Nuclear-Cytoplasmic Shuttling of a RING-IBR Protein RBCK1 and Its Functional Interaction with Nuclear Body Proteins*

The intracellular localization of a RING-IBR protein, RBCK1, possessing DNA binding and transcriptional activities, has been investigated. The endogenous RBCK1 was found in both the cytoplasm and nucleus. Particularly in the nucleus, it was localized in the granular structures, most likely nuclear bodies. In contrast, the over-expressed RBCK1 was detected exclusively in the cytoplasm. When the cells were treated with leptomycin B, the over-expressed RBCK1 accumulated in the nuclear bodies. These results suggest that RBCK1 possesses the signal sequences responsible for the nuclearcytoplasmic translocation. Mutational analysis of RBCK1 has indicated that an N-terminal region containing Leu-142 and Leu-145 and a C-terminal one containing the RING-IBR domain serve as the nuclear export and localization signals, respectively. Thus, RBCK1 is a transcription factor dynamically shuttling between cytoplasm and nucleus. Furthermore, RBCK1 was found to interact with nuclear body proteins, CREB-binding protein (CBP), and promyelocytic leukemia protein (PML). Coexpression of RBCK1 with CBP significantly enhanced the transcriptional activity of RBCK1. Although PML per se showed no effect on the transcriptional activity of RBCK1, the CBP-enhanced activity was repressed by coexpression with PML, presumably through the interaction of PML and CBP. Taken together, our data demonstrate that RBCK1 is involved in transcriptional machinery in the nuclear bodies, and its transcriptional activity is regulated by nucleocytoplasmic shuttling.

The RING finger is a protein motif that binds two zinc ions in a Cys/His-rich region and mediates protein-protein or protein-DNA interactions (1). More than 2,000 RING finger-containing proteins have been reported so far (2), which possess both or either one of the transcriptional and ubiquitin ligase (E3) activities. For example, an acute promyelocytic leukemia protein (PML) 1 (3,4), a Ret finger protein (5), and a lung cancer-associated gene product (6) are the RING finger proteins with transcriptional activity. A RING finger LIM domain-binding protein (7), a mouse double minute-2 protein (8), and a cellular Casitas B-lineage lymphoma protein (9) are those with E3 activity. A breast cancer 1 protein (BRCA1) (10,11) and a ubiquitin-conjugating enzyme Ubc5B-interacting protein AO7 (10,12) are the proteins with both activities. An advanced protein motif containing two RING fingers with an additional Cys/His-rich region, placed in between the RING fingers (IBR), is called a RING-IBR domain (13) and has been found in a human homologue of ariadne and an autosomal recessive juvenile parkinsonism-related gene product.
We previously identified a novel RING-IBR protein, RBCK1, by the yeast two-hybrid screening of a rat brain cDNA library using protein kinase C␤ as bait (14). RBCK1 has been found to possess transcriptional activity and consist of a ubiquitin-like sequence, two coiled-coil regions, and a RING-IBR domain, arranged from N to C terminus (see Fig. 1A). RBCK1 mRNA is ubiquitously expressed in normal rat tissues. The RING-IBR domain of RBCK1 interacts with DNA fragments containing a TGG-rich sequence. The RING finger motif occurring at the N-terminal side (RING1) is essential for the transcriptional activity of RBCK1, which is enhanced by coexpression with protein kinase A and significantly repressed by coexpression with extracellular signal-regulated kinase activator kinase 1 (MEK1) and MEK kinase 1 (15). A ubiquitin-like-truncated form of RBCK1, also known as a hepatitis B virus X-associating protein 3 (XAP3), is generated by alternative splicing of human RBCK1 gene and trans-activates Rous sarcoma virus long terminal repeat (RSV LTR) promoter (16). RBCK1 also has been suggested to act as E3, a UbcM4 (a ubiquitin conjugating enzyme, E2)-interacting protein, UIP28 (17), and a ubiquitin ligase-1 (HOIL-1) for a heme-oxidized iron regulatory protein-2 (IRP2) (18) are the mouse and human splice variants of RBCK1, respectively. Collectively, it is concluded that RBCK1 is a ubiquitin ligase and a transcriptional factor having a RING-IBR domain. However, RBCK1 is usually present in the cytoplasm unlike other conventional transcriptional factors.
To further elucidate cellular functions of RBCK1, we investigated its intracellular localization. In this paper, we report the identification of the nuclear export signal (NES) and the nuclear localization signal (NLS) in RBCK1, which are responsible for shuttling between the cytoplasm and nucleus. Furthermore, we demonstrate here interactions of RBCK1 with nuclear body proteins, CBP and PML, and their up-and downregulation of the transcriptional activity of RBCK1.
Preparation of Anti-RBCK1 Polyclonal Antibody-A synthetic 24residue oligopeptide (LTLQPRGPLEPVLPKPRTHQETGQ) corresponding to rat RBCK1 (from Leu-150 to Glu-173) was conjugated to keyhole limpet hemocyanin with m-maleimidebenzoyl N-hydroxysuccinimide ester (Wako, Osaka, Japan) and used as an antigen. The oligopeptide was also conjugated with the activated HiTrap NH resins (Amersham Biosciences) according to the manufacture's protocol. Antisera were raised in female Japanese rabbits (2.5-3.5 kg) by subcutaneous injections of the keyhole limpet hemocyanin-conjugated oligopeptide. Antiserum was applied onto the oligopeptide-conjugated column and washed with PBS. The anti-RBCK1 antibody was eluted from the column with 0.2 M glycine buffer, pH 2.5, dialyzed against PBS, concentrated by using a Centricon YM-10 cartridge (Millipore, Bedford, MA), and stored at Ϫ20°C until use.
Immunocytochemical Analysis-Cells were fixed with 100% (v/v) methanol, washed twice with PBS, and permeabilized with PBS containing 0.25% (v/v) Triton X-100 for 30 min at room temperature. A nuclear body protein PML was visualized with an anti-human PML mouse monoclonal antibody (PG-M3) (Santa Cruz Biotechnology) as a primary antibody (dilution 1:1,000) and an anti-mouse IgG goat antibody conjugated with Cy3 (Amersham Biosciences) as a secondary antibody (dilution 1:1,000). CBP was detected with an anti-human CBP rabbit polyclonal antibody (C-20) (Santa Cruz Biotechnology) as a primary antibody (dilution 1:500) and an anti-rabbit IgG goat antibody conjugated with Cy3 (Amersham Biosciences) as a secondary antibody (dilution 1:500). FLAG-RBCK1 was detected with an anti-FLAG rabbit polyclonal antibody (Sigma) as a primary antibody (dilution 1:1,000) and an anti-rabbit IgG goat antibody conjugated with Cy2 (Amersham Biosciences) as a secondary antibody (dilution 1:1,000). Endogenous RBCK1 was detected by using an anti-RBCK1 rabbit polyclonal antibody (described above) as a primary antibody (dilution 1:500) and an anti-rabbit IgG goat antibody conjugated with Cy2 as a secondary antibody (dilution 1:1,000).
Microinjection of GST⅐GFP⅐RBCK1-A cell lysate of Escherichia coli BL21 expressing GST⅐GFP⅐RBCK1 or GST⅐GFP⅐RBCK1(L142A/L145A) was applied onto a small column of glutathione-Sepharose 4B (Amersham Biosciences), and the GST-fused protein was eluted according to the manufacture's protocol. The eluted protein was concentrated and dialyzed against PBS by using a Centricon YM-10 cartridge and used for microinjection into the nuclei of baby hamster kidney 21 cells (ϳ1 ϫ 10 4 cells) cultured on a cover glass in Dulbecco's modified Eagle's medium supplemented with 5% (v/v) FBS at 37°C under humidified air with 5% CO 2 . Each protein was mixed with rhodamine B-isothiocyanate-conjugated bovine serum albumin and microinjected by using a glass capillary. The cells were incubated at 37°C for 30 min under humidified air with 5% CO 2 , washed twice with PBS, and fixed with 3.7% (w/v) formaldehyde in PBS at room temperature for 30 min. Fluorescence of the cells was observed under an LSM510 confocal laser scan microscope.
Luciferase Reporter Assay-Transcriptional activities of RBCK1 were measured by the luciferase reporter gene assay with the RSV3Ј-LTR promoter. HEK293 cells (ϳ1 ϫ 10 6 cells) were cotransfected using FuGENE 6 with the reporter plasmid pGL3-RSV-LTR (10 ng) and either pTB701-FLAG-RBCK1 (50 ng) or pTB701-FLAG-RBCK1(L142A/ L145A) (50 ng). The transcriptional activities of RBCK1 were also measured by the one-hybrid reporter assay using GAL4 DNA-binding domain. HEK293 cells (ϳ1 ϫ 10 6 cells) were cotransfected using Fu-GENE 6 with the reporter plasmid pFR-Luc (Stratagene) (1 g) containing the firefly-derived luciferase gene 3Ј-downstream of the synthetic promoter consisting of five repeats of the GAL4-recognition site (17-mer), pM-RBCK1 (50 ng), and either pRc/RSV-mCBP⅐HA⅐RK (50 ng) or pCMX-PML (50 ng). After 24 h, the cells were washed once with PBS and lysed with 200 l of passive lysis buffer (Promega). In both of the luciferase reporter gene assays, the cell lysate was assayed with a luciferase assay kit (Promega) according to the manufacture's protocol. Transcriptional activities were shown by the relative luciferase unit divided by protein concentration, which was measured with a BCA (bicinchoninic acid) assay kit (Sigma) using bovine serum albumin as a standard.
Pull-down Assay-COS7 cells (ϳ5 ϫ 10 7 cells) transfected with pRc/ RSV-mCBP⅐HA⅐RK or pCMX-PML were suspended in 500 l of lysis buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5 mM dithiothreitol, 10 mM NaF, 1 mM Na 3 VO 4 , 1 tablet/50 ml of the complete protease inhibitor mixture (Roche Diagnostics), and 0.5% (v/v) Nonidet P-40. Cleared lysate was incubated with 50 l of glutathione-Sepharose 4B beads (50% slurry) immobilized with the purified RBCK1-GST (30 g). After incubation at 4°C for 1 h, the beads were washed twice with the lysis buffer and subjected to SDS-PAGE. Western blot analyses were carried out with an anti-human CBP rabbit polyclonal antibody (C-20) (dilution 1:200) or an anti-human PML mouse antibody (PG-M3) (dilution 1:200) as a primary antibody and an anti-mouse or anti-rabbit IgG antibody conjugated with horse radish peroxidase as a secondary antibody (Amersham Biosciences) (dilution 1:3,000). Immunoreactive bands were visualized by the enhanced chemiluminescence method with an ECL Plus (Amersham Biosciences) according to the manufacture's protocol.

Effect of LMB on Intracellular Localization of RBCK1-We
first investigated the intracellular localization of the over-expressed RBCK1. As shown in Fig. 1B, the GFP-fused RBCK1 was detected almost exclusively in the cytoplasm of HEK293 cells (a). When the cells were treated with leptomycin B (LMB), an inhibitor for the CRM1-dependent nuclear export (21), most RBCK1 translocated from the cytoplasm to the nucleus at 1 h after the LMB treatment (Fig. 1B, b). Although at 4 h after the treatment the protein still remained evenly in the nucleus (Fig.  1B, c), it accumulated in small granular structures inside the nucleus at 8 h after the treatment (d). Localization of the control GFP was unaffected by the LMB treatment (Fig. 1B,  e-h). These results suggest that RBCK1 has an LMB-sensitive NES sequence. However, the predominant existence of RBCK1 in the nucleus after the LMB treatment is unusual for NEScontaining proteins, which are generally present in both the nucleus and cytoplasm in the presence of LMB (22), and suggests that RBCK1 possesses not only NES but also NLS sequences, like Smad1 (23) and p53 (24,25).
Colocalization of RBCK1 and PML in Nuclear Bodies-PML is a major protein in the nuclear bodies (known also as NB, nuclear domain 10, or PML oncogenic domain) (20), which have granular structures of 250 -500 nm in diameter and are present in the nuclei of most cells. The localization of RBCK1 in the nuclear body-like structures (Fig. 1B, d) was further examined by immunocytochemical analysis. HEK293 cells expressing RBCK1-GFP were treated with LMB, and the endogenous PML was visualized with an anti-PML antibody. In the nucleus, more than 70% of fluorescent spots derived from RBCK1-GFP overlapped with those of the nuclear bodies containing PML ( Fig. 2A). Neither over-expression of RBCK1 nor LMB treatment affected the localization of PML in the nuclear bodies.
Next, the localization of endogenous RBCK1 was studied using an anti-rat RBCK1 rabbit antibody purified by affinity chromatography. Upon Western blotting of HEK293 cell lysate, the antibody could detect a 56-kDa protein (data not shown), whose size corresponds to the molecular mass (56, 380) calculated from the amino acid sequence of human RBCK1 (NCB Accession number NP_112506). When the antibody was pretreated with excess of the 24-residue oligopeptide that was used as an antigen, the 56-kDa-protein band was not observed, indicating that the 56-kDa protein is an endogenous RBCK1. As shown in Fig. 2B, the endogenous RBCK1 was detected in both the cytoplasm and nucleus, unlike the over-expressed one mostly detected in the cytoplasm (cf. Fig. 1B, a). Nevertheless, RBCK1 present in the nucleus colocalized with PML at the nuclear bodies (Fig. 2B, d).
Identification of NES and NLS-Truncated forms of RBCK1 were used to delineate the positions of NES and NLS. The Nterminal half (from Met-1 to Glu-269) and C-terminal half (from Cys-270 to His-498) of RBCK1 were expressed as FLAG-tagged proteins in HEK293 cells, and the immunocytochemical analyses were carried out with anti-FLAG and anti-PML antibodies. The N-terminal half of RBCK1 was entirely observed in the cyto-   10 m (A, a and B, a). plasm (Fig. 3a). In marked contrast, the C-terminal half was detected only in the dot-like structures within the nucleus (Fig.  3d), which mostly colocalized with the nuclear bodies (f) similarly to the full-length RBCK1 in the LMB-treated cells. These results show that the NES and nuclear-localizing function are contained in the N-and C-terminal halves of RBCK1, respectively. It is reasonable that the C-terminal half of RBCK1, also containing the transcriptional activation domain of RBCK1 (15), is translocated to the nuclear bodies where genes are actively transcribed (26) and activates the transcription by interacting with the transcriptional machineries.
Truncated forms of RBCK1 (from Thr-151 to Glu-269 and from Cys-270 to His-498) were also prepared as N-terminally GST⅐GFP-fused proteins. These proteins were microinjected into the nuclei of baby hamster kidney-21 cells together with the rhodamine B-isothiocyanate-labeled bovine serum albumin. Even 1 h after microinjection, these proteins remained in the nuclei (data not shown), whereas the GST⅐GFP-fused fulllength RBCK1 was exported from the nucleus within 30 min (Fig. 4C, a), corroborating that the NES sequence of RBCK1 is contained in the N-terminal region (from Met-1 to Leu-150). The consensus sequence for the LMB-sensitive NES has been reported as LX 2-3 (L/I/V/F/M)X 2-3 LX(L/I) (where X is any amino acid residue; Fig. 4B), which is recognized by an NES-receptor exportin-1/CRM1 (27). By comparing the entire RBCK1 sequence with the consensus sequence, a putative NES sequence was found at the region including Leu-142 and Leu-145 (Fig.  4A). It has been reported that the replacement of Leu by Ala in the NES sequence leads to disruption of the NES function (28). Indeed, when the GST⅐GFP-fused RBCK1(L142A/L145A) in which both Leu-142 and Leu-145 were substituted by Ala was injected into the nuclei, the protein did not translocate from the nucleus to the cytoplasm at 30 min after the microinjection (Fig. 4C, c). Also, the GFP-fused RBCK1(L142A/L145A) protein over-expressed in HEK293 cells spontaneously colocalized with PML in the nucleus as dot-like structures without the LMB treatment (Fig. 4D). These results indicate that Leu-142 and Leu-145 are essential for the NES function of RBCK1.
Consistent with the previous report that RBCK1 plays as a transcriptional activator for the RSV LTR promoter (16), RBCK1 activated the transcription of RSV LTR about 2.5-fold in the luciferase reporter assay (Fig. 4E). The NES mutant, RBCK1(L142A/L145A), localizing predominantly in the nuclear bodies, showed further enhancement of the transcriptional activity of RSV LTR promoter. A similar result has been obtained in the one-hybrid reporter assay using the GAL4 DNA-binding domain-fused RBCK1(L142A/L145A) (see Fig. 7), suggesting that the transcriptional activity of RBCK1 is down-regulated by NES.
Interaction of RBCK1 with PML and CBP-CBP has recently been revealed to scaffold and activate various transcription factors in the nuclear bodies (26,29). PML, a main constituent of the nuclear bodies, was also shown to participate in the transcription mechanism involving either CBP or p53 (30,31). In HEp-2 (26) and SK-N-SH cells (32), endogenous CBP is mostly detected in the nuclear body. However, the localization of CBP depends on cell lines; in HEK293 cells, it accumulates in nuclear body only when PML is over-expressed (32). Thus, colocalization of RBCK1 with PML in the nuclear bodies prompted us to examine the colocalization of RBCK1 with CBP. GFP-fused RBCK1 was expressed in HEK293 cells, and the cells were treated with LMB. The transfection efficiency was ϳ15%. In the cells, in which RBCK1-GFP was not over-expressed, the endogenous CBP was detected diffusely in the nucleus. However, in the cells, in which RBCK1-GFP was overexpressed, both RBCK1-GFP and CBP were detected in dotlike structures (Fig. 5A). Similar localization was observed with the cells over-expressing RBCK1(L142A/L145A)-GFP (Fig. 5B). These results suggest that RBCK1 interacts with not only PML but also CBP.
The interactions of RBCK1 with PML and CBP were further examined by a GST pull-down assay. The purified RBCK1-GST protein was adsorbed onto a glutathione-Sepharose 4B resin, mixed with the lysate of COS7 cells over-expressing either PML or CBP, and subjected to the pull-down assay. As shown in Fig. 6A, CBP (ϳ265 kDa) was found to interact with RBCK1. When the lysate of COS7 cells expressing PML was analyzed by Western blotting, two bands (ϳ58 and 62 kDa) were observed (Fig. 6B). Because the calculated molecular weight of PML is 62,006, the 58-kDa protein might be generated by proteolysis of the 62-kDa PML protein. In the GST pull-down assay, the 62-kDa PML protein was found to interact with RBCK1.
Regulation of Transcriptional Activity of RBCK1 by CBP and PML-CBP was shown to act as a coactivator for various transcriptional factors. PML was also shown to act as a coactivator (3,31) or a corepressor (33,34). It is assumed that the transcriptional activity of RBCK1 is affected by the coexpression of either CBP or PML. Thus the effects of CBP and PML on the RBCK1-dependent transcription were examined by using a luciferase reporter gene assay. The transcriptional activity of RBCK1 was enhanced by 3.6-fold by the coexpression with CBP but was unaffected by the coexpression with PML (Fig. 7). Furthermore, the CBP-enhanced transcriptional activity of RBCK1 was repressed to ϳ0.5 by the coexpression with PML. Similar effects of CBP and PML were observed in the transcriptional activity of the NES-disrupted RBCK1 mutant, RBCK1(L142A/L145A), which localizes exclusively in the nuclear bodies. Taken together, RBCK1 interacts functionally with PML and CBP in the nuclear bodies, and its transcriptional activity is up-or down-regulated by the interaction. It is likely that PML acts as a transcriptional repressor by interacting with the RBCK1⅐CBP complex, but not with the sole RBCK1. DISCUSSION A transcription factor, RBCK1, was revealed to possess NES and NLS concurrently. The NES of RBCK1 includes the leucine-rich consensus sequence, and this NES function was inhibited by the CRM1 inhibitor leptomycin B. Therefore, it was clarified that the NES of RBCK1 is involved in the CRM1 dependent nuclear-export machinery. The consensus sequences for NLS, which interacts with the importin ␣/␤ complex, are classified into two types. One type is a single cluster of basic residues, for example the simian virus 40 large T antigen-derived NLS (35). The other type consists of two basic regions separated by a 10-amino acid spacer (36). Although the C-terminal half of RBCK1 is preferentially localized in the nucleus, RBCK1 does not possess a typical NLS. Endogenous RBCK1 in HEK293 cells was found to localize in both cytoplasm and nucleus, whereas the over-expressed RBCK1 in the same cells localized only in the cytoplasm. We hereby propose that the cytoplasmic localization of over-expressed RBCK1 is caused by the failure of either an RBCK1 anchoring protein in the nucleus or a cytoplasmic cargo protein for the transport of RBCK1 to the nucleus. In the case of BRCA1, a RING proteincontaining NES (37), and NLS (38), its nuclear localization is regulated by BRCA1-interacting protein BARD1. The RING finger of BRCA1 mediates the import to the nucleus through association with the RING finger of BARD1, and it has been revealed that BARD1 retains BRCA1 in the nucleus by masking its NES (39). It is therefore presumed that an unidentified RBCK1-interacting protein masks the NES of RBCK1 and fa-  cilitates the nuclear translocation of RBCK1. RBCK1 was first identified as a protein kinase C␤-and protein kinase C-interacting protein. Because parts of protein kinase C␤ and -are known to translocate to the nuclear body-like domains in phorbol ester-treated cells (40), it is hypothesized that protein kinase C␤ and -may be RBCK1-anchoring proteins in nuclear bodies. We demonstrated that RBCK1 colocalizes with PML in nucleus and interacts with PML and CBP. PML or CBP is also a possible candidate for a nuclear anchoring protein for RBCK1. The RBCK1-recruiting protein, which controls the localization of RBCK1, may regulate its interaction with RBCK1 via a phosphorylation signal. The forked head of the rhabdomyosarcoma protein is known as a transcription factor responsible for nucleocytoplasmic shuttling, of which the nuclear localization is mediated by phosphorylation with protein kinase B (41) and interaction with 14-3-3 protein (42). In the previous study, the transcriptional activity of RBCK1 was modulated by the expression of protein kinase A, MEK1, and MEK kinase 1 (15). Unfortunately, the subcellular localization of RBCK1 was not changed by the coexpression of these protein kinases (data not shown), but it cannot be entirely denied that other protein kinase affects the localization of RBCK1. Recently, RBCK1 has been shown to possess a ubiquitin ligase E3 activity. UbcM4interacting protein (UIP28) (17) and heme-oxidized IRP2 ubiquitin ligase-1 (HOIL-1) (18) are the mouse and human homologue of RBCK1, respectively. It is postulated therefore that RBCK1 possesses a ubiquitin ligase E3 activity. Iron regulatory protein 2 (IRP2) interacts with and stabilizes a specific mRNA in the cytosol. The ubiquitination of IRP2 by HOIL-1 is supposed to be executed in the cytosol, whereas RBCK1 localizes in not only cytoplasm but also nucleus, and the nuclear RBCK1 possesses the transcriptional activating ability. These facts indicate that the role of RBCK1 may be regulated by the switch of the NLS and NES.
The nuclear body is a granule structure of 250 -500 nm in diameter, and its main component protein is PML, which was shown to exert a transcriptional function in the nuclear body (31). A growing number of proteins have been demonstrated to colocalize with PML, including speckled 100-kDa protein (Sp100) (43), death domain-associated protein (44), retinoblastoma protein (45), CBP (46), p53 (4), and BRCA1 (47). These proteins are involved in the transcriptional mechanism. The chromatin-surrounding nuclear bodies are highly acetylated, and the nascent RNA is associated with the periphery of the nuclear body (26). The nuclear body is thus considered to be an active site of transcription mechanisms (48). In this report, RBCK1 interacts with PML and CBP, and was demonstrated to be a novel component protein of the nuclear body. CBP connects the DNA-binding transcriptional factors with the general transcription machinery (29,49), and RBCK1 associates with the specific DNA (14). CBP might serve to promote the interaction of RBCK1 with the transcription machinery in the nuclear body. PML has been shown to exert two distinct effects on the transcriptional mechanisms. One is transcriptional enhancement; PML acts as a transcriptional coactivator in the p53-mediated apoptotic signaling pathway (30) and enhances p53-dependent transcription via interaction with CBP (4). The other is transcriptional repression; PML is a component of the multiple corepressor complex, which includes N-CoR, c-Ski, HDAC1, and mSin3A (32). PML acts as a transcriptional corepressor with c-Ski and the mouse homologue of yeast suppressor interacting protein 3. Although PML has been recognized so far as a transcriptional coactivator for CBP in the CBP⅐p53 complex (4), our results indicate that PML, by contrast, represses the transcriptional activity of the CBP⅐RBCK1 complex. It is important to elucidate how p53 and RBCK1 share the transcriptional function of the CBP⅐PML complex in the nuclear body at the molecular level.
The functions of autoimmune regulator are very similar to that of RBCK1. Autoimmune regulator binds to CBP (50) and the specific nucleotide sequence (51) and activates the transcription (52). The DNA-associating ability of autoimmune regulator is dependent on its homodimer or homotetramer formation. In a previous experiment, the complexes of RBCK1 and the RBCK1-specific nucleotide sequences were observed as two bands by using electrophoretic mobility shift assay (14). This result suggested that RBCK1 also binds to the specific nucleotides with a monomer and dimer. The autoimmune regulator has two plant homeodomain-type zinc-finger motif, which binds zinc in a cross-brace topology between anti-parallel ␤-strands reminiscent of RING finger (53), and the N terminus plant homeodomain is important domain for the ubiquitin ligase activity of the autoimmune regulator (54). Some RING proteins, such as BRCA1 (10,11) and AO7 (10,12), were also reported to possess both transcriptional and ubiquitin ligase activity. Thus, RBCK1 and some ubiquitin ligases may regulate transcription via its ubiquitin ligase activity. In some cases, ubiquitination of the transcription factor is the important step of the transcriptional activation (55,56). However, it is unknown how the two distinct activities of RBCK1 are involved in the function of nuclear bodies. It is important to more precisely define the function of nuclear bodies in transcription and ubiquitination.