical to Ro52a. Ro52b functions as an E3 ubiquitin ligase in vitro We recently reported that Ro52a acts as an E3 ubiquitin ligase and ubiquitinates itself in vitro. Importantly, the E3 activity depends on the function of the RING-finger motif [3]. Although the RING-finger motif is conserved in Ro52b, it lacks the leucine zipper found in Ro52a (Fig. 1). To test whether this deletion affects the E3 activity, we performed an in vitro ubiquitination assay, as described previously [3]. In this assay, Ro52a was used as a positive control and a Ro52a mutant (C16A), which lacks E3 activity as a result of a single substitution in the RING-finger domain [3], was used as a negative control. Specifically, we tested the self-ubiquitination of Ro52b when catalyzed by itself. MBPfused Ro52a, its C16A mutant, or Ro52b was expressed in bacteria and purified using beads. MBP-Ro52 that was immobilized on the beads was then incubated with RH-ubiquitin, recombinant E1 enzyme, and recombinant E2 enzyme (UbcH5B). After the incubation, MBP-Ro52 was solubilized and analyzed by Western blotting using anti-RH antibody and anti-MBP antibody. As shown in Fig. 2, Ro52b ubiquitinated itself (lane 5) as did Ro52a (lane 4), whereas the C16A mutant of Ro52a did not ubiquitinate itself at all (lane 6). These results indicate that Ro52b, like Ro52a, is functional as an E3 ubiquitin ligase in vitro. Ro52b is ubiquitinated when overexpressed with ubiquitin in vivo Recently, we showed in vivo the self-ubiquitination of Ro52a [3]. Briefly, wild-type Ro52a was strongly monoubiquitinated and weakly polyubiquitinated when overexpressed with ubiquitin in HEK293T cells. In contrast, its C16A mutant was faintly ubiquitinated when overexpressed with ubiquitin in HEK293T cells. This faint ubiquitination of the C16A mutant appeared to be catalyzed by the wild-type Ro52a that was endogenously expressed in HEK293T cells. These results indicate that Ro52a ubiquitinates itself in HEK293T cells through the function of its RING-finger domain [3]. Based on these findings and the fact that Ro52b is functional as an E3 ubiquitin ligase in vitro (Fig. 2), we hypothesized that Ro52b functions as an E3 ubiquitin ligase in vivo and ubiquitinates itself. To test this hypothesis,

Fig. 2. E3 activity of Ro52a and Ro52b in vitro. The UbcH5B-dependent self-ubiquitination of Ro52a and Ro52b was tested in vitro. MBP-fused Ro52 (a or b isoform) was purified using beads and incubated with the reaction mixture containing RH-tagged ubiquitin, recombinant E1 enzyme, and poly-His-tagged UbcH5B. MBP-fused Ro52a mutant (C16A) was used as a negative control of the self-ubiquitination. This mutant possesses a single substitution at Cys-16, which abolishes the E3 activity. After the reaction, MBP-Ro52 immobilized on beads was washed to remove the reaction mixture and solubilized in SDS solution. MBPRo52 was then analyzed by Western blotting using anti-MBP antibody to detect both unubiquitinated and ubiquitinated MBP-Ro52 (lanes 1–3) and anti-RH antibody to detect ubiquitinated MBP-Ro52 (lanes 4–6). Molecular size markers are shown on the left in kilodaltons (kDa).

we performed an in vivo self-ubiquitination assay, as described previously [3], using Ro52a as a positive control (lanes 1–3, Fig. 3). In this assay, RH-tagged Ro52a or Ro52b was expressed with or without HA-tagged ubiquitin in HEK293T cells. The cells were then harvested and lysed under denaturing conditions. Afterwards, Ro52-RH (a or b isoform) in the lysate was precipitated by TALON beads, solubilized, and then analyzed by Western blotting using anti-HA antibody to detect ubiquitinated Ro52-RH and anti-RH antibody to detect both unubiquitinated and ubiquitinated Ro52-RH. As shown in Fig. 3A, Ro52a was strongly monoubiquitinated and weakly polyubiquitinated when overexpressed with HA-ubiquitin in HEK293T cells (lane 3). Similarly, as shown in Fig. 3B, Ro52b was monoubiquitinated and polyubiquitinated when overexpressed with HA-ubiquitin in HEK293T cells (lane 3). Of note, the monoubiquitination of Ro52b was not as strong as that of Ro52a. These results thus support our hypothesis that Ro52b functions as an E3 ubiquitin ligase in vivo and ubiquitinates itself. Stability of Ro52b is regulated by the ubiquitin-proteasome system in vivo As described previously, the ubiquitin modification of Ro52a is predominantly of the monoubiquitination type,

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Ro52b was not as strong as that of Ro52a (see lane 3 in the lower panels of Figs. 3A and B). This raised the question whether the ubiquitination of Ro52b targets it to the proteasome for degradation. To determine this, we performed an in vivo ubiquitination assay in conjunction with the last experiment (Figs. 3A and B). Specifically, using the proteasome inhibitor MG132, we inhibited the proteasomal degradation in HEK293T cells to determine whether Ro52b was stabilized (lanes 4–6, Fig. 3B). Ro52a was used as a control (lanes 4–6, Fig. 3A). As shown in Fig. 3A, the MG132 did not clearly increase the expression of either ubiquitinated or unubiquitinated Ro52a-RH (lane 3 vs. lane 6), as noted previously [3]. These results suggest that the self-ubiquitination of Ro52a does not efficiently lead to its proteasomal degradation. In contrast, Ro52b was stabilized by the MG132. Namely, as shown in Fig. 3B, the MG132 increased the expression of unubiquitinated Ro52b-RH by approximately threefold (lane 3 vs. lane 6, lower panel). It also increased the ubiquitinated Ro52bRH (lane 3 vs. lane 6, upper panel) by approximately twofold. This was because the MG132 inhibited the proteasomal degradation of the ubiquitinated Ro52b-RH, resulting in its accumulation. These results suggest that the ubiquitination of Ro52b targets it for proteasomal degradation. Ro52b is deubiquitinated by UnpEL in vivo

Fig. 3. E3 activity of Ro52a and Ro52b in HEK293T cells. In vivo selfubiquitination of Ro52a (A) and Ro52b (B) was examined in HEK293T cells. RH-tagged Ro52 (a or b isoform) was expressed with or without HA-tagged ubiquitin in HEK293T cells by plasmid transfection. Sixteen hours after transfection, the HEK293T cells were further cultured for 6 h in the absence (lanes 1–3) or presence (lanes 4–6) of the proteasome inhibitor MG132. After incubation, the cells were harvested and lysed under denaturing conditions. Ro52-RH in the lysate was precipitated by cobalt-coated TALON beads and solubilized in 2% SDS solution. The solubilized Ro52-RH was then analyzed by Western blotting using antiHA antibody to detect ubiquitinated Ro52-RH (upper panel) and anti-RH antibody to detect both unubiquitinated and ubiquitinated Ro52-RH (lower panel). A nonspecific band is indicated by an asterisk.

which does not target Ro52a to the proteasome for degradation [3]. In the experiment described in the preceding section, however, we found that the monoubiquitination of

Ro52a is ubiquitinated by itself [3] and it is deubiquitinated by a Ro52a-interacting protein UnpEL (also known as Usp4) [4]. Because a yeast two-hybrid assay previously revealed that UnpEL interacts with Ro52a but not with Ro52b [12], we hypothesized that UnpEL deubiquitinates Ro52a but not Ro52b. To test our hypothesis, we performed an in vivo deubiquitination assay, as described previously [4]. Specifically, Ro52 (a or b isoform) and ubiquitin were expressed with empty vector, wild-type UnpEL, or mutant UnpEL (C311A) in HEK293T cells. Ro52 was then precipitated, solubilized, and analyzed by Western blotting to examine its deubiquitination (see Figs. 4A and B for Ro52a and Ro52b, respectively). As shown in the middle panel of Fig. 4A, we clearly detected the ubiquitination of Ro52a when Ro52a and ubiquitin were coexpressed with empty vector (lane 4). In contrast, their coexpression with wild-type UnpEL greatly reduced the level of ubiquitinated Ro52a because UnpEL removed ubiquitin from Ro52a by its isopeptidase activity (lane 5). The coexpression of Ro52a and ubiquitin with a mutant UnpEL (C311A), however, did not affect the ubiquitination of Ro52a (lane 6). This is because a catalytic site Cys-311 was substituted with Ala in the UnpEL (C311A), which abolished the catalytic activity of UnpEL [4]. Thus, UnpEL deubiquitinated Ro52a, also as described previously [4]. In Fig. 4B, we showed the activity of UnpEL to Ro52b. As shown in the middle panel, UnpEL deubiquitinated Ro52b (lane 5) but its mutant UnpEL (C311A) did not (lane 6). Thus, although the interaction between Ro52b and UnpEL was not detected in yeast cells [12],

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Ro52b is diffusely or reticulately located in both the cytoplasm and the nucleus Ro52a predominantly localized to the cytoplasmic rodlike structures in HEK293 cells [4]. In this section, we examined the subcellular location of Ro52b in HEK293 cells and compared it with that of Ro52a. Specifically, we expressed EGFP-fused Ro52a or Ro52b in HEK293 cells to determine its subcellular location under a fluorescence microscope. As shown in Fig. 5A, Ro52a-EGFP mainly localized to the cytoplasmic rod-like structures (panels c and d). In addition, although the signal was weak, Ro52a-EGFP was diffusely located in the cytoplasm. However, the Ro52a-EGFP signal was hardly detectable in the nucleus. In contrast to Ro52a-EGFP, Ro52b-EGFP was diffusely or reticulately located in both the cytoplasm and the nucleus (panels e and f). Thus, the subcellular location of Ro52b-EGFP was totally different from that of Ro52aEGFP. Since both Ro52a and Ro52b have the activity of E3 ubiquitin ligase in mammalian cells, our results suggest that these molecules function at the different locations. Recently, Wang et al. [11] showed that Ro52a forms homodimers but Ro52b does not. In addition, Di Donato et al. [12] showed that Ro52a forms heterodimers with UnpEL in yeast cells but Ro52b does not. Since Ro52b structurally lacks the leucine zipper domain found in Ro52a (Fig. 1), these observations indicate that the leucine zipper domain of Ro52a is required for its homodimerization and heterodimerization. In this section, we further demonstrated that Ro52a localizes to the cytoplasmic rod-like structures but Ro52b does not (Fig. 5A), suggesting that the homodimerization or heterodimerization of Ro52a through the leucine zipper domain is essential for its subcellular localization to the cytoplasmic rod-like structures. Subcellular location of UnpEL is not changed when it is coexpressed with Ro52b

Fig. 4. In vivo deubiquitination of Ro52a and Ro52b by isopeptidase activity of human UnpEL. The isopeptidase activity of UnpEL was tested for ubiquitinated Ro52a (A) and Ro52b (B). RH-tagged Ro52 (a or b isoform) was expressed with HA-ubiquitin (lanes 4–6) or without HAubiquitin (lanes 1–3) in HEK293T cells. In addition, 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 coexpressed. Part of the cells were lysed for Western blot analysis using anti-FLAG antibody to detect FLAG-UnpEL (upper panel). The rest of the cells were lysed in 6 M guanidine–HCl. Ro52-RH in the lysate was then precipitated by cobalt-coated TALON beads and analyzed by Western blotting using anti-HA antibody to detect ubiquitinated Ro52RH (middle panel) and anti-RH antibody to detect all derivatives of Ro52-RH (lower panel). A nonspecific band is indicated by an asterisk.

Ro52b, like Ro52a, was deubiquitinated by UnpEL in HEK293T cells. This suggests that the stable interaction between Ro52b and UnpEL is not required for the UnpEL-mediated deubiquitination of Ro52b.

UnpEL was originally described as a nuclear protein [13]. However, Soboleva et al. [14] recently reported that UnpEL possesses both a nuclear-exporting signal (NES) and a nuclear localization signal (NLS), which enable it to shuttle between the nucleus and the cytoplasm in cells. Indeed, we previously observed that UnpEL translocates to the cytoplasmic rod-like structures and colocalizes with Ro52a when overexpressed with Ro52a [4]. We examined here the translocation of UnpEL when it is overexpressed with Ro52b. Specifically, we coexpressed FLAG-tagged UnpEL with EGFP alone (negative control), Ro52a-EGFP (positive control for nuclear-cytoplasmic shuttling), or Ro52b-EGFP in HEK293 cells and determined the subcellular translocation of FLAG-UnpEL. As shown in panel b of Fig. 5B, FLAG-UnpEL was located diffusely or reticulately in both the cytoplasm and the nucleus when coexpressed with EGFP (panel a). In contrast, when we coexpressed FLAG-UnpEL with Ro52a-EGFP in HEK293 cells, FLAG-UnpEL was relocated to the

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Fig. 5. Subcellular location of Ro52a, Ro52b, and their regulator UnpEL in HEK293 cells. (A) Subcellular location of Ro52a and Ro52b. EGFP alone or EGFP-fused Ro52 (a or b isoform) was expressed in HEK293 cells by plasmid transfection. After 24 h, the cells were fixed and then treated with DAPI to stain the nucleus. Afterwards, the cells were analyzed under a fluorescence microscope. The localization of EGFP alone (panels a and b), Ro52a-EGFP (panels c and d), or Ro52b-EGFP (panels e and f) was shown by the green fluorescence of EGFP. Nuclear counterstaining was shown by the blue fluorescence of DAPI (panels b, d, and f). (B) Nuclear-cytoplasmic shuttling of UnpEL induced by overexpressing Ro52a or Ro52b. FLAG-UnpEL was expressed in HEK293 cells with EGFP alone (panels a–d), Ro52a-EGFP (panels e–h), or Ro52b-EGFP (panels i–l). After 24 h, the cells were fixed and immunostained with mouse antiFLAG antibody. After washing, the cells were labeled with Alexa Fluor 594-conjugated anti-mouse IgG antibody. Finally, the cells were counterstained with DAPI and analyzed under a fluorescence microscope. The localization of EGFP alone or Ro52-EGFP was shown by the green fluorescence of EGFP (panels a, e, and i), and the localization of FLAG-UnpEL was shown by the red fluorescence of Alexa Fluor 594 (panels b, f, and j). Their colocalization was shown by the merging of both fluorescences (panels c, g, and k). Nuclear staining was shown by the blue fluorescence of DAPI (panels d, h, and l).

cytoplasmic rod-like structures (panel f). Of note, the level of FLAG-UnpEL in the nucleus was highly reduced (panel f vs. panel b) as a result of its shuttling between the nucleus

and the cytoplasm [14]. At the cytoplasmic rod-like structures, FLAG-UnpEL colocalized with Ro52a-EGFP (panel g). When we coexpressed FLAG-UnpEL with

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Ro52b-EGFP in HEK293 cells, the subcellular location of FLAG-UnpEL was not changed (panel j vs.panel b). FLAG-UnpEL and Ro52b-EGFP were located diffusely or reticulately in both the cytoplasm and the nucleus (panel j and panel i). Thus, we did not clearly observe the translocation of UnpEL when it was coexpressed with Ro52b. However, because the subcellular location of UnpEL (panel b, Fig. 5B) is similar to that of Ro52b (panel e, Fig. 5A), the translocation of UnpEL might not be observed when coexpressed with Ro52b. In conclusion, our biochemical studies showed that Ro52b, like Ro52a, functions as an E3 ubiquitin ligase and ubiquitinates itself. Unlike Ro52a, however, the self-ubiquitination of Ro52b seems to target it to the proteasome for degradation. Interestingly, Ro52b is deubiquitinated by UnpEL, indicating that UnpEL is a regulator of Ro52b. Our cytological studies further suggested that Ro52b ubiquitinates its substrates both in the cytoplasm and the nucleus, whereas Ro52a ubiquitinates its substrates at the cytoplasmic rod-like structures but not in the nucleus. Thus, although both Ro52a and Ro52b are functional E3 ubiquitin ligases, each isoform localizes at different subcellular locations to ubiquitinate its particular substrates.

[3] [4]

[5]

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[7] [8]

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[10]

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Acknowledgment This work was supported by National Institutes of Health Grant R01 DK56298 (to T.K.).

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