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Cytoplasmic tethering of a RING protein RBCK1 by its splice variant lacking the RING domain
BBRC Biochemical and Biophysical Research Communications 335 (2005) 550–557 www.elsevier.com/locate/ybbrc
Cytoplasmic tethering of a RING protein RBCK1 by its splice variant lacking the RING domain q Nobuo Yoshimoto a, Kenji Tatematsu a,*, Tomoyoshi Koyanagi a, Toshihide Okajima b, Katsuyuki Tanizawa a, ShunÕichi Kuroda a a
b
Department of Structural Molecular Biology, Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan Department of Nanobiology, Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan Received 8 July 2005
Abstract RBCC protein interacting with C-kinase (RBCK1) is a transcription factor belonging to the RING-IBR protein family and has been shown to shuttle between the nucleus and cytoplasm, possessing both the nuclear export and localization signals within its amino acid sequence. RBCK2, lacking the C-terminal half of RBCK1 including the RING-IBR domain, has also been identified as an alternative splice variant of RBCK1. RBCK2 shows no transcriptional activity and instead it represses the transcriptional activity of RBCK1. Here, we show that RBCK2 is present usually in the cytoplasm containing two Leu-rich regions that presumably serve as a nuclear export signal (NES). Moreover, an NES-disrupted RBCK1 that is mostly localized within the nucleus is translocated to the cytoplasm when coexpressed with RBCK2, suggesting that RBCK2 serves as a cytoplasmic tethering protein for RBCK1. We propose a novel and general function of RING-lacking splice variants of RING proteins to control the intracellular localization and functions of the parental RING proteins by forming a hetero-oligomeric complex. 2005 Elsevier Inc. All rights reserved. Keywords: RING protein; Splice variant; Transcription factor; Nuclear export signal
RBCC protein interacting with C-kinase 1 (RBCK1) consists of 498 amino acid residues harboring a ubiquitin-like (UBL) sequence, two coiled-coil regions, and a RING-IBR domain, arranged from N- to C-terminus (Fig. 1A). The RING-IBR domain is composed of two RING fingers and an Ôin-between-RING fingersÕ (IBR) domain. The protein was originally identified as a protein q Abbreviations: RBCK1, RBCC protein interacting with PKC 1; PKC, protein kinase C; IBR, in-between-RING fingers; NES, nuclear export signal; NLS, nuclear localization signal; PML, promyelocytic leukemia; CRE, cyclic AMP response element; CREB, CRE-binding protein; CBP, CREB-binding protein; HEK293, human embryonic kidney 293; GAL4DBD, GAL4 DNA-binding domain; LMB, leptomycin B; BRCA1, breast cancer-linked tumor suppressor. * Corresponding author. Fax: +81 6 6879 8464. E-mail address: [email protected] (K. Tatematsu).
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.07.104
kinase C (PKC) b-interacting protein by the yeast twohybrid screening and shown to be a transcription factor possessing both transcriptional and DNA-binding activities [1]. The transcriptional activity of RBCK1, residing in the RING-IBR domain, is significantly enhanced by protein kinase A (PKA) and repressed by extracellular signal-regulated kinase activator kinase 1 (ERK1) [2]. RBCK1 possesses a classical Leu-rich nuclear export signal (NES) in the segment containing Leu-142 and Leu-145 as well as the nuclear localization signal (NLS) activity in the C-terminal half of its sequence. We have shown recently that these intracellular localization signals facilitate the nucleocytoplasmic shuttling of RBCK1 [3]. Furthermore, intra-nuclear RBCK1 colocalizes with a promyelocytic leukemia protein (PML) and a CREBbinding protein (CBP) present in the nuclear bodies and
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Fig. 1. (A) Schematic structures of RBCK1, RBCK2, and their truncated mutants. Amino acid residue numbers of the N- and C-termini are given for the whole fragments and specific motifs: Ubl, ubiquitin-like; C-C, coiled-coil; RING1, the first RING finger; IBR, in-between-RING fingers; and RING2, the second RING finger. Amino acid sequences of Leu-rich regions and the region that differs between RBCK1 and RBCK2 are indicated by one-letter codes. (B) Intracellular localization of RBCK2 and NES-disrupted mutants. HEK293 cells were transfected with an expression plasmid for the N-terminally FLAG-tagged RBCK1 and NES-disrupted mutants, and incubated with (+) or without () LMB. The FLAG-tagged proteins were visualized by indirect immunofluorescence with the anti-FLAG antibody.
interacts with them. PML, a main constituent of the nuclear body, enhances the tumor suppressor gene product p53-dependent transcription via the interaction with CBP [4]. The transcriptional activity of RBCK1 is thus modulated by interaction with PML and CBP in the nuclear bodies [3]. A 260-residue protein RBCK2 has been identified as an alternative splice variant of RBCK1 [5]. The N-terminal 240-residue sequence of RBCK2 is identical with that of RBCK1, while the remaining C-terminal 20-residue sequence is unique for RBCK2 (Fig. 1A). Unlike RBCK1, RBCK2 shows no transcriptional activity without the RING-IBR domain and instead it represses the transcriptional activity of RBCK1 in a dose-dependent manner [5]. Based on the observation that RBCK2 interacts with RBCK1 but not with itself in the in vitro binding assay, it is postulated that RBCK2 represses the transcriptional activity of RBCK1 by forming an RBCK1/2 hetero-oligomeric complex. In the present studies reported herein, we have analyzed the intracellu-
lar localization of RBCK2, particularly in relation to that of the parental protein RBCK1, and show that RBCK2 represses the transcriptional activity of RBCK1 by tethering it within the cytoplasm. Such a function may be general for other RING-lacking splice variants reported recently for various RING proteins, including an autosomal recessive juvenile parkinsonism-related gene product, Parkin, whose molecular organization is very similar to that of RBCK1 with the N-terminal UBL sequence and the C-terminal RING-IBR domain [6].
Materials and methods Construction of plasmids. Mammalian expression plasmids pTB701-FLAG and pTB701-HA were described previously for expression of N-terminally FLAG- and haemagglutinin (HA)-tagged proteins, respectively [7]. For expression of N-terminally HA- or FLAG-tagged wild-type and truncated forms of RBCK1 [HA- or FLAG-RBCK1, HA- or FLAG-RBCK1(1–120), HA- or FLAG-
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RBCK1(1–240), HA- or FLAG-RBCK1(1–269), and HA- or FLAGRBCK1(270–498)], plasmids pTB701-HA (or FLAG)-RBCK1, pTB701-HA (or FLAG)-RBCK1(1–120), pTB701-HA (or FLAG)RBCK1(1–240), pTB701-HA (or FLAG)-RBCK1(1–269), and pTB701-HA (or FLAG)-RBCK1(270–498) were constructed by inserting DNA fragments encoding a full length, a ubiquitin-like sequence (from Met-1 to Leu-120), an N-terminal 240 amino acid sequence (from Met-1 to Gln-240), N-terminal (from Met-1 to Glu-269), and C-terminal (from Cys-270 to His-498) halves of RBCK1 into pTB701-HA (or FLAG), respectively [3]. For expression of N-terminally HA- or FLAG-tagged wild-type RBCK2 (HA- or FLAGRBCK2), plasmid pTB701-HA (or FLAG)-RBCK2 was constructed by inserting DNA fragment encoding a full length of RBCK2 into pTB701-HA (or FLAG). Plasmids pTB701-FLAG-RBCK1 (L118A/ L121A), pTB701-FLAG-RBCK2(L118A/L121A), pTB701-FLAGRBCK2(L142A/L145A), and pTB701-FLAG-RBCK2(L118A/L121A/ L142A/L145A) were constructed by site-directed mutagenesis for expression of RBCK1(L118A/L121A), RBCK2(L118A/L121A), RBCK 2(L142A/L145A), and RBCK2(L118A/L121A/ L142A/L145A), respectively. A mammalian expression plasmid pM (Promega, Madison, WI) for the N-terminally GAL4DBD (GAL4 DNA-binding domain)fused protein was used for the transcriptional assay. Plasmids pMRBCK1, pM-RBCK1(L142A/L145A), and pM-RBCK1(270–498) were described previously [1,3]. Cell culture. Human embryonic kidney 293 (HEK293) cells were cultured in DulbeccoÕs modified EagleÕs medium supplemented with 10% (v/v) fetal bovine serum at 37 C under humidified air with 5% CO2. Immunocytochemical analysis. HEK293 cells (approximately 5 · 104 cells) were transfected with the mammalian expression plasmids by using a FuGENE 6 transfection reagent (Roche diagnostics, Basel, Switzerland). The cells were grown on a coverglass (diameter, 2.7 cm) for 72 h, washed twice with phosphate-buffered saline (PBS, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, and 1 mM KH2PO4), and fixed with 100% (v/v) methanol at 20 C for 10 min. These cells were permeabilized with 0.15% (v/v) Triton X-100 in PBS for 15 min at room temperature, washed twice with PBS, incubated with blocking buffer [PBS containing 0.03% (v/v) Triton X-100, 2% (w/v) bovine serum albumin (BSA), and 3% (v/v) normal goat serum] for 30 min at room temperature, and incubated with the blocking buffer containing a primary antibody for 30 min at room temperature. The cells were washed four times with PBS containing 0.03% (v/v) Triton X-100 for 10 min at room temperature and mounted with 90% (v/v) glycerol in PBS. Fluorescence of the cells was observed under an LSM 5 PASCAL confocal laser scan microscope (Carl Zeiss, Oberkochen, Germany). The HA-tagged proteins were detected with an anti-HA rabbit polyclonal antibody (MBL, Nagoya, Japan) (dilution, 1:400) or an anti-HA mouse monoclonal antibody (12CA5) (Roche Diagnostics) (dilution, 1:600) as a primary antibody. The FLAG-tagged proteins were reacted with an anti-FLAG mouse monoclonal antibody (M2) (Sigma, St. Louis, MO) (dilution, 1:1,000) or an anti-FLAG rabbit polyclonal antibody (Sigma) (dilution, 1:250) as a primary antibody. A Cy2-labeled anti-rabbit IgG goat polyclonal antibody (KPL, Gaithersburg, MD) (dilution, 1:600) or a Cy3-labeled anti-mouse IgG goat polyclonal antibody (Amersham Biosciences, Uppsala, Sweden) (dilution, 1:800) was used as a secondary antibody. Immunoprecipitation and Western blotting. HEK293 cells (approximately 1 · 107 cells) were cotransfected with pTB701-HA-RBCK2 (5 lg) and pTB701-FLAG-RBCK1 (2–6 lg) by electroporation, and cultured for 48 h. The cells were washed twice with PBS and suspended in 1 ml of the lysis buffer [50 mM Tris–HCl at pH 7.5, 150 mM NaCl, 0.1% (v/v) Nonidet P-40, 1 mM dithiothreitol, and 1 tablet of Complete protein inhibitor cocktail (Roche diagnostics) per 50 ml]. The cleared lysate was incubated with 10 lg of an anti-HA mouse monoclonal antibody on ice for 60 min and incubated with 30 ll of protein G–Sepharose 4 [50% (v/v) slurry] (Amersham Biosciences) at 4 C for 30 min. The beads were washed four times with 1 ml of lysis buffer, resuspended
in 35 ll of LaemmliÕs sample buffer, subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA). Western blot analyses were carried out with a peroxidase-conjugated anti-HA mouse monoclonal antibody (Roche Diagnostics) (dilution, 1:5000) or an anti-FLAG mouse monoclonal antibody (Sigma) (dilution, 1:5000). Immunoreactive bands were visualized by the enhanced chemiluminescence method with an ECL Plus (Amersham Biosciences) according to the manufacturerÕs protocol. Luciferase assay. Transcriptional activities of GAL4DBD-fused RBCK1 or its mutants were measured by luciferase reporter-gene assays. HEK293 cells (about 1 · 106 cells) were cotransfected using FuGENE 6 with the reporter plasmid pFR-Luc (Stratagene, La Jolla, CA) (1 lg) containing the firefly-derived luciferase gene 3 0 -downstream of the synthetic promoter consisting of five repeats of the GAL4-recognition site (17-mer), pM-RBCK1 (25 ng), and pTB701-FLAGRBCK2. After 24 h, the cells were washed once with PBS and lysed with 200 ll of passive lysis buffer (Promega). The cell lysate was assayed with a luciferase assay kit (Promega) according to the manufacturerÕs protocol. Transcriptional activities were indicated by the relative luciferase unit (RLU) divided by protein concentration, which was measured with a bicinchoninic acid (BCA) assay kit (Sigma) using BSA as a standard.
Results Identification of NES sequences in RBCK2 We first investigated the intracellular localization of RBCK2. When RBCK2 was expressed in HEK293 cells, the protein was exclusively localized in the cytoplasm (Fig. 1B-a). Upon treatment with leptomycin B (LMB), an inhibitor for the CRM1-dependent nuclear export [8], a considerable portion of RBCK2 was translocated from the cytoplasm to the nucleus and was detected as dot-like structures (Fig. 1B-b), indicating that RBCK2 possesses an NES sequence(s) recognized by the CRM1-dependent nuclear export system. We have already reported that RBCK1 possesses a conventional Leu-rich NES in the region containing Leu-142 and Leu-145 [3]. Since RBCK2 has an identical amino acid sequence with RBCK1 for residues 1–240 (Fig. 1A), the same region (Leu-142 to Leu-145) has been assumed to serve as a potential NES of RBCK2. However, when the two Leu residues were mutated into Ala, the mutant protein RBCK2(L142A/L145A) was mostly retained in the cytoplasm (Fig. 1B-c). This result contrasts with the previous result for RBCK1, in which mutation of the same two Leu residues into Ala resulted in the complete translocation of RBCK1 into the nucleus [3], and suggests that RBCK2 possesses an additional NES sequence(s) that can function independently of the NES at Leu-142 and Leu-145. By inspecting the RBCK2 sequence, the segment containing Leu-118 and Leu-121 was found to match moderately to the consensus Leurich NES sequence [9] (Fig. 1A) and besides this segment there was no Leu-rich region. Therefore, these two Leu residues (Leu-118 and Leu-121) were then replaced by
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Ala by site-specific mutagenesis and the intracellular localization of the mutant protein RBCK2(L118A/ L121A) was analyzed. As shown in Fig. 1B-e, the mutant protein of RBCR2 was still detected in the cytoplasm, although enriched in the outer perinuclear region, suggesting that the former NES sequence (Leu142 to Leu-145) is alternately functioning in this mutant. Hence, finally the four Leu residues (Leu-118, 121, 142, and 145) were all replaced by Ala. The resultant quadruple mutant, RBCK2(L118A/L121A/L142A/L145A), was present evenly in both the cytoplasm and nucleus (Fig. 1B-g). These results suggest that both of the two Leu-rich regions in RBCK2 can serve independently as NES. A considerable portion of the RBCK2 mutant observed in the nucleus is presumably derived from free diffusion of RBCK2 that has no NLS. It has been reported that proteins smaller than 40 kDa could be
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translocated into the nucleus by free diffusion, when their functional NES is destroyed [10]. The newly identified NES (Leu-118 to Leu-121) in RBCK2 is also contained in the parental protein, RBCK1 (Fig. 1A). However, disruption of only the previously identified NES (Leu-142 to Leu-145) was sufficient for the complete intra-nuclear localization of RBCK1 [3]; the NES at Leu-118 and Leu-121 does not function in the RBCK1(L142A/L145A) mutant. Moreover, mutation of Leu-118 and Leu-121 in RBCK1 little affected its localization in the cytoplasm (Fig. 1B-i), suggesting that the NES at Leu-142 and Leu-145 is working in this mutant. Presumably, the potential NES region containing Leu-118 and Leu-121 may be structurally masked in RBCK1 not to function as NES. Similar masking of nucleocytoplasmic translocation signals has been reported for the NES of BRCA1 [11] and the
Fig. 2. Immunoprecipitation assays for identification of the region of RBCK1 interacting with RBCK2. HEK293 cells were transfected with an expression plasmid for the N-terminally HA- and FLAG-tagged proteins. The top panels in (A)–(D) show the detection of FLAG-tagged proteins in the immunoprecipitates obtained with an anti-HA antibody. The middle panels in (A)–(D) show the detection of FLAG-tagged proteins in the cell lysates. The bottom panels in (A)–(D) show the detection of HA-tagged proteins in the immunoprecipitates obtained with an anti-HA antibody.
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NLS of NF-jB p65 subunit [12]. It is noteworthy that treatment with LMB led to the considerable intra-nuclear localization of wild-type and mutant proteins of RBCK2, particularly in the dot-like structures (Figs. 1B-b, -d, -f, and -h). This may be caused by interaction of RBCK2 with an unidentified nuclear protein(s), such as endogenous RBCK1 that has already been shown to associate with the nuclear bodies [3]. Identification of region of RBCK1 interacting with RBCK2 We previously showed that RBCK1 interacts with itself and also with RBCK2, but the latter does not interact with itself [5]. To identify the region(s) of RBCK1 interacting with RBCK2, N- and C-terminally truncated mutants of RBCK1 were constructed (see Fig. 1A) and coexpressed with RBCK2 in HEK293 cells. Immunoprecipitation assays have revealed that RBCK2 interacts with the N-terminal half of RBCK1 [from Met-1 to Glu-269; RBCK1(1–269)] but not with the C-terminal half [from Cys-270 to His-498; RBCK1(270–498)] (Fig. 2A), indicating that the N-terminal half of RBCK1 is involved in the interaction with RBCK2. Although RBCK2 having the same amino acid sequence as RBCK1 for residues 1–240 did not interact with itself as reported previously [5] (see also Fig. 2A), the N-terminal half of RBCK1 (1–269) interacted with
itself (Fig. 2C). This suggests that the C-terminal unique 20-residue sequence of RBCK2 (residues 240–260) has an interfering effect on the self-interaction of RBCK2. On the other hand, RBCK1(1–240) interacted with itself, with RBCK2, and also with RBCK1(1–269) (Fig. 2D). In addition, the UBL region (residues 1– 120) of RBCK1/2 was found not to be involved in the interaction between RBCK1 and RBCK2 (data not shown). Collectively, these results show that the region covering residues 121–240 is commonly participating in the self-interaction of RBCK1 and the heterologous interaction between RBCK1 and RBCK2. Cytoplasmic tethering of RBCK1 by RBCK2 When the wild-type proteins of RBCK1 and RBCK2 were coexpressed in HEK293 cells, they colocalized in the cytoplasm (Fig. 3D), suggesting that the two proteins are interacting with each other within the cells, as shown above by immunoprecipitation assays. The effect of coexpression of RBCK2 on the intracellular localization of RBCK1 was further examined using the NESdisrupted and N-terminally truncated forms of RBCK1. As already reported [3], the NES-disrupted RBCK1 [RBCK1(L142A/L145A)] was predominantly localized in the nucleus without coexpression of RBCK2 (Fig. 3E). Significantly, coexpression of RBCK2 prevented completely the translocation of the NES-disrupt-
Fig. 3. Intracellular localization of RBCK1 and its mutant coexpressed with RBCK2. Cytoplasmic tethering of RBCK1 by RBCK2. (A–L) HEK293 cells were transfected with an expression plasmid for the N-terminally HA-tagged wild-type RBCK1, RBCK1(L142A/L145A), or RBCK1(270–498) with (B, F, and J) or without (A, E, and I) an expression plasmid for the FLAG-tagged RBCK2. The HA- and FLAG-tagged proteins were visualized by indirect immunofluorescence with an anti-HA antibody (A, B, E, F, I, and J) and an anti-FLAG antibody (C, G, and K), respectively. Merge, merged images (D, H, and L).
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ed RBCK1 into the nucleus (Fig. 3F) and the RBCK1 mutant retained in the cytoplasm colocalized with RBCK2 (Fig. 3H). The C-terminal half of RBCK1 [RBCK1(270–498)], which does not interact with RBCK2, was unaffected in its nuclear localization by the coexpression of RBCK2 (Figs. 3I, J, and L). These results clearly show that the cytoplasmic RBCK2 tethers the nucleocytoplasmic shuttling protein RBCK1 in the cytoplasm by interacting presumably through the regions containing residues 121–240.
ase reporter-gene assays (Fig. 4). In marked contrast, the transcriptional activity of the C-terminal half of RBCK1 [RBCK1(270–498)], existing in the nucleus irrespectively of the presence of RBCK2, was almost unaffected by the coexpression of RBCK2. Based on these results, we conclude that RBCK2 represses the transcriptional activity of RBCK1 by tethering it within the cytoplasm and preventing its translocation into the nucleus.
Repression of transcriptional activity of RBCK1 by RBCK2
Discussion
Previously, the transcriptional activity of RBCK1 was shown to be repressed by RBCK2 depending on the intracellular amount of expressed RBCK2 protein [5] (see also Fig. 4). The effect of coexpression of RBCK2 on the transcription activity of RBCK1 was further analyzed using the NES-disrupted and N-terminally truncated forms of RBCK1, expressed as a GAL4DBD-fused protein in HEK293 cells. The transcriptional activity of the NES-disrupted RBCK1 mutant, localizing usually in the nucleus in the absence of RBCK2 and exhibiting a very high transcriptional activity, was significantly repressed depending on the amount of expression plasmid for RBCK2 applied in the lucifer-
Fig. 4. Effect of coexpression of RBCK2 on the transcriptional activity of RBCK1. An expression plasmid for GAL4DBD (Mock) or the GAL4DBD-fused form of RBCK1, RBCK1(L142A/L145A), or RBCK1(270–498) was cotransfected to HEK293 cells with an indicated amount (ng) of the expression plasmid for FLAG-RBCK2 and the plasmid containing the luciferase reporter gene. Transcriptional activities of the GLA4DBD-fused proteins were measured by the luciferase assay and indicated as relative values against the negative control (GAL4DBD = 1). Each value was the mean of those obtained from six independent measurements and error bars indicate a 95% confidence interval.
Many transcription factors are known as nucleocytoplasmic proteins, whose transcriptional activities are closely associated with their nuclear localization, which, in turn, is governed by the cooperative functions of NES and NLS and also regulated by a number of intracellular events, such as phosphorylation, ubiquitination, sumoylation, and so on. For example, NF-jB p65 subunit, one of the major inflammatory responsive transcriptional factors, is normally localized in the cytoplasm with its NLS sequence masked by IjBa containing NES. When IjBa is phosphorylated by inflammation-activated IKK (IjBa kinase), it undergoes polyubiquitination and the following degradation, leading to unmasking of the NLS of NF-jB p65 subunit and then translocation into the nucleus for initiating the transcription [12]. Another example is a cytoplasmic RING protein the breast cancer-linked tumor suppressor 1 (BRCA1). When the NES of BRCA1 is inactivated by association with BRCA1-associated RING domain protein 1 (BARD1), the BRCA1–BARD1 complex is translocated into the nucleus by the NLS contained in BRCA1 [11]. Thus, the intracellular localization of a nucleocytoplasmic shuttling protein is often regulated by its associating protein, and the protein–protein interaction is significantly affected by modification of the partner protein as well as the balance of intracellular amounts of each protein. As revealed in the present studies using an overexpression system, RBCK2, a splice variant of RBCK1, functions as an anchoring protein for the parental RBCK1 and represses its transcriptional activity by tethering it within the cytoplasm. For exhibiting the transcriptional activity, RBCK1 must be released from the RBCK1–RBCK2 complex and translocated into the nucleus. However, it remains to be investigated what kind of modification occurs on RBCK1 or RBCK2 and in which signal cascade it leads to the dissociation of the RBCK1–RBCK2 complex and the translocation of RBCK1 into the nucleus. As for the intracellular expression levels of RBCK1 and RBCK2, RBCK1/2 mRNA is expressed ubiquitously in all adult rat tissues with the mRNA ratio of RBCK2 over RBCK1 being about 10% [5] and it is unknown if this ratio changes depending on the cell states. Thus, more detailed studies are cer-
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Fig. 5. Schematic structures of various RING proteins and their RING-lacking splice variants. The GenBank accession numbers, when available, are indicated below each protein name. Amino acid residue numbers of the N- and C-termini are provided for each protein. Abbreviations for specific motifs are the same as in Fig. 1, except for BRCT (BRCA1 C-terminus domain) and FHA (fork head-associated domain). Closed triangles indicate the point of exon junction causing a frameshift, which introduces a stop codon at a point different from the parental protein. Open triangles indicate the point of exon deletion occurring by alternative splicing.
tainly needed to elucidate the in vivo function of RBCK1 and its splice variant RBCK2. Shown in Fig. 5 are the schematic structures of various RING-IBR proteins and their RING-lacking splice variants recently identified. The full-length BARD1 forms a complex with BRCA1 and colocalizes in the nucleus, whereas a RING-lacking splice variant of BARD1 does not affect the cytoplasmic localization of BRCA1 without interacting with it [13]. Also, BRCA1 shows an ubiquitin ligase activity only in the complex with the full-length BARD1. Furthermore, some RING proteins possessing a transcriptional activity, such as RING finger protein 8 (RNF8) [14] and BRCA1 [15], also have their own RING-lacking splice variants. RNF8 is localized in the nucleus, while the RING-lacking variant is localized in the cytoplasm [14]. BRCA1 itself has both NLS and NES, but its RING-lacking variant lacks NES. Parkin, having the same molecular organization as RBCK1 with an N-terminal UBL domain and a C-terminal RINGIBR domain, also has a RING-lacking splice variant similar to RBCK2 [6]. On the basis of the findings reported in this paper, we propose that these RINGlacking splice variants of RING proteins generally have a function to control the intracellular localization of the parental full-length RING proteins by forming a hetero-oligomeric complex and thereby regulate their cellular functions.
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Cytoplasmic tethering of a RING protein RBCK1 by its splice variant lacking the RING domain q Nobuo Yoshimoto a, Kenji Tatematsu a,*, Tomoyoshi Koyanagi a, Toshihide Okajima b, Katsuyuki Tanizawa a, ShunÕichi Kuroda a a
b
Department of Structural Molecular Biology, Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan Department of Nanobiology, Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan Received 8 July 2005
Abstract RBCC protein interacting with C-kinase (RBCK1) is a transcription factor belonging to the RING-IBR protein family and has been shown to shuttle between the nucleus and cytoplasm, possessing both the nuclear export and localization signals within its amino acid sequence. RBCK2, lacking the C-terminal half of RBCK1 including the RING-IBR domain, has also been identified as an alternative splice variant of RBCK1. RBCK2 shows no transcriptional activity and instead it represses the transcriptional activity of RBCK1. Here, we show that RBCK2 is present usually in the cytoplasm containing two Leu-rich regions that presumably serve as a nuclear export signal (NES). Moreover, an NES-disrupted RBCK1 that is mostly localized within the nucleus is translocated to the cytoplasm when coexpressed with RBCK2, suggesting that RBCK2 serves as a cytoplasmic tethering protein for RBCK1. We propose a novel and general function of RING-lacking splice variants of RING proteins to control the intracellular localization and functions of the parental RING proteins by forming a hetero-oligomeric complex. 2005 Elsevier Inc. All rights reserved. Keywords: RING protein; Splice variant; Transcription factor; Nuclear export signal
RBCC protein interacting with C-kinase 1 (RBCK1) consists of 498 amino acid residues harboring a ubiquitin-like (UBL) sequence, two coiled-coil regions, and a RING-IBR domain, arranged from N- to C-terminus (Fig. 1A). The RING-IBR domain is composed of two RING fingers and an Ôin-between-RING fingersÕ (IBR) domain. The protein was originally identified as a protein q Abbreviations: RBCK1, RBCC protein interacting with PKC 1; PKC, protein kinase C; IBR, in-between-RING fingers; NES, nuclear export signal; NLS, nuclear localization signal; PML, promyelocytic leukemia; CRE, cyclic AMP response element; CREB, CRE-binding protein; CBP, CREB-binding protein; HEK293, human embryonic kidney 293; GAL4DBD, GAL4 DNA-binding domain; LMB, leptomycin B; BRCA1, breast cancer-linked tumor suppressor. * Corresponding author. Fax: +81 6 6879 8464. E-mail address: [email protected] (K. Tatematsu).
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.07.104
kinase C (PKC) b-interacting protein by the yeast twohybrid screening and shown to be a transcription factor possessing both transcriptional and DNA-binding activities [1]. The transcriptional activity of RBCK1, residing in the RING-IBR domain, is significantly enhanced by protein kinase A (PKA) and repressed by extracellular signal-regulated kinase activator kinase 1 (ERK1) [2]. RBCK1 possesses a classical Leu-rich nuclear export signal (NES) in the segment containing Leu-142 and Leu-145 as well as the nuclear localization signal (NLS) activity in the C-terminal half of its sequence. We have shown recently that these intracellular localization signals facilitate the nucleocytoplasmic shuttling of RBCK1 [3]. Furthermore, intra-nuclear RBCK1 colocalizes with a promyelocytic leukemia protein (PML) and a CREBbinding protein (CBP) present in the nuclear bodies and
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Fig. 1. (A) Schematic structures of RBCK1, RBCK2, and their truncated mutants. Amino acid residue numbers of the N- and C-termini are given for the whole fragments and specific motifs: Ubl, ubiquitin-like; C-C, coiled-coil; RING1, the first RING finger; IBR, in-between-RING fingers; and RING2, the second RING finger. Amino acid sequences of Leu-rich regions and the region that differs between RBCK1 and RBCK2 are indicated by one-letter codes. (B) Intracellular localization of RBCK2 and NES-disrupted mutants. HEK293 cells were transfected with an expression plasmid for the N-terminally FLAG-tagged RBCK1 and NES-disrupted mutants, and incubated with (+) or without () LMB. The FLAG-tagged proteins were visualized by indirect immunofluorescence with the anti-FLAG antibody.
interacts with them. PML, a main constituent of the nuclear body, enhances the tumor suppressor gene product p53-dependent transcription via the interaction with CBP [4]. The transcriptional activity of RBCK1 is thus modulated by interaction with PML and CBP in the nuclear bodies [3]. A 260-residue protein RBCK2 has been identified as an alternative splice variant of RBCK1 [5]. The N-terminal 240-residue sequence of RBCK2 is identical with that of RBCK1, while the remaining C-terminal 20-residue sequence is unique for RBCK2 (Fig. 1A). Unlike RBCK1, RBCK2 shows no transcriptional activity without the RING-IBR domain and instead it represses the transcriptional activity of RBCK1 in a dose-dependent manner [5]. Based on the observation that RBCK2 interacts with RBCK1 but not with itself in the in vitro binding assay, it is postulated that RBCK2 represses the transcriptional activity of RBCK1 by forming an RBCK1/2 hetero-oligomeric complex. In the present studies reported herein, we have analyzed the intracellu-
lar localization of RBCK2, particularly in relation to that of the parental protein RBCK1, and show that RBCK2 represses the transcriptional activity of RBCK1 by tethering it within the cytoplasm. Such a function may be general for other RING-lacking splice variants reported recently for various RING proteins, including an autosomal recessive juvenile parkinsonism-related gene product, Parkin, whose molecular organization is very similar to that of RBCK1 with the N-terminal UBL sequence and the C-terminal RING-IBR domain [6].
Materials and methods Construction of plasmids. Mammalian expression plasmids pTB701-FLAG and pTB701-HA were described previously for expression of N-terminally FLAG- and haemagglutinin (HA)-tagged proteins, respectively [7]. For expression of N-terminally HA- or FLAG-tagged wild-type and truncated forms of RBCK1 [HA- or FLAG-RBCK1, HA- or FLAG-RBCK1(1–120), HA- or FLAG-
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RBCK1(1–240), HA- or FLAG-RBCK1(1–269), and HA- or FLAGRBCK1(270–498)], plasmids pTB701-HA (or FLAG)-RBCK1, pTB701-HA (or FLAG)-RBCK1(1–120), pTB701-HA (or FLAG)RBCK1(1–240), pTB701-HA (or FLAG)-RBCK1(1–269), and pTB701-HA (or FLAG)-RBCK1(270–498) were constructed by inserting DNA fragments encoding a full length, a ubiquitin-like sequence (from Met-1 to Leu-120), an N-terminal 240 amino acid sequence (from Met-1 to Gln-240), N-terminal (from Met-1 to Glu-269), and C-terminal (from Cys-270 to His-498) halves of RBCK1 into pTB701-HA (or FLAG), respectively [3]. For expression of N-terminally HA- or FLAG-tagged wild-type RBCK2 (HA- or FLAGRBCK2), plasmid pTB701-HA (or FLAG)-RBCK2 was constructed by inserting DNA fragment encoding a full length of RBCK2 into pTB701-HA (or FLAG). Plasmids pTB701-FLAG-RBCK1 (L118A/ L121A), pTB701-FLAG-RBCK2(L118A/L121A), pTB701-FLAGRBCK2(L142A/L145A), and pTB701-FLAG-RBCK2(L118A/L121A/ L142A/L145A) were constructed by site-directed mutagenesis for expression of RBCK1(L118A/L121A), RBCK2(L118A/L121A), RBCK 2(L142A/L145A), and RBCK2(L118A/L121A/ L142A/L145A), respectively. A mammalian expression plasmid pM (Promega, Madison, WI) for the N-terminally GAL4DBD (GAL4 DNA-binding domain)fused protein was used for the transcriptional assay. Plasmids pMRBCK1, pM-RBCK1(L142A/L145A), and pM-RBCK1(270–498) were described previously [1,3]. Cell culture. Human embryonic kidney 293 (HEK293) cells were cultured in DulbeccoÕs modified EagleÕs medium supplemented with 10% (v/v) fetal bovine serum at 37 C under humidified air with 5% CO2. Immunocytochemical analysis. HEK293 cells (approximately 5 · 104 cells) were transfected with the mammalian expression plasmids by using a FuGENE 6 transfection reagent (Roche diagnostics, Basel, Switzerland). The cells were grown on a coverglass (diameter, 2.7 cm) for 72 h, washed twice with phosphate-buffered saline (PBS, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, and 1 mM KH2PO4), and fixed with 100% (v/v) methanol at 20 C for 10 min. These cells were permeabilized with 0.15% (v/v) Triton X-100 in PBS for 15 min at room temperature, washed twice with PBS, incubated with blocking buffer [PBS containing 0.03% (v/v) Triton X-100, 2% (w/v) bovine serum albumin (BSA), and 3% (v/v) normal goat serum] for 30 min at room temperature, and incubated with the blocking buffer containing a primary antibody for 30 min at room temperature. The cells were washed four times with PBS containing 0.03% (v/v) Triton X-100 for 10 min at room temperature and mounted with 90% (v/v) glycerol in PBS. Fluorescence of the cells was observed under an LSM 5 PASCAL confocal laser scan microscope (Carl Zeiss, Oberkochen, Germany). The HA-tagged proteins were detected with an anti-HA rabbit polyclonal antibody (MBL, Nagoya, Japan) (dilution, 1:400) or an anti-HA mouse monoclonal antibody (12CA5) (Roche Diagnostics) (dilution, 1:600) as a primary antibody. The FLAG-tagged proteins were reacted with an anti-FLAG mouse monoclonal antibody (M2) (Sigma, St. Louis, MO) (dilution, 1:1,000) or an anti-FLAG rabbit polyclonal antibody (Sigma) (dilution, 1:250) as a primary antibody. A Cy2-labeled anti-rabbit IgG goat polyclonal antibody (KPL, Gaithersburg, MD) (dilution, 1:600) or a Cy3-labeled anti-mouse IgG goat polyclonal antibody (Amersham Biosciences, Uppsala, Sweden) (dilution, 1:800) was used as a secondary antibody. Immunoprecipitation and Western blotting. HEK293 cells (approximately 1 · 107 cells) were cotransfected with pTB701-HA-RBCK2 (5 lg) and pTB701-FLAG-RBCK1 (2–6 lg) by electroporation, and cultured for 48 h. The cells were washed twice with PBS and suspended in 1 ml of the lysis buffer [50 mM Tris–HCl at pH 7.5, 150 mM NaCl, 0.1% (v/v) Nonidet P-40, 1 mM dithiothreitol, and 1 tablet of Complete protein inhibitor cocktail (Roche diagnostics) per 50 ml]. The cleared lysate was incubated with 10 lg of an anti-HA mouse monoclonal antibody on ice for 60 min and incubated with 30 ll of protein G–Sepharose 4 [50% (v/v) slurry] (Amersham Biosciences) at 4 C for 30 min. The beads were washed four times with 1 ml of lysis buffer, resuspended
in 35 ll of LaemmliÕs sample buffer, subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA). Western blot analyses were carried out with a peroxidase-conjugated anti-HA mouse monoclonal antibody (Roche Diagnostics) (dilution, 1:5000) or an anti-FLAG mouse monoclonal antibody (Sigma) (dilution, 1:5000). Immunoreactive bands were visualized by the enhanced chemiluminescence method with an ECL Plus (Amersham Biosciences) according to the manufacturerÕs protocol. Luciferase assay. Transcriptional activities of GAL4DBD-fused RBCK1 or its mutants were measured by luciferase reporter-gene assays. HEK293 cells (about 1 · 106 cells) were cotransfected using FuGENE 6 with the reporter plasmid pFR-Luc (Stratagene, La Jolla, CA) (1 lg) containing the firefly-derived luciferase gene 3 0 -downstream of the synthetic promoter consisting of five repeats of the GAL4-recognition site (17-mer), pM-RBCK1 (25 ng), and pTB701-FLAGRBCK2. After 24 h, the cells were washed once with PBS and lysed with 200 ll of passive lysis buffer (Promega). The cell lysate was assayed with a luciferase assay kit (Promega) according to the manufacturerÕs protocol. Transcriptional activities were indicated by the relative luciferase unit (RLU) divided by protein concentration, which was measured with a bicinchoninic acid (BCA) assay kit (Sigma) using BSA as a standard.
Results Identification of NES sequences in RBCK2 We first investigated the intracellular localization of RBCK2. When RBCK2 was expressed in HEK293 cells, the protein was exclusively localized in the cytoplasm (Fig. 1B-a). Upon treatment with leptomycin B (LMB), an inhibitor for the CRM1-dependent nuclear export [8], a considerable portion of RBCK2 was translocated from the cytoplasm to the nucleus and was detected as dot-like structures (Fig. 1B-b), indicating that RBCK2 possesses an NES sequence(s) recognized by the CRM1-dependent nuclear export system. We have already reported that RBCK1 possesses a conventional Leu-rich NES in the region containing Leu-142 and Leu-145 [3]. Since RBCK2 has an identical amino acid sequence with RBCK1 for residues 1–240 (Fig. 1A), the same region (Leu-142 to Leu-145) has been assumed to serve as a potential NES of RBCK2. However, when the two Leu residues were mutated into Ala, the mutant protein RBCK2(L142A/L145A) was mostly retained in the cytoplasm (Fig. 1B-c). This result contrasts with the previous result for RBCK1, in which mutation of the same two Leu residues into Ala resulted in the complete translocation of RBCK1 into the nucleus [3], and suggests that RBCK2 possesses an additional NES sequence(s) that can function independently of the NES at Leu-142 and Leu-145. By inspecting the RBCK2 sequence, the segment containing Leu-118 and Leu-121 was found to match moderately to the consensus Leurich NES sequence [9] (Fig. 1A) and besides this segment there was no Leu-rich region. Therefore, these two Leu residues (Leu-118 and Leu-121) were then replaced by
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Ala by site-specific mutagenesis and the intracellular localization of the mutant protein RBCK2(L118A/ L121A) was analyzed. As shown in Fig. 1B-e, the mutant protein of RBCR2 was still detected in the cytoplasm, although enriched in the outer perinuclear region, suggesting that the former NES sequence (Leu142 to Leu-145) is alternately functioning in this mutant. Hence, finally the four Leu residues (Leu-118, 121, 142, and 145) were all replaced by Ala. The resultant quadruple mutant, RBCK2(L118A/L121A/L142A/L145A), was present evenly in both the cytoplasm and nucleus (Fig. 1B-g). These results suggest that both of the two Leu-rich regions in RBCK2 can serve independently as NES. A considerable portion of the RBCK2 mutant observed in the nucleus is presumably derived from free diffusion of RBCK2 that has no NLS. It has been reported that proteins smaller than 40 kDa could be
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translocated into the nucleus by free diffusion, when their functional NES is destroyed [10]. The newly identified NES (Leu-118 to Leu-121) in RBCK2 is also contained in the parental protein, RBCK1 (Fig. 1A). However, disruption of only the previously identified NES (Leu-142 to Leu-145) was sufficient for the complete intra-nuclear localization of RBCK1 [3]; the NES at Leu-118 and Leu-121 does not function in the RBCK1(L142A/L145A) mutant. Moreover, mutation of Leu-118 and Leu-121 in RBCK1 little affected its localization in the cytoplasm (Fig. 1B-i), suggesting that the NES at Leu-142 and Leu-145 is working in this mutant. Presumably, the potential NES region containing Leu-118 and Leu-121 may be structurally masked in RBCK1 not to function as NES. Similar masking of nucleocytoplasmic translocation signals has been reported for the NES of BRCA1 [11] and the
Fig. 2. Immunoprecipitation assays for identification of the region of RBCK1 interacting with RBCK2. HEK293 cells were transfected with an expression plasmid for the N-terminally HA- and FLAG-tagged proteins. The top panels in (A)–(D) show the detection of FLAG-tagged proteins in the immunoprecipitates obtained with an anti-HA antibody. The middle panels in (A)–(D) show the detection of FLAG-tagged proteins in the cell lysates. The bottom panels in (A)–(D) show the detection of HA-tagged proteins in the immunoprecipitates obtained with an anti-HA antibody.
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NLS of NF-jB p65 subunit [12]. It is noteworthy that treatment with LMB led to the considerable intra-nuclear localization of wild-type and mutant proteins of RBCK2, particularly in the dot-like structures (Figs. 1B-b, -d, -f, and -h). This may be caused by interaction of RBCK2 with an unidentified nuclear protein(s), such as endogenous RBCK1 that has already been shown to associate with the nuclear bodies [3]. Identification of region of RBCK1 interacting with RBCK2 We previously showed that RBCK1 interacts with itself and also with RBCK2, but the latter does not interact with itself [5]. To identify the region(s) of RBCK1 interacting with RBCK2, N- and C-terminally truncated mutants of RBCK1 were constructed (see Fig. 1A) and coexpressed with RBCK2 in HEK293 cells. Immunoprecipitation assays have revealed that RBCK2 interacts with the N-terminal half of RBCK1 [from Met-1 to Glu-269; RBCK1(1–269)] but not with the C-terminal half [from Cys-270 to His-498; RBCK1(270–498)] (Fig. 2A), indicating that the N-terminal half of RBCK1 is involved in the interaction with RBCK2. Although RBCK2 having the same amino acid sequence as RBCK1 for residues 1–240 did not interact with itself as reported previously [5] (see also Fig. 2A), the N-terminal half of RBCK1 (1–269) interacted with
itself (Fig. 2C). This suggests that the C-terminal unique 20-residue sequence of RBCK2 (residues 240–260) has an interfering effect on the self-interaction of RBCK2. On the other hand, RBCK1(1–240) interacted with itself, with RBCK2, and also with RBCK1(1–269) (Fig. 2D). In addition, the UBL region (residues 1– 120) of RBCK1/2 was found not to be involved in the interaction between RBCK1 and RBCK2 (data not shown). Collectively, these results show that the region covering residues 121–240 is commonly participating in the self-interaction of RBCK1 and the heterologous interaction between RBCK1 and RBCK2. Cytoplasmic tethering of RBCK1 by RBCK2 When the wild-type proteins of RBCK1 and RBCK2 were coexpressed in HEK293 cells, they colocalized in the cytoplasm (Fig. 3D), suggesting that the two proteins are interacting with each other within the cells, as shown above by immunoprecipitation assays. The effect of coexpression of RBCK2 on the intracellular localization of RBCK1 was further examined using the NESdisrupted and N-terminally truncated forms of RBCK1. As already reported [3], the NES-disrupted RBCK1 [RBCK1(L142A/L145A)] was predominantly localized in the nucleus without coexpression of RBCK2 (Fig. 3E). Significantly, coexpression of RBCK2 prevented completely the translocation of the NES-disrupt-
Fig. 3. Intracellular localization of RBCK1 and its mutant coexpressed with RBCK2. Cytoplasmic tethering of RBCK1 by RBCK2. (A–L) HEK293 cells were transfected with an expression plasmid for the N-terminally HA-tagged wild-type RBCK1, RBCK1(L142A/L145A), or RBCK1(270–498) with (B, F, and J) or without (A, E, and I) an expression plasmid for the FLAG-tagged RBCK2. The HA- and FLAG-tagged proteins were visualized by indirect immunofluorescence with an anti-HA antibody (A, B, E, F, I, and J) and an anti-FLAG antibody (C, G, and K), respectively. Merge, merged images (D, H, and L).
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ed RBCK1 into the nucleus (Fig. 3F) and the RBCK1 mutant retained in the cytoplasm colocalized with RBCK2 (Fig. 3H). The C-terminal half of RBCK1 [RBCK1(270–498)], which does not interact with RBCK2, was unaffected in its nuclear localization by the coexpression of RBCK2 (Figs. 3I, J, and L). These results clearly show that the cytoplasmic RBCK2 tethers the nucleocytoplasmic shuttling protein RBCK1 in the cytoplasm by interacting presumably through the regions containing residues 121–240.
ase reporter-gene assays (Fig. 4). In marked contrast, the transcriptional activity of the C-terminal half of RBCK1 [RBCK1(270–498)], existing in the nucleus irrespectively of the presence of RBCK2, was almost unaffected by the coexpression of RBCK2. Based on these results, we conclude that RBCK2 represses the transcriptional activity of RBCK1 by tethering it within the cytoplasm and preventing its translocation into the nucleus.
Repression of transcriptional activity of RBCK1 by RBCK2
Discussion
Previously, the transcriptional activity of RBCK1 was shown to be repressed by RBCK2 depending on the intracellular amount of expressed RBCK2 protein [5] (see also Fig. 4). The effect of coexpression of RBCK2 on the transcription activity of RBCK1 was further analyzed using the NES-disrupted and N-terminally truncated forms of RBCK1, expressed as a GAL4DBD-fused protein in HEK293 cells. The transcriptional activity of the NES-disrupted RBCK1 mutant, localizing usually in the nucleus in the absence of RBCK2 and exhibiting a very high transcriptional activity, was significantly repressed depending on the amount of expression plasmid for RBCK2 applied in the lucifer-
Fig. 4. Effect of coexpression of RBCK2 on the transcriptional activity of RBCK1. An expression plasmid for GAL4DBD (Mock) or the GAL4DBD-fused form of RBCK1, RBCK1(L142A/L145A), or RBCK1(270–498) was cotransfected to HEK293 cells with an indicated amount (ng) of the expression plasmid for FLAG-RBCK2 and the plasmid containing the luciferase reporter gene. Transcriptional activities of the GLA4DBD-fused proteins were measured by the luciferase assay and indicated as relative values against the negative control (GAL4DBD = 1). Each value was the mean of those obtained from six independent measurements and error bars indicate a 95% confidence interval.
Many transcription factors are known as nucleocytoplasmic proteins, whose transcriptional activities are closely associated with their nuclear localization, which, in turn, is governed by the cooperative functions of NES and NLS and also regulated by a number of intracellular events, such as phosphorylation, ubiquitination, sumoylation, and so on. For example, NF-jB p65 subunit, one of the major inflammatory responsive transcriptional factors, is normally localized in the cytoplasm with its NLS sequence masked by IjBa containing NES. When IjBa is phosphorylated by inflammation-activated IKK (IjBa kinase), it undergoes polyubiquitination and the following degradation, leading to unmasking of the NLS of NF-jB p65 subunit and then translocation into the nucleus for initiating the transcription [12]. Another example is a cytoplasmic RING protein the breast cancer-linked tumor suppressor 1 (BRCA1). When the NES of BRCA1 is inactivated by association with BRCA1-associated RING domain protein 1 (BARD1), the BRCA1–BARD1 complex is translocated into the nucleus by the NLS contained in BRCA1 [11]. Thus, the intracellular localization of a nucleocytoplasmic shuttling protein is often regulated by its associating protein, and the protein–protein interaction is significantly affected by modification of the partner protein as well as the balance of intracellular amounts of each protein. As revealed in the present studies using an overexpression system, RBCK2, a splice variant of RBCK1, functions as an anchoring protein for the parental RBCK1 and represses its transcriptional activity by tethering it within the cytoplasm. For exhibiting the transcriptional activity, RBCK1 must be released from the RBCK1–RBCK2 complex and translocated into the nucleus. However, it remains to be investigated what kind of modification occurs on RBCK1 or RBCK2 and in which signal cascade it leads to the dissociation of the RBCK1–RBCK2 complex and the translocation of RBCK1 into the nucleus. As for the intracellular expression levels of RBCK1 and RBCK2, RBCK1/2 mRNA is expressed ubiquitously in all adult rat tissues with the mRNA ratio of RBCK2 over RBCK1 being about 10% [5] and it is unknown if this ratio changes depending on the cell states. Thus, more detailed studies are cer-
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Fig. 5. Schematic structures of various RING proteins and their RING-lacking splice variants. The GenBank accession numbers, when available, are indicated below each protein name. Amino acid residue numbers of the N- and C-termini are provided for each protein. Abbreviations for specific motifs are the same as in Fig. 1, except for BRCT (BRCA1 C-terminus domain) and FHA (fork head-associated domain). Closed triangles indicate the point of exon junction causing a frameshift, which introduces a stop codon at a point different from the parental protein. Open triangles indicate the point of exon deletion occurring by alternative splicing.
tainly needed to elucidate the in vivo function of RBCK1 and its splice variant RBCK2. Shown in Fig. 5 are the schematic structures of various RING-IBR proteins and their RING-lacking splice variants recently identified. The full-length BARD1 forms a complex with BRCA1 and colocalizes in the nucleus, whereas a RING-lacking splice variant of BARD1 does not affect the cytoplasmic localization of BRCA1 without interacting with it [13]. Also, BRCA1 shows an ubiquitin ligase activity only in the complex with the full-length BARD1. Furthermore, some RING proteins possessing a transcriptional activity, such as RING finger protein 8 (RNF8) [14] and BRCA1 [15], also have their own RING-lacking splice variants. RNF8 is localized in the nucleus, while the RING-lacking variant is localized in the cytoplasm [14]. BRCA1 itself has both NLS and NES, but its RING-lacking variant lacks NES. Parkin, having the same molecular organization as RBCK1 with an N-terminal UBL domain and a C-terminal RINGIBR domain, also has a RING-lacking splice variant similar to RBCK2 [6]. On the basis of the findings reported in this paper, we propose that these RINGlacking splice variants of RING proteins generally have a function to control the intracellular localization of the parental full-length RING proteins by forming a hetero-oligomeric complex and thereby regulate their cellular functions.
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