Báo cáo khoa học: Accumulation of polyubiquitinated proteins by overexpression of RBCC protein interacting with docx

Accumulation of polyubiquitinated proteins by overexpression of RBCC protein interacting with protein kinase C2, a splice variant of ubiquitin ligase RBCC protein interacting with protein kinase C1 Nobuo Yoshimoto 1,2 , Kenji Tatematsu 1 , Toshihide Okajima 3 , Katsuyuki Tanizawa 1 and Shun’ichi Kuroda 1,2 1 Department of Structural Molecular Biology, Institute of Scientific and Industrial Research, Osaka University, Japan 2 Laboratory of Industrial Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Japan 3 Department of Nanobiology, Institute of Scientific and Industrial Research, Osaka University, Japan Keywords 26S proteasome; RING-IBR; S5a; splice variant; ubiquitin Correspondence K. Tatematsu and S. Kuroda, Laboratory of Industrial Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8601, Japan Fax: +81 52 789 5227 Tel: +81 52 789 5227 E-mail: ktatematsu@ucsd.edu and skuroda@agr.nagoya-u.ac.jp (Received 10 June 2009, revised 26 August 2009, accepted 3 September 2009) doi:10.1111/j.1742-4658.2009.07350.x The nuclear–cytoplasmic shuttling protein RBCC protein interacting with protein kinase C1 (RBCK1) possesses transcriptional and ubiquitin ligase activities. We have recently reported that RBCC protein interacting with protein kinase C2 (RBCK2), a RING–in-between-RING fingers domain- lacking splice variant of RBCK1, lacks transcriptional activity, but rather represses the RBCK1-mediated transcriptional activity as a cytoplasmic tethering protein for RBCK1. In this study, we have found that RBCK2 overexpressed in human embryonic kidney 293 cells interacts with the poly- ubiquitin chain and the polyubiquitin-interacting subunit S5a, and signifi- cantly increases the intracellular amount of polyubiquitinated proteins. These results strongly suggested that RBCK2 functions as an adaptor protein for the polyubiquitinated protein and the S5a subunit in 26S proteasome through its novel zinc finger motif and ubiquitin-like domain, respectively, presumably delivering the polyubiquitinated proteins to the entrance of the 26S proteasome catalytic domain for degradation. Structured digital abstract l MINT-7261426: S5a (uniprotkb:P55036) binds (MI:0407)toRbck1 (uniprotkb:Q62921)by pull down ( MI:0096) l MINT-7261435: S5a (uniprotkb:P55036) physically interacts (MI:0915) with Rbck1 (uniprotkb: Q62921)byanti bait coimmunoprecipitation (MI:0006) l MINT-7261448: S5a (uniprotkb:P55036) physically interacts (MI:0915) with Rbck1 (uni- protkb: Q62921)byanti tag coimmunoprecipitation (MI:0007) l MINT-7261462: Rbck1 (uniprotkb:Q62921) physically interacts (MI:0915) with Ubiquitin (uniprotkb: P62988)byanti tag coimmunoprecipitation (MI:0007) l MINT-7261503: S5a (uniprotkb:P55036) binds (MI:0407)toUbiquitin (uniprotkb:P62988)by pull down ( MI:0096) l MINT-7261477: Rbck1 (uniprotkb:Q62921) binds (MI:0407)toUbiquitin (uniprotkb:P62988) by pull down ( MI:0096) Abbreviations CBP, CREB-binding protein; E3, ubiquitin ligase; GST, glutathione S-transferase; HA, hemagglutinin; HEK293, human embryonic kidney 293; HHR23, human homolog of Rad23; HRP, horseradish peroxidase; IBR, in-between-RING fingers; NES, nuclear export signal; NLS, nuclear localization signal; NZF, novel zinc finger; PML, promyelocytic leukemia protein; RBCK1, RBCC protein interacting with protein kinase C1; RBCK2, RBCC protein interacting with protein kinase C2; UBA, ubiquitin-associated; UBL, ubiquitin-like; UIM, ubiquitin-interacting motif. FEBS Journal 276 (2009) 6375–6385 ª 2009 The Authors Journal compilation ª 2009 FEBS 6375 Introduction RBCC protein interacting with protein kinase C1 (RBCK1), consisting of 498 amino acids, has been iso- lated from a rat brain cDNA library by using a yeast two-hybrid system with the regulatory region of protein kinase Cb as bait [1] (Fig. 1A). The protein contains a ubiquitin-like (UBL) domain, a novel zinc finger (NZF) motif between two coiled-coil regions, a first RING finger (RING1), an ‘in-between-RING fingers (IBR) motif, and a second RING finger (RING2) from the N-terminus to the C-terminus [1–3]. A RING finger coordinating two zinc ions is found in many transcription factors and ubiquitin ligases (E3s). The RING finger in the transcription factors plays a pivotal role in either sequence-specific DNA binding [4] or transcriptional regulation [5,6]. RBCK1 has been shown to possess both DNA-binding and transcrip- tional activities [1]. On the other hand, the RING finger in E3s functions as a scaffold for either its specific substrate or ubiquitin-conjugating enzymes [7,8]. Recently, it was revealed that RBCK1 possesses an E3 activity in addition to the transcriptional and DNA-binding activities [9]. RBCC protein interacting with protein kinase C2 (RBCK2), a splice variant of RBCK1 (Fig. 1A), con- sists of 260 amino acids and shares the N-terminal 240 amino acids with RBCK1. RBCK2 contains the UBL domain, two coiled-coil regions, and the NZF motif, but not the RING1–IBR–RING2 domain (RING– IBR domain). The C-terminal 20 amino acid sequence of RBCK2 is generated by the alternative splicing of the RBCK1 mRNA, and shows no similarity to RBCK1 [10]. RBCK2 and RBCK1 mRNAs are ubiqui- tously expressed in adult rat tissues. The amount of RBCK2 mRNA was estimated to be about 10% that of RBCK1 mRNA in all tissues [10]. RBCK2 interacts with the N-terminal half of RBCK1 but not with itself [11]. Whereas RBCK1 possesses a nuclear export signal (NES) and a nuclear localization signal (NLS) concurrently, and shuttles between the cytoplasm and the nucleus [12], RBCK2 has two distinct NESs and A B Fig. 1. Effect of overexpression of RBCK2 on accumulation of polyubiquitinated pro- teins. (A) Schematic drawing of RBCK1 and RBCK2. Amino acid residue numbers of the N-termini and C-termini are indicated for the whole fragments and their domains: UBL, ubiquitin-like; C-C, coiled-coil; RING1, first RING finger; IBR, in-between-RING fingers; RING2, second RING finger; and NZF, novel zinc finger. The consensus amino acids of the NZF motif are indicated in the gray box. The cysteine residues essential for the formation of zinc clusters are indicated in the black box. (B) Effects of overexpression of RBCK2 on the amount of polyubiquitinat- ed protein in cells. An expression plasmid for the N-terminally HA-tagged RBCK2 was cotransfected into HEK293 cells with an expression plasmid for FLAG-tagged ubiquitin. The upper and lower panels show the detection of polyubiquitinated proteins and overexpressed RBCK2 in the cell lysate, respectively. MG132, a specific inhibitor of the proteasome, was used. Potential polyubiquitinated protein carrier RBCK2 N. Yoshimoto et al. 6376 FEBS Journal 276 (2009) 6375–6385 ª 2009 The Authors Journal compilation ª 2009 FEBS localizes preferentially in the cytoplasm [11]. When RBCK2 is coexpressed with RBCK1 in human embry- onic kidney 293 (HEK293) cells, RBCK1 localizes preferentially in the cytoplasm, indicating that RBCK2 functions as a cytoplasmic tethering protein specific for RBCK1. RBCK2 per se does not show transcriptional activity [10]. However, RBCK2 represses the RBCK1- mediated transcriptional activity in a dose-dependent manner by forming a hetero-oligomeric complex with RBCK1 in the cytoplasm [10,11]. When RBCK2 has been expressed in HEK293 cells, it has been unexpectedly found that RBCK2, not RBCK1, solely induces the significant accumulation of polyubiq- uitinated proteins (Fig. 1B). This result has led us to investigate the function of RBCK2 in the ubiquitin– proteasome system. In this study, we have found that RBCK2 directly and concurrently interacts with poly- ubiquitin chains and the S5a subunit of the 19S regulatory subcomplex of the 26S proteasome through its NZF motif and UBL domain, respectively. We here propose that RBCK2 contributes to the efficient delivery of polyubiquitinated substrates to the 26S proteasome. Results Accumulation of polyubiquitinated proteins by overexpressed RBCK2 When hemagglutinin (HA)-tagged RBCK2 and FLAG-tagged ubiquitin were coexpressed in HEK293 cells, the lysates were subjected to western blotting using antibody against FLAG. Whereas endogenous proteins were polyubiquitinated, which was not detected under reduced sensitivity (Fig. 1B, lane 1), a significant amount of polyubiquitinated proteins was found to unexpectedly accumulate in the cells (Fig. 1B, lane 3). A comparable amount of polyubiquitinated proteins was also observed in the HEK293 cells treated with MG132, a proteasomal inhibitor (Fig. 1B, lane 2). As RBCK2 lacks a RING domain, which plays a piv- otal role in the E3 activity (Parkin [13], MDM2 [14], and RNF8 [15]), the overexpressed RBCK2 was postu- lated to participate in either the enhancement of the activity of endogenous E3s or the inhibition of prote- asomal degradation of polyubiquitinated proteins. Colocalization of RBCK2 with the 26S proteasome The subcellular localization of endogenous RBCK1 ⁄ 2 (RBCK1 and RBCK2) was investigated in HEK293 cells. As described previously [12], RBCK1 ⁄ 2 resides in the cytoplasm and nuclear bodies (Fig. 2A). Immuno- chemical observations showed that endogenous RBCK1 ⁄ 2 colocalizes with the 20S catalytic core sub- complex of the 26S proteasome mainly in the cyto- plasm (Fig. 2A, upper panels). After the MG132 treatment, the 20S subcomplex was translocated from the cytoplasm to the nucleus, as reported previously (Fig. 2B,E) [16]. The RBCK1 ⁄ 2 proteins were also translocated from the cytoplasm to the nucleus (Fig. 2A,D), suggesting that RBCK1 ⁄ 2 is involved in the 20S subcomplex. Direct interaction of RBCK2 with S5a The UBL domains from Parkin, an autosomal-recessive juvenile parkinsonism-related protein [13], and a human homolog of Rad23 (HHR23) directly interact with 19S proteasomal subunit S5a (a subunit of the 19S regula- tory subcomplex of the 26S proteasome) [17–19]. We therefore examined whether or not RBCK2 containing a UBL domain (Fig. 1A) interacts with S5a. Bacterially expressed RBCK2 was subjected to an in vitro pull- down assay using purified N-terminally glutathione S-transferase (GST)-fused S5a (GST–S5a; Fig. 3A). RBCK2 was pulled down with GST–S5a but not with GST, indicating that RBCK2 directly interacts with S5a in vitro. In the immunoprecipitation assay using HEK293 cells overexpressing RBCK2, interaction of endogenous S5a with RBCK2 was observed (Fig. 3B, lane 2). To delineate the region of RBCK2 that is essen- tial for the interaction with S5a, HA-tagged human S5a was coexpressed with FLAG-tagged RBCK2 or FLAG-tagged RBCK2DUBL (131–260 amino acids of RBCK2) in HEK293 cells, and subjected to an immu- noprecipitation analysis. Whereas RBCK2 interacted with S5a (Fig. 3C, lane 2), the deletion of the UBL domain resulted in the abolishment of the S5a–RBCK2 interaction (Fig. 3C, lane 3). The UBL domain of HHR23 efficiently interacts with a ubiquitin-interacting motif (UIM) 2 in S5a [20,21]. Thus, the UBL domain of RBCK2 might be essential for the interaction with the S5a UIM2. The interaction was slightly weakened by MG132 treatment (Fig. 3B, lane 4). As polyubiqu- itin chains (larger than tetraubiquitin chains) directly interact with both UIM1 and UIM2 in S5a [22], the RBCK2–S5a interaction may be disturbed by the competition of the S5a UIM2 with newly synthesized polyubiquitin chains (Fig. 1B, lane 2). Direct interaction of RBCK2 with polyubiquitin chains The NZF motif of Npl4, an endoplasmic reticulum- associated degradation-related protein [3], directly N. Yoshimoto et al. Potential polyubiquitinated protein carrier RBCK2 FEBS Journal 276 (2009) 6375–6385 ª 2009 The Authors Journal compilation ª 2009 FEBS 6377 interacts with polyubiquitinated proteins [3]. The NZF motif of RBCK2 was also assumed to interact with polyubiquitinated proteins. The lysate of HEK293 cells overexpressing RBCK2 was subjected to immuno- precipitation analysis. Whereas a small amount of polyubiquitinated protein was observed in the immu- noprecipitates of RBCK2 (Fig. 4A, lane 2), the amount was significantly increased by the MG132 treatment (Fig. 4A, lane 4), indicating that RBCK2 interacts with polyubiquitinated proteins. Polyubiquitin chains linked with Lys48 of ubiquitin are efficiently and specifically recognized by the 26S proteasome [23]. Therefore, the direct interaction of the Lys48-linked polyubiquitin chain with RBCK2 was examined by an in vitro pull-down assay with bacterially expressed purified GST-fused proteins (Fig. 4B,C). As reported previously [22], polyubiquitin chains larger than tetra- ubiquitins directly interact with purified GST–S5a (positive control; Fig. 4B, lane 2). RBCK2 was shown to interact directly with polyubiquitin chains larger than triubiquitin chains (Fig. 4B, lane 4). The ubiqu- itin–RBCK2 interaction was abolished by quadruple mutation of the NZF motif of RBCK2 (Fig. 4B, lane 5), which is the replacement of four cysteines essential for the NZF motif with serines (Fig. 1A). Taken together, these findings show that RBCK2 interacts with poly- ubiquitin chains through its NZF motif. Discussion RBCK2 as a potential polyubiquitinated protein carrier RBCK2 has been shown to interact directly with S5a and polyubiquitin chains (larger than triubiquitin chains). On the basis of previous studies [3,17,20,21] and this study, the interactions of S5a and polyubiqu- itin chains are mediated by the UBL domain and the NZF motif of RBCK2, respectively (Fig. 5A, upper model). This finding indicates that the ternary complex consisting of RBCK2, S5a and a polyubiquitin chain is involved in the ubiquitin–proteasome system. HHR23, containing a UBL domain and two ubiquitin-associated (UBA) domains (UBA1 and UBA2), was found to interact with S5a and ubiquitin chains (larger than monoubiquitin), respectively [18,24]. In primary human fibroblasts, HHR23 was shown to form the ternary complex of HHR23 (HHR23–S5a–ubiquitin chain; Fig. 5A, lower model), and this was followed by the delivery of p53 ubiquitinated by an E3 enzyme, MDM2, to the 26S proteasome [25]. The overexpres- sion of hPlic-2 (a human homolog of yeast Dsk2) protein, containing a UBL domain and a UBA domain, also induces the accumulation of p53 in HeLa cells [26]. Overexpressed RBCK2 might play a similar role. A B C D E F Fig. 2. Localization of endogenous RBCK1 ⁄ 2 and the 20S proteasome. (A–F) The intracellular localization of endogenous RBCK1 ⁄ 2 and the 20S proteasome was detected by indirect immunofluorescence. HEK293 cells were cultured with or without MG132 and stained with an antibody against RBCK (A, D) or an antibody against the 20S proteasome (B, E). Merge, merged images (C, F). Potential polyubiquitinated protein carrier RBCK2 N. Yoshimoto et al. 6378 FEBS Journal 276 (2009) 6375–6385 ª 2009 The Authors Journal compilation ª 2009 FEBS Overexpression of either HHR23, hPlic-2 or RBCK2 was considered to disturb the delivery of polyubiquiti- nated proteins to the 26S proteasome by depriving the polyubiquitin chain of the ternary complex containing the ‘ubiquitin carrier protein’, S5a, and the polyubiqu- itin chain. On the basis of these findings, RBCK2 was postulated to be a ‘polyubiquitinated protein carrier’ facilitating the delivery of polyubiquitinated proteins to the 26S proteasome by forming a ternary complex con- taining RBCK2, S5a, and the polyubiquitin chain (Fig. 5A, upper model). RBCK1, harboring both an NES and an NLS, shut- tles between the cytoplasm and the nucleus and inter- acts with the transcription factor promyelocytic leukemia protein (PML) and the coactivator, CREB- binding protein (CBP), in nuclear bodies [12]. RBCK2 is constitutively excluded from the nucleus by its NES activity, and is a cytoplasmic tethering protein for RBCK1 [11]. In this study, we found that RBCK2 induces the accumulation of polyubiquitinated proteins in cooperation with the 26S proteasome. These results strongly suggested that RBCK2 is a potential cytoplas- mic ‘polyubiquitinated protein carrier’ facilitating the delivery of polyubiquitinated proteins to the 26S pro- teasome in the cytoplasm. RBCK2 might be excluded from the nucleus so as not to disturb the RBCK1-med- iated cellular functions in the nucleus, such as tran- scriptional activation involving PML and CBP. As RBCK1 harbors DNA-binding activity per se, transcriptional activity [1], and E3 activity [9], it is still unknown whether the E3 activity of RBCK1 is operative with the proteasome in the nucleus or not. A B C Fig. 3. Interaction of RBCK2 with the 19S proteasome regulatory subunit S5a. (A) In vitro binding between S5a and RBCK2. N-termi- nally GST-fused S5a was bound to glutathione–agarose beads, incu- bated with purified RBCK2, and subjected to a GST pull-down assay. RBCK2 precipitated with GST–S5a was detected by western blotting with an antibody against RBCK. (B) In vivo binding between endogenous S5a and RBCK2. HEK293 cells were transfected with an expression plasmid for the N-terminally FLAG-tagged RBCK2, and cultured with or without MG132. The top panel shows the FLAG-tagged RBCK2 coimmunoprecipitated with an antibody against S5a. The middle panel shows the FLAG-tagged RBCK2 in the cell lysate. The bottom panel shows the endogenous S5a in the immunoprecipitates obtained with an antibody against S5a. (C) Requirement of the UBL domain for the S5a interaction. HEK293 cells were cotransfected with expression plasmids for the N-termi- nally HA-tagged S5a and either the N-terminally FLAG-tagged RBCK2 or RBCK2DUBL. The top panel shows the FLAG-tagged proteins coimmunoprecipitated with an antibody against HA. The middle panel shows the FLAG-tagged RBCK2 and RBCK2DUBL in the cell lysate. The bottom panel shows HA-tagged S5a in the immunoprecipitates obtained with an antibody against HA. N. Yoshimoto et al. Potential polyubiquitinated protein carrier RBCK2 FEBS Journal 276 (2009) 6375–6385 ª 2009 The Authors Journal compilation ª 2009 FEBS 6379 RBCK2-interacting proteins The 19S proteasomal subunit S5a and a yeast homo- log, Rpn10, have two UIMs (UIM1 and UIM2) and one UIM, respectively. Each UIM of S5a interacts with the polyubiquitin chain [22]. In particular, the C-terminal UIM (UIM2) of S5a interacts with the UBL domains of HHR23 and hPlic-2 [20,21,26]. On the basis of the NMR analysis for the complex of the S5a UIM2 with the HHR23 UBL domain, Leu38, Ile44, Gly47 and Val70 in the UBL domain are essen- tial for the association with S5a (Fig. 5B) [20,27]. Three hydrophobic residues and glycine are well con- served in HHR23, RBCK1, and RBCK2 (Fig. 5B). It is plausible that these residues of RBCK2 play crucial roles in the interaction with the UIM2 of S5a. This, in turn, corroborates the formation of the ternary RBCK2–polyubiquitinated protein–S5a complex in the vicinity of the 26S proteasome. In this study, the NZF motif of RBCK2 was found to interact directly with the Lys48-linked polyubiquitin chain (Fig. 4B), which is exclusively involved in prote- asomal degradation [22,23]. Recently, various lysine residues of ubiquitin (e.g. Lys6, Lys11, Lys27, Lys29, Lys33, and Lys63) were revealed to be involved in the formation of polyubiquitin chains [28–32]. Unlike the Lys48-linked polyubiquitin chain, these chains function as switching molecules for various cellular functions, such as endocytic sorting, transcriptional regulation, and modulation of protein–protein interactions. A pre- liminary experiment indicated that RBCK2 directly interacts with the Lys63-linked polyubiquitin chain in vitro (data not shown), suggesting that RBCK2 acts not only as a carrier of Lys48-linked polyubiquitinated substrates to the 26S proteasome, but also as a modu- lator of cellular functions mediated by Lys63-linked polyubiquitinated proteins. Splice variants of RING proteins More than 1000 RING proteins have so far been found in eukaryotic cells, and many of them are presumed to participate in the ubiquitin–proteasome system. Recently, some of the RING proteins were shown to accompany the RING-lacking splice vari- ants, such as the mRNAs of breast cancer-linked A B C Fig. 4. Interaction of RBCK2 with Lys48-linked polyubiquitin chains. (A) Interaction of RBCK2 with polyubiquitinated proteins in cells. HEK293 cells were transfected with an expression plasmid for the N-terminally FLAG-tagged RBCK2, and cultured with or without MG132. The top panel shows the endogenous polyubiquitinated proteins coimmunoprecipitated with an antibody against FLAG. The bottom panel shows the FLAG-tagged RBCK2 in the immunoprecipitates obtained with an antibody against FLAG. Asterisks indicate nonspecific bands. (B) In vitro pull-down assay of RBCK2 and Lys48-linked polyubiquitin chains. N-terminally GST-fused S5a, RBCK2,or RBCK2DNZF was bound to glutathione–Sepharose beads, incubated with Lys48-linked polyubiquitin chains, and subjected to a GST pull-down assay. The polyubiquitin chains pulled down with the GST-fused proteins were detected by western blotting with an antibody against ubiquitin. (C) Purified GST-fused proteins. GST-fused proteins used in the pull-down assay were subjected to SDS ⁄ PAGE and detected by Coomassie brilliant blue (CBB) staining. The numbers shown below the panel are corresponded to those of Fig. 4B. Potential polyubiquitinated protein carrier RBCK2 N. Yoshimoto et al. 6380 FEBS Journal 276 (2009) 6375–6385 ª 2009 The Authors Journal compilation ª 2009 FEBS tumor suppressor 1 (BRCA1) [33] and Parkin [34], and the BRCA1-associated RING domain 1 protein [35]. However, the biochemical analyses of these splice vari- ants have not been fully pursued yet. This study dem- onstrated for the first time that the splice variant of the RING–IBR protein RBCK1, RBCK2, is expressed endogenously in various tissues, and participates not only in the transcriptional system [11], but also in the ubiquitin–proteasome system. On the other hand, Parkin, a RING–IBR protein, was shown to accom- pany many splice variants in the transcripts of rat and fetal human brain [34]. Interestingly, two of these splice variants (TV4 and TV5 in [34]) retain the UBL domain and lack the RING–IBR domain, as does RBCK2. Although these variants possess neither the NZF domain nor the UIM ⁄ UBA domain, they interact with polyubiquitinated proteins through the N-terminal region [13]. It is postulated that these splice variants of Parkin serve, like RBCK2, as polyubiquitinated pro- tein carriers proximal to the 26S proteasome. Another Parkin splice variant, which also retains the UBL domain and lacks the RING–IBR domain, was origi- nally expressed in the peripheral leukocytes of normal humans. In patients with sporadic Parkinson’s disease, the same splice variant was frequently found in brain [36]. Moreover, the Parkin mutant generated by the guanine 535 microdeletion, which harbors the UBL domain only, was found to be closely associated with the onset of autosomal-recessive juvenile parkinsonism A B C Fig. 5. Schematic drawing of the RBCK2–S5a–polyubiquitinated protein ternary complex. (A) Possible mode of binding of the ternary com- plex of RBCK2, S5a, and polyubiquitinated protein. The 19S proteasome regulatory subunit S5a possesses two UIM motifs, which are known to bind to the polyubiquitin chain. The C-terminal UIM motif (UIM2) is found to interact with both the ubiquitin chain and the UBL domain of HHR23. RBCK2 interacts with S5a and polyubiquitinated proteins with the UBL domain and the NZF motif, respectively. (B) Align- ment of amino acid sequences of UBL domains. The UBL domains of mammalian proteins are shown. The consensus amino acids of these domains are indicated on the gray background. Amino acids thought to be essential for the interaction with S5a are indicated on the black background. (C) The NZF motifs of mammalian proteins are shown. The consensus amino acids of these motifs are indicated on the gray background. Cysteines essential for the formation of a zinc cluster are indicated on the black background. N. Yoshimoto et al. Potential polyubiquitinated protein carrier RBCK2 FEBS Journal 276 (2009) 6375–6385 ª 2009 The Authors Journal compilation ª 2009 FEBS 6381 [37]. These data implied that the splice variants of RING proteins, including Parkin, are expressed in a tissue-specific manner, and that the abnormal expres- sion of these variants may lead to the derangement of cellular functions. In the transcriptional system, RBCK1 functions as a transcriptional factor and RBCK2 represses RBCK1- mediated transcription as a cytoplasmic tethering pro- tein specific to RBCK1. In the ubiquitin–proteasome system, RBCK1 acts as an E3 enzyme for the synthesis of polyubiquitinated proteins, whereas RBCK2 acts as a polyubiquitinated protein carrier for the degradation. Together, these findings show that RBCK2 has the ability to exhibit opposite functions to RBCK1, although it is still unclear why RBCK1 and RBCK2 are involved in both the transcriptional system and the ubiquitin–proteasome system. Experimental procedures Plasmids Deletion and substitution mutants of the rat RBCK2 gene were obtained with a QuickChange site-directed mutagene- sis kit (Stratagene, La Jolla, CA, USA). For the expression of N-terminally HA-tagged rat RBCK2 (HA–RBCK2), N-terminally HA-tagged human S5a (HA–S5a), N-terminally FLAG-tagged RBCK2 (FLAG–RBCK2) and the N-termi- nally FLAG-tagged UBL domain (1–130 amino acids)- truncated RBCK2 mutant (FLAG–RBCK2DUBL) in mammalian cells, the plasmids pTB701–HA–RBCK2 [11], pTB701–HA–S5a, pTB701–FLAG–RBCK2 and pTB701– FLAG–RBCK2DUBL were constructed using pTB701– FLAG or pTB701–HA [38]. The expression plasmid pcDNA3.1–FLAG–ubiquitin [9] was used for the expres- sion of N-terminally FLAG-tagged human ubiquitin (FLAG–ubiquitin). The RBCK2DNZF gene was generated by replacing the sequences encoding four cysteines (Cys187, Cys190, Cys201, and Cys204) in the NZF motif with sequences encoding serines. For bacterial expression of GST-fused RBCK2 (GST–RBCK2) and RBCK2DNZF (GST–RBCK2DNZF), plasmids pGEX-6P-1–RBCK2 and pGEX-6P-1–RBCK2DNZF were constructed by inserting cDNA fragments encoding full-length RBCK2 and RBCK2DNZF into pGEX-6P-1 (GE Healthcare, Chalfont St Giles, UK). Western blot analysis HEK293 cells were cultured in DMEM supplemented with 10% (v ⁄ v) fetal bovine serum at 37 °C under humidified air with 5% (v ⁄ v) CO 2 . HEK293 cells (approximately 1 · 10 7 cells) were transfected with pTB701–HA–RBCK2 (5 lg) and pcDNA3.1–FLAG–ubiquitin (5 lg) by electro- poration, and cultured for 48 h. When MG132 (Peptide Institute, Osaka, Japan) was applied, the cells were pre- treated with 50 lm MG132 at 37 °C for 4 h. The cells were washed twice with NaCl ⁄ P i (pH 7.2), and lysed in 1 mL of lysis buffer [50 mm Tris ⁄ HCl (pH 7.5), 150 mm NaCl, 1mm EGTA, 0.1% (v ⁄ v) Triton X-100, 1 mm dithiothreitol, and one tablet per 50 mL of Complete protease inhibitor cocktail (Roche, Basel, Switzerland)]. The cleared lysates (10 lL) were mixed with an equal volume of Laemmli’s sample buffer, and subjected to SDS ⁄ PAGE. The samples were blotted onto a poly(vinylidene difluoride) membrane (Millipore, Billerica, MA, USA), and western blot analysis was performed with a horseradish peroxidase (HRP)-conju- gated mouse monoclonal antibody against FLAG (clone M2; Sigma, St Louis, MO, USA) (dilution, 1 : 5000) and an HRP-conjugated antibody against HA (clone 12CA5; Roche) (dilution, 1 : 3500). Immunoreactive bands were visualized by the enhanced chemiluminescence method with ECL Plus Western Blotting Detection Reagents (GE Healthcare) under a luminescence image analyzer LAS-4000mini (Fujifilm, Tokyo, Japan). Immunofluorescence analysis Approximately 5 · 10 4 HEK293 cells were grown on a 2.7 cm cover glass, washed twice with NaCl ⁄ P i (pH 7.2), 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 NaCl ⁄ P i (pH 7.2) for 15 min at room tempera- ture, washed twice with NaCl ⁄ P i (pH 7.2), incubated with blocking buffer [NaCl ⁄ P i (pH 7.2) containing 0.03% (v ⁄ v) Triton X-100, 2% (w ⁄ v) BSA, and 3% (v ⁄ v) normal goat serum] for 30 min at room temperature, and incubated with the blocking buffer containing a primary antibody (a rabbit polyclonal antibody against RBCK1 ⁄ 2 (dilution, 1 : 200) [12] and a mouse monoclonal antibody against the 20S proteasome (clone HP810; BIOMOL, Plymouth Meeting, PA, USA) (dilution, 1 : 50)] for 30 min at room tempera- ture. The cells were washed four times with NaCl ⁄ P i (pH 7.2) containing 0.03% (v ⁄ v) Triton X-100 for 10 min at room temperature, incubated with the blocking buffer con- taining a secondary antibody [an Alexa Fluor 488-labeled anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA) (dilution, 1 : 400) and a Cy3-labeled anti-mouse IgG (GE Healthcare) (dilution, 1 : 500)] for 30 min at room temperature, and mounted with 90% (v ⁄ v) glycerol in NaCl ⁄ P i (pH 7.2). Cell fluorescence was observed under an LSM 5 PASCAL confocal laser scan microscope (Carl Zeiss, Oberkochen, Germany). Pull-down assay using GST–S5a Purified GST (3.5 lg) or GST–S5a (BIOMOL) (10 lg) was added to 800 lL of binding buffer [50 mm Tris ⁄ HCl (pH Potential polyubiquitinated protein carrier RBCK2 N. Yoshimoto et al. 6382 FEBS Journal 276 (2009) 6375–6385 ª 2009 The Authors Journal compilation ª 2009 FEBS 7.2), 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 2 mm dithiothreitol, 0.1% (v ⁄ v) Triton X-100, and one tablet per 50 mL of Complete protease inhibitor cocktail] and immo- bilized on 30 lL of 50% slurry glutathione–agarose beads (Sigma). GST–RBCK2 was overexpressed in Escherichi- a coli BL21-CodonPlus-RP (Stratagene) and purified with Glutathione Sepharose 4B (GE Healthcare), according to the manufacturer’s protocol. RBCK2 protein was isolated from GST–RBCK2 by removing the GST portion with Pre- Scission protease (GE Healthcare). The resin was mixed with purified RBCK2 (5 lg), incubated with rotation at 4 °C for 30 min, washed four times with the binding buffer, and resuspended in 35 lL of Laemmli’s sample buffer. Western blot analysis was carried out with a rabbit poly- clonal antibody against RBCK (dilution, 1 : 3000) as a primary antibody, and an HRP-conjugated anti-rabbit IgG (GE Healthcare) (dilution, 1 : 10 000) as a secondary antibody. Immunoprecipitation assay HEK293 cells (approximately 1 · 10 7 cells) were transfected with pTB701–FLAG–RBCK2 (5 lg), pTB701–FLAG– RBCK2DUBL (5 lg) or pTB701–HA–S5a (2 lg) by electro- poration, and cultured for 48 h. When MG132 was applied, the cells were pretreated with 50 lm MG132 at 37 °C for 4 h. The cells were washed twice with NaCl ⁄ P i (pH 7.2), and lysed in 1 mL of lysis buffer. The cleared lysate was mixed with a mouse monoclonal antibody against S5a (clone S5a-18; BIOMOL) (2 lg), a mouse monoclonal anti- body against HA (clone 12CA5; Roche) (10 lg), or a mouse monoclonal antibody against FLAG (clone M2; Sigma) (10 lg), incubated on ice for 60 min, mixed with 30 lL of protein G Sepharose 4 Fast Flow [50% (v ⁄ v) slurry] (GE Healthcare), and incubated with rotation at 4 °C for 30 min. The beads were washed four times with lysis buffer, resuspended in 35 lL of Laemmli’s sample buffer, and subjected to SDS ⁄ PAGE. The samples were blotted onto a poly(vinylidene difluoride) membrane, and western blot analysis was carried out using an HRP-conju- gated antibody against FLAG, an antibody against S5a (dilution, 1 : 500), an HRP-conjugated antibody against HA, or a mouse monoclonal antibody against ubiquitin (clone P4D1; Santa Cruz Biotechnology, Santa Cruz, CA, USA) (dilution, 1 : 1000) as a primary antibody, and an HRP-conjugated anti-mouse IgG (GE Healthcare) (dilution, 1 : 10 000) as secondary antibody. Pull-down assay using GST–S5a and GST–RBCK2 Either purified GST (1.5 lg), GST–RBCK2 (3 lg), GST– RBCK2DNZF (3 lg) or GST–S5a (4.5 lg) was added to 800 lL of binding buffer and immobilized on 25 lL of 50% (v ⁄ v) slurry Glutathione Sepharose 4B beads, and this was followed by the addition of Lys48-linked ubiquitin chains (Boston Biochem, Boston, MA, USA) (0.5 lg). 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Yoshimoto et al. 6384 FEBS Journal 276 (2009) 6375–6385 ª 2009 The Authors Journal compilation ª 2009 FEBS [...]... Molecular genetic analysis of a novel Parkin gene in Japanese families with autosomal recessive juvenile parkinsonism: evidence for variable homozygous deletions in the Parkin gene in affected individuals Ann Neurol 44, 935–941 38 Kuroda S, Tokunaga C, Kiyohara Y, Higuchi O, Konishi H, Mizuno K, Gill GN & Kikkawa U (1996) Protein protein interaction of zinc finger LIM domains with protein kinase C J Biol... (2005) A truncated splice variant of human BARD1 that lacks the RING finger and ankyrin repeats Cancer Lett 233, 108–116 36 Tan EK, Shen H, Tan JM, Lim KL, Fook-Chong S, Hu WP, Paterson MC, Chandran VR, Yew K, Tan C et al (2005) Differential expression of splice variant and wild-type parkin in sporadic Parkinson’s disease Neurogenetics 6, 179–184 Potential polyubiquitinated protein carrier RBCK2 37 Hattori . Accumulation of polyubiquitinated proteins by overexpression of RBCC protein interacting with protein kinase C2, a splice variant of ubiquitin ligase RBCC protein interacting with protein. shuttling protein RBCC protein interacting with protein kinase C1 (RBCK1) possesses transcriptional and ubiquitin ligase activities. We have recently reported that RBCC protein interacting with protein. protein; RBCK1, RBCC protein interacting with protein kinase C1; RBCK2, RBCC protein interacting with protein kinase C2; UBA, ubiquitin-associated; UBL, ubiquitin-like; UIM, ubiquitin-interacting