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Faculty of Science and Technology, Department of Applied Biological Science, Tokyo University of Science, Noda, Chiba 278-8510, Japan

	Abstract

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Bood POZ containing gene type 2 (BPOZ-2) is involved in the growth suppressive effect of the phosphatase and tensin homologue (PTEN). We showed that BPOZ-2 is a human counterpart of yeast Btb3p, which is a putative adaptor for Pcu3p-based ubiquitin ligase. BPOZ-2 bound to E3 ligase CUL3 in vitro and in vivo. BPOZ-2 itself was ubiquitinated through the CUL3-based E3 ligase mainly within the nucleus and degraded by the 26S proteasome. Although BPOZ-2 was mainly expressed within the cytoplasm, it accumulated within the nucleus in the presence of the specific 26S proteasome inhibitor MG132. BPOZ-2 may be recruited to the nucleus from the cytoplasm. Terminal deoxynucleotidyltransferase (TdT) was detected as a BPOZ-2-binding protein using a yeast two-hybrid system by screening a human thymus cDNA library. TdT, BPOZ-2, and CUL3 formed a ternary complex in vivo. TdT was ubiquitinated only within the nucleus and degraded by the 26S proteasome. The ubiqutination or degradation of TdT was markedly promoted by co-expression of BPOZ-2 and CUL3 or BPOZ-2 in 293T cells, respectively.

	Introduction

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The ubiquitin–proteasome system directs target proteins to 26S-proteasome-mediated degradation by polyubiquitination on lysine residues in the proteins (Hershko & Ciechanover 1998; Pickart 2004). Three enzymes are ubiquitously involved in ubiquitin transfer: E1, ATP-dependent activation enzyme of ubiquitin; E2, ubiquitin-conjugating enzyme, which transfers activated ubiquitin to the target protein; and E3, ubiquitin ligase. Two classes of E3, that is, RING- and HECT-type E3s, have been identified according to the structure of the E2 binding region (Glickman & Ciechanover 2002). RING-type E3 has monomer and complex types as subtypes. The complex type includes a large subgroup called Cullin-Rbx-type E3. At least seven Cullin family members, that is, CUL1, CUL2, CUL3, CUL4A, CUL4B, CUL5, and CUL7, have been identified and partially characterized from yeasts to humans. Recent findings suggest that adaptor proteins with the BTB/POZ domain function as substrate-specific adaptors for CUL3-based E3 ligases (Furukawa et al. 2003). Thus CUL3-Rbx1-type E3 consists of CUL3, Rbx1 and an adaptor protein with the BTB/POZ domain. Schizosaccharomyces pombe Btb3p, which has two ankyrin repeats and two BTB/POZ domains at the N- and C-terminals, respectively, was reported to function as a putative substrate-specific adaptor for Pcu3p-based ubiquitin ligase (Geyer et al. 2003). Evolutionary conservation is evidenced by the close similarity of the amino acid sequence of human Bood POZ containing gene type 2 (BPOZ-2) to that of yeast Btb3p. BPOZ-2 shares a 29% amino acid identity with Btb3p (Fig. 1). BPOZ-2 was initially isolated from a human leukocyte cDNA library (Dai et al. 2000). BPOZ-2 expression is activated by the phosphatase and tensin homologue, a tumor suppressor. Since BPOZ-2 over-expression inhibits cell cycle progression during the G1/S transition, BPOZ-2 may be involved in the growth suppressive effect of phosphatase and tensin homologue (Unoki & Nakamura 2001). On the basis of these facts, we speculated that BPOZ-2 is a human counterpart of yeast Btb3p and functions as a substrate-specific adaptor for CUL3-based E3 ligase. Thus, to confirm our speculation, we first examined whether there is binding between BPOZ-2 and CUL3 and observed such binding. We next identified the substrate of BPOZ-2 by the yeast two-hybrid screening of a human thymus cDNA library using BPOZ-2 as the bait and isolated the terminal deoxynucleotidyltransferase (TdT) gene. We also found that BPOZ-2 promotes TdT degradation by the 26S proteasome.

View larger version (21K): [in this window] [in a new window]   Figure 1  Identity and similarity between human BPOZ-2 and yeast Btb3p, and predicted secondary structure. Human BPOZ-2 and yeast Btb3p have two ankyrin repeats, two BTB/POZ domains (1 and BTB2) and a coiled-coil structure in the N- and C-terminal half regions, respectively. BPOZ-2 and Btb3p have a 29.9% amino acid identity and a 56.2% similarity, as determined using ClustalW (matrix; BLOSUM 80). *, identical residues; :, residue showing that the "strong" group is fully conserved; ., residue showing that the "weak" group is fully conserved. The strong and weak groups are defined as having scores > 0.5 and  0.5, respectively. The secondary structure was predicted using PREDICTPROTEIN. Tubes and notches show -helices and β-sheets, respectively.

	Results

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Association of BPOZ-2 and CUL3

Yeast Btb3p is ubiquitinated by Pcu3p-based E3 ligase (Geyer et al. 2003). From the amino acid sequence homology and presumed organization of functional domains, we expected that BPOZ-2 and CUL3 are human counterparts of yeast Btb3p and Pcu3p, respectively. We thus determined whether there is the binding between BPOZ-2 and CUL3 by GST pull-down assay using His-tagged BPOZ-2 and GST-CUL3 in vitro. We expressed recombinant GST-CUL3 in Escherichia coli. The GST-CUL3 expressed was coupled to glutathione Sepharose 4B. Escherichia coli cell lysate expressing His-BPOZ-2 was then added to the reaction mixture containing GST-CUL3-bound beads. The binding between BPOZ-2 and CUL3 was analyzed by Western blotting using a rabbit anti-BPOZ-2 antibody. As shown in Fig. 2A, although His-BPOZ did not bind to GST-bound beads (lane 1), it bound to GST-CUL3-bound beads (lane 2), indicating that BPOZ-2 binds to CUL3. To further verify the association in mammalian cells, we expressed Myc-BPOZ-2, Flag-CUL3, and Flag-Rbx1 in 293T cells and carried out immunoprecipitation analysis using an anti-BPOZ-2 antibody. The proteins co-immunoprecipitated with Myc-BPOZ-2, were detected using an anti-Myc or anti-Flag antibody. As shown in Fig. 2B, Flag-CUL3 was detected in the immunoprecipitant. We also detected Rbx1, which directly binds to E2 and CUL3-based E3 ligase, in the immunoprecipitant in the presence of CUL3. These results strongly suggest that BPOZ-2, CUL3, and Rbx1 form a ternary complex in mammalian cells and that BPOZ-2 is a human counterpart of yeast Btb3p, as expected.

View larger version (20K): [in this window] [in a new window]   Figure 2  BPOZ directly binds to CUL3. (A) BPOZ-2 binds to CUL3 in vitro. The cell lysate of E. coli expressing recombinant His-BPOZ-2 was incubated with GST-bound (lane 1) or GST-CUL3-bound (lane 2) Glutathione Sepharose 4B. Proteins bound to the beads were eluted with an SDS-PAGE sample buffer by boiling. The eluate was subjected to SDS-PAGE and then analyzed by immunoblotting using an anti-BPOZ-2 or anti-GST antibody. His-BPOZ-2 expressed in the cell lysate was detected using an anti-BPOZ-2 antibody (lane 3). (B) BPOZ-2 associates with CUL3 in vivo. Expression vectors for Myc-BPOZ-2 (lanes 1, 3, and 4), and Flag-CUL3 (lanes 2 and 3), Flag-Rbx1 (lanes 2–4) or control vectors without target genes were co-transfected into 293T cells as indicated (–, vector without target gene was transfected; +, vector containing target gene was transfected). Myc-BPOZ-2 and Flag-CUL3 or Flag-Rbx expressed in the cell lysate were detected using anti-Myc and anti-Flag antibodies, respectively. Immunoprecipitants were subjected to immunoblotting using an anti-Myc or anti-Flag antibody. Asterisk (*) shows a smeary background. (C) CUL3 binding region in BPOZ-2. The vectors with genes encoding GAL4BD-fused BPOZ-2 deletion mutants and either GAL4AD-fused CUL3 or GAL4AD were transformed in the yeast strain Y190. To examine protein–protein interactions, the LacZ activity of the transformants was determined by β-galactosidase colony lift-filter assay. The mutants expressed are schematically shown (+, positive interation; –, no interaction; ±, weak interaction). Amino acid residues 1–31 and 35–64 are predicted to form ankyrin repeats. Residues 115–212 and 272–376 are predicted to form BTB/POZ domains. (D) Binding between CUL3 and BPOZ-2 with mis-sense mutation. The cell lysate of E. coli expressing recombinant His-H129L (lanes 1 and 4), His-H286L (lanes 2 and 5) or His-BPOZ-2 (lanes 3 and 6) was incubated with GST-bound (lanes 1–3) or GST-CUL3-bound (lanes 4–6) Glutathione Sepharose 4B. Proteins bound to the beads were eluted in the SDS-PAGE sample buffer by boiling. The eluate was subjected to SDS-PAGE and then analyzed by immunoblotting using an anti-BPOZ-2 or anti-GST antibody.

BPOZ-2 itself is ubiquitinated and degraded through the CUL3-based ubiquitin–proteasome system

Given that BPOZ-2 is a human counterpart of yeast Btb3p, BPOZ-2 is expected to be ubiquitinated and degraded through the CUL3-based ubiquitin–proteasome system, because human CUL3 is also expected to be a human counterpart of yeast E3 ligase Pcu3p. Thus, to determine whether BPOZ-2 is actually ubiquitinated, we expressed Myc-BPOZ-2, Flag-CUL3, Flag-Rbx1, and His-Ub in 293T cells. After precipitating ubiquitinated proteins with Ni²⁺ beads, ubiquitinated Myc-BPOZ-2 was detected using an anti-Myc antibody. As shown in Fig. 3A, BPOZ-2 ubiquitination was markedly promoted in the presence of the 26S-proteasome-specific inhibitor MG132 (lanes 5 and 6). Such ubiquitination promotion was also detected in the absence of MG132 (lanes 2 and 3). These results show that BPOZ-2 is ubiquitinated through the CUL3-based ubiquitination system. We also determined whether BPOZ-2 is degraded through the 26S proteasome system using MG132. As shown in Fig. 3B, BPOZ-2 was degraded within 3 h in the absence of MG132, but was not degraded in the presence of MG132, indicating that BPOZ-2 is degraded by the 26S proteasome.

View larger version (28K): [in this window] [in a new window]   Figure 3  Proteasome-dependent degradation of BPOZ-2 through CUL3-based ubiquitination system. (A) Ubiquitination of BPOZ-2 in vivo. 293T cells were transfected with plasmids encoding 6xHis-ubiquitin (lanes 1–6), Myc-BPOZ-2 (lanes 2, 3, 5, and 6), Flag-CUL3 (lanes 3 and 6), and Flag-Rbx1 (lanes 3 and 6) in the indicated combinations. After incubation for 36 h, the cells were treated with 20 µM MG132 (lanes 4–6) or DMSO (lanes 1–3) for another 7 h. The cells were lysed under denaturing condition, and ubiquitin-containing complexes were affinity-purified and subjected to SDS-PAGE. Ubiquitinated BPOZ-2 was detected by immunoblotting using an anti-Myc antibody. Myc-BPOZ-2, Flag-CUL3 and Flag-Rbx1 in the lysate were detected using an anti-Myc or anti-Flag antibody. (B) BPOZ-2 degradation in 293T cells. 293T cells were transfected with plasmids encoding Flag-BPOZ-2. 36 h after transfection, the cells were treated with 100 µg/mL cycloheximide (CHX) in the presence of dimethyl sulfoxide (DMSO) (lanes 1–5) or MG132 (20 µM) (lanes 6–10) for 10 min to 3 h. The cells were lysed and the total lysate (10 µg) was subjected to SDS-PAGE, followed by immunoblotting (WB) using an anti-Flag antibody. The figure for the quantification of BPOZ-2 degradation is also shown. The intensities of the bands of BPOZ-2 were quantified from the NIH image and normalized to that of actin. (C) Ubiquitinated Myc-BPOZ-2 is detected mainly within the nucleus. 293T cells were transfected with plasmids encoding 6xHis-ubiquitin (lane 1–9), Myc-BPOZ-2 (lanes 2, 3, 5, 6, 8, and 9), Flag-CUL3 (lanes 1, 3, 4, 6, 7, and 9), and Flag-Rbx1 (lane 1, 3, 4, 6, 7, and 9) in the indicated combinations. After incubation for 36 h, the cells were treated with 20 µM MG132 for another 7 h. The cells were fractionated into NF and CF. The nuclear and cytosolic fractions (NF and CF, respectively) were suspended in a denaturing buffer, and ubiquitinated proteins were affinity-purified and subjected to SDS-PAGE. Ubiquitinated BPOZ-2 was determined by immunoblotting using an anti-Myc antibody. The expressions of Myc-BPOZ-2, Flag-CUL3, and Flag-Rbx1 in the transfected cells were detected by immunoblotting using an anti-Myc or anti-Flag antibody. β-Tubulin and HP1 in NF and CF were detected by immunoblotting as control.

View larger version (82K): [in this window] [in a new window]   Figure 9  Localization of TdT or BPOZ-2 expressed in 293T cells in the presence or absence of MG132. (A) Localization of BPOZ-2 in the presence of the 26S proteasome inhibitor MG132. 293T cells transfected with plasmids encoding EGFP-BPOZ-2 were treated with DMSO (1) or MG132 (2). BPOZ-2 was detected within the nucleus and perinuclear region. The cells were detected by immunocytochemistry (ICC) using immunofluorescence microscopy with anti-Ub and Alexa Fluor 546-conjugated antibodies. Nuclei were counter-stained with DAPI. (B) Localization of TdT and BPOZ-2 in 293T cells. 293T cells were transfected with plasmids encoding EGFP and DsRed-TdT (1), EGFP-BPOZ-2 and DsRed (2). The DNA was stained with DAPI. The cells were observed under immunofluorescence microscopy. (C) TdT accumulated within the nucleus as speckles in the presence of MG132. 293T cells transfected with plasmids encoding EGFP ((1) and (2)) or EGFP-TdT ((3) and (4)) were treated with MG132 ((2) and (4)) or DMSO ((1) and (3)). The cells were observed under immunofluorescence microscopy using anti-Ub and Alexa Fluor 546-conjugated antibodies. Nuclei were counter-stained with DAPI. (D) EGFP-BPOZ-2 and DsRed-TdT co-localize in the presence of MG132. 293T cells transfected with plasmids encoding EGFP-BPOZ and DsRed-TdT were treated with MG132 ((3) and (5)) or DMSO ((2) and (4)). The cells were observed under immunofluorescence microscopy ((1), (2), and (3)) or confocal microscopy ((4) and (5)) using anti-Ub and Alexa Fluor 350-conjugated antibodies ((2) and (3)). Nuclei were counter-stained with DAPI ((1), (4), and (5)). (E) CUL3, BPOZ-2 and TdT are co-expressed as nuclear speckles. 293T cells were co-transfected with plasmids encoding both EGFP-CUL3 and DsRed ((1) and (2)), EGFP-CUL3 and DsRed-TdT ((3) and (4)) or EGFP-BPOZ-2 and DsRed-TdT ((5) and (6)). The cells were treated with MG132 ((2), (4), and (6)) or DMSO ((1), (3) and (5). The cells were detected by ICC using either immunofluorescence microscopy ((1), (2), (4), and (5)) with an anti-Flag antibody or confocal microscopy using anti-Flag and Alexa Fluor 350-conjugated antibodies ((4) and (5)). Nuclei were counter-stained with DAPI ((1) and (2)).

From the findings showing that BPOZ-2 binds to CUL3, BPOZ-2 was expected to be an adaptor for CUL3-based E3 ligase. We then attempted to identify the protein substrates of BPOZ-2 using a yeast two-hybrid system. The full-length human cDNA for BPOZ-2 was fused to the GAL4-DNA-binding domain and a human thymus cDNA library was screened. Fifty candidate clones were isolated by screening 1.4 x 10⁷ clones on Leu, His, and Trp dropout plates. All the plasmids were transformed in E. coli (DH5) and their DNA sequences were determined. When the DNA sequences were compared with those in the NCBI sequence database using the BLASTN and BLASTP algorithm (Altschul et al. 1997), two (SM25 and SM50) of the 50 clones were found to be TdT cDNAs. The clones isolated contained C-terminal regions, namely, residues 299-509 (SM25) or 279-509 (SM50) in TdT.

To confirm the direct binding between BPOZ-2 and full-length TdT in vitro, we constructed a vector that expresses GST-BPOZ-2 in E. coli. After expressing GST-BPOZ-2, it was bound to glutathione Sepharose 4B beads. Purified His-TdT was then reacted with GST-BPOZ-2- or GST-bound beads. After washing the beads thoroughly, we determined whether TdT binds to BPOZ-2-bound beads by Western blotting using a polyclonal antibody against TdT. As shown in Fig. 4A, TdT bound to GST-BPOZ-2-bound beads (lane 3), although it did not bind to GST-bound beads (lane 2), indicating that TdT binds to BPOZ-2 in vitro. To further determine the association of BPOZ-2 and TdT in COS7 cells, immunoprecipitation analysis was carried out. pME18s-Flag-BPOZ-2 and pCMV-Myc-TdT were co-transfected into COS7 cells with lipofectamine, and proteins that bound to BPOZ-2 were immunoprecipitated using an anti-BPOZ-2 antibody. As shown in Fig. 4B (lane 4), TdT was immunoprecipitated together with BPOZ-2, indicating that BPOZ-2 associates with TdT over-expressed in COS7 cells. We further confirmed their association by changing the bait from BPOZ-2 to TdT. As shown in Fig. 4C (lane 4), when Myc-TdT was used as the bait, Flag-BPOZ-2 was also detected in the immunoprecipitant. We finally determined whether BPOZ-2 associates with TdT in the bovine thymocytes by immunoprecipitation using an anti-TdT antibody as the bait. As shown in Fig. 4D, BPOZ-2 was immunorecipitated together with TdT. Collectively, these results indicate that BPOZ-2 binds to TdT in vitro and in vivo.

View larger version (20K): [in this window] [in a new window]   Figure 4  Binding between TdT and BPOZ-2. (A) BPOZ-2 binds to TdT in vitro. Purified recombinant His-TdT was incubated with GST- (lane 2) or GST-BPOZ-2-bound (lane 3) glutathione Sepharose 4B. Proteins bound to the beads were eluted in the SDS-PAGE sample buffer by boiling. The eluate was subjected to SDS-PAGE and then analyzed by immunoblotting using an anti-TdT or anti-GST antibody. Purified His-TdT was detected using an anti-TdT antibody (lane 1). (B) BPOZ-2 associates with TdT in COS7 cells. Vectors with genes encoding Myc-TdT and Flag-BPOZ-2 (lanes 3 and 4) or a control vector (lanes 1 and 2) was transiently transfected into COS7 cells. BPOZ-2 or TdT expressed in the cell lysate was analyzed by immunoblotting using anti-Flag and anti-TdT antibodies, respectively. Immunoprecipitation was performed using an anti-BPOZ-2 antibody. Western blotting was carried out using anti-TdT and anti-Flag antibodies. (C) TdT associates with BPOZ-2 in COS7 cells. Vectors with genes encoding Myc-TdT and Flag-BPOZ-2 (lanes 3 and 4) or a control vector (lanes 1 and 2) was transiently transfected into COS7 cells. BPOZ-2 or TdT expressed in the cell lysate was analyzed by immunoblotting using anti-Flag and anti-TdT antibodies, respectively. Immunoprecipitation was performed using an anti-TdT antibody. Western blotting was carried out using anti-Flag and anti-TdT antibodies. (D) BPOZ-2 associates with TdT in the bovine thymocytes. Immunoprecipitation was carried out using an anti-TdT antibody (lane 3) or rabbit preimmune serum (pre IS) (lane 2) as the bait. Immunoprecipitants were subjected to SDS-PAGE and following Western blotting using an anti-TdT or anti-BPOZ-2 antibody. One mg of the whole cell lysate was used for each immunoprecipitation. (E) BPOZ-2 binding region in TdT. Vectors with genes encoding GAL4BD-fused TdT deletion mutants and either GAL4AD-fused BPOZ-2 or GAL4AD were transformed in the yeast strain Y190. The LacZ activity of the transformants was determined by β-galactosidase colony lift-filter assay. The mutants expressed are schematically shown. +, interaction was detected as a blue color; –, no interaction was detected. (F) TdT binding region in BPOZ-2. Vectors with genes encoding GAL4BD-fused BPOZ-2 deletion mutants and either GAL4AD-fused SM25 (a.a. 299-509 of TdT) or GAL4AD were transformed in the yeast strain Y190. To examine protein–protein interactions, the LacZ activity of the transformants was determined by β-galactosidase colony lift-filter assay. +, interaction was detected as a blue color; –, no interaction was detected. (G) Binding between CUL3 and BPOZ-2 with mis-sense mutation. The lysate of E. coli expressing recombinant His-H129L (lanes 1 and 4), His-H286L (lanes 2 and 5) or His-BPOZ-2 (lanes 3 and 6) was incubated with GST-bound (lanes 1–3) or GST-TdT-bound (lanes 4–6) glutathione Sepharose 4B. Proteins that bound to the beads were eluted in the SDS-PAGE sample buffer by boiling. The eluate was subjected to SDS-PAGE and then analyzed by immunoblotting using an anti-BPOZ-2 or anti-GST antibody. His-H129L, His-H286L and His-BPOZ-2 in the cell lysate were detected using an anti-BPOZ-2 antibody (lanes 7–9, respectively).

We also attempted to confine the TdT binding region in BPOZ-2 by constructing eight BPOZ-2 deletion mutants using a yeast two-hybrid system. The binding was detected using BPOZ-2 deletion mutants as the bait. As shown in Fig. 4F, the C-terminal region del 6 was found to be the confined TdT binding region in BPOZ-2. Given that the C-terminal region without the two BTB/POZ domains is the TdT binding region in BPOZ-2, the mutants H129L and H286L are expected to bind to TdT. To determine whether the two BTB/POZ domains are not involved in the TdT binding, we carried out GST pull-down assay using GST-TdT as the bait. As shown in Fig. 4G (lanes 4 and 5), H129L and H286L bound to TdT as expected, indicating that the TdT binding region is structurally separated from the N-terminal half region containing the two BTB/POZ domains.

TdT, BPOZ-2, and CUL3 form a ternary complex

From the findings that BPOZ-2 binds to TdT and CUL3, we suspected that BPOZ-2, TdT and CUL3 form a ternary complex in a mammalian cell. To test this possibility, we expressed Flag-CUL3, EGFP-BPOZ-2, and Myc-TdT in 293T cells, and carried out immunoprecipitation analysis with Flag-CUL3 as the bait. As shown in Fig. 5A (lane 4), CUL3 associated with TdT together with BPOZ-2, indicating that BPOZ-2, TdT, and CUL3 over-expressed in 293T cells form a ternary complex. Because TdT did not bind to CUL3 (lane 3), the ternary complex may be TdT–BPOZ-2–CUL3. When we changed the bait from Flag-CUL3 to Myc-TdT, Flag-CUL3, and Flag-BPOZ-2 were also detected in the immunoprecipitant as TdT-associating proteins (Fig. 5B; lane 3). We further determined whether CUL3 associates with TdT and BPOZ-2 in the bovine thymocytes by immunoprecipitation using an anti-CUL3 antibody as the bait. As shown in Fig. 5C, TdT and BPOZ-2 were co-immunoprecipitated together with CUL3. Collectively, these results indicate that CUL3, TdT, and BPOZ-2 form a ternary complex in vivo.

View larger version (16K): [in this window] [in a new window]   Figure 5  CUL3, BPOZ and TdT form a CUL3-BPOZ-TdT ternary complex. (A) Immunoprecipitation using Flag-CUL3 as bait. 293T cells were co-transfected with expression vectors encoding Flag-CUL3 (lanes 2–4), EGFP-BPOZ-2 (lanes 1, 2 and 4), EGFP (lane 3) and Myc-TdT (lanes 1, 3, and 4). Immunoprecipitation was carried out using Flag-CUL3 as the bait. CUL3, BPOZ-2 and TdT in the cell lysates were detected using anti-Flag, anti-GFP, and anti-TdT antibodies (lower three panels), respectively. Immunoprecipitants were subjected to immunoblotting using anti-Flag, anti-GFP and anti-TdT antibodies (upper three panels). (B) Immunoprecipitation using Myc-TdT as bait. 293T cells were co-transfected with expression vectors encoding Flag-CUL3 (lanes 1–3), Flag-BPOZ-2 (lanes 1 and 3), and Myc-TdT (lanes 2 and 3). Immunoprecipitation was carried out using Myc-TdT as the bait. CUL3 and BPOZ-2 or TdT in the cell lysate were detected using anti-Flag and anti-Myc antibodies, respectively. Immunoprecipitants were subjected to immunoblotting using an anti-Flag or anti-TdT antibody. (C) CUL3 associates with BPOZ-2 and TdT in the bovine thymocytes. Immunoprecipitation was carried out using an anti-CUL3 antibody (lane 3) or anti-rabbit IgG antibody (lane 2). Immunoprecipitants were subjected to SDS-PAGE and following Western blotting using an anti-CUL3, anti-BPOZ-2, or anti-TdT antibody. One mg of the whole cell lysate was used for each immunoperecipitation.

Because TdT forms a ternary complex together with BPOZ-2 and CUL3, we expected that TdT is a protein substrate of BPOZ-2 for ubiquitination. We then determined whether TdT is ubiquitinated through the CUL3-based ubiquitination system by expressing TdT, BPOZ-2, CUL3, Rbx1, and Ub in 293T cells in the presence of MG132. Since Rbx1 is essential for the CUL3 binding to E2, Flag-Rbx1 was also co-expressed with Myc-BPOZ-2 and Flag-CUL3. As shown in Fig. 6, when TdT and Ub were co-expressed in the cells, TdT was ubiquitinated (lane 3), whereas when only TdT or Ub was expressed, no ubiquitinated TdT was detected (lanes 1 and 2). When BPOZ-2 was additionally expressed together with TdT and Ub, unexpectedly, only a slight enhancement of TdT ubiquitination was observed (lane 4). However, when BPOZ-2 and CUL3 were co-expressed, TdT ubiquitination was notably promoted (lane 5). These results strongly suggest that TdT is a substrate of BPOZ-2 and is ubiquitinated by the CUL3-based E3 ligase. As shown in Fig. 6 (lane 3), TdT was ubiquitinated without over-expressing BPOZ-2 and CUL3. TdT might have been ubiquitinated using the endogenous BPOZ-2/CUL3 ubiquitination system.

View larger version (29K): [in this window] [in a new window]   Figure 6  TdT ubiquitination in vivo. 293T cells were transfected with plasmids encoding 6xHis-ubiquitin (lanes 2–5), Myc-TdT (lanes 1, 3–5), Flag-BPOZ-2 (lanes 4 and 5), Flag-CUL3 (lane 5), and Flag-Rbx1 (lane 5) in the indicated combinations. After incubation for 36 h, the cells were treated with 20 µM MG132 for another 7 h. The cells were lysed under denaturing condition. Ubiquitin-containing complexes were affinity-purified and subjected to SDS-PAGE. Ubiquitinated TdT was detected by immunoblotting using an anti-Myc antibody. Myc-TdT and Flag-BPOZ-2, Flag-CUL3 or Flag-Rbx1 were detected using anti-Myc and anti-Flag antibodies, respectively.

Since we observed TdT ubiquitination, we next attempted to determine whether TdT degradation through the 26S proteasome system is promoted by BPOZ-2. After inhibiting protein synthesis with cycloheximide, TdT expressed in 293T cells was chased. As shown in Fig. 7A(1), although no TdT degradation was detected until 3 h, TdT was gradually degraded when BPOZ-2 began to be expressed in the cells (Fig. 7A(2)); however, no TdT was detected 1 h later. When MG132 was added to the culture medium, no TdT degradation was observed even in the presence of BPOZ-2. As shown in Fig. 7B, the expression of the BPOZ-2 gene in 293T cells was confirmed by RT-PCR analysis. These results indicate that TdT degradation through the 26S proteasome system is promoted by BPOZ-2. To further confirm the promoted TdT degradation by BPOZ-2, we analyzed TdT degradation using J.EcoR cells, which constitutively express TdT (Fig. 7D). The retrovirus carrying the BPOZ-2 gene was used to infect J.EcoR cells to observe TdT degradation. As shown in Fig. 7C(1), TdT was gradually degraded in the absence of MG132 when cycloheximide was added to the culture medium, whereas no TdT degradation was observed in the presence of MG132, indicating that TdT is degraded through the endogenous 26S proteasome system. When BPOZ-2 was over-expressed in the cells, TdT was rapidly degraded within 30 min in the absence of MG132 (Fig. 7C(2)). These results indicate that TdT degradation through the 26S proteasome system is promoted by BPOZ-2. The reason no TdT degradation through an endogenous 26S proteasome system was observed in 293T cells (Fig. 7A(1)) compared with that in J.EcoR cells (Fig. 7C(1)) may be the high or low expression level of TdT and BPOZ-2, respectively, in 293T cells.

View larger version (27K): [in this window] [in a new window]   Figure 7  BPOZ-2 promotes TdT degradation. (A) Myc-TdT degradation in 293T cells. Myc-TdT was expressed in 293T cells with (2) or without Flag-tagged BPOZ-2 (1). 36 h after transfection, cycloheximide (CHX) (100 µg/mL) was added to the medium containing dimethyl sulfoxide (DMSO) (lanes 1–5) or MG132 (20 µM) (lanes 6–10), and the culture was incubated for 10 min to 3 h. The cells were lysed and 10 µg of total lysate was subjected to SDS-PAGE, followed by immunoblotting (WB) using an anti-Myc, anti-Flag or anti-actin antibody. The figure for the quantification of TdT degradation is also presented. The intensities of the bands of TdT were quantified from the NIH image and normalized to that of actin. (B) Flag-BPOZ-2 mRNA expression in 293T cells. 293T cells were transfected with pME18s-2xFlag (empty vector) (lane 1) or pME18s-2xFlag-BPOZ-2 (lane 2). The cells were incubated for 36h and harvested. Flag-BPOZ-2 mRNA was detected by RT-PCR analysis. The integrity of each RNA template was controlled by GAPDH amplification. (C) TdT degradation in J.EcoR cells. J.EcoR cells were infected either with retrovirus vectors encoding BPOZ-2 (2) or with an empty vector alone (1). The cells endogenously express TdT. Cycloheximide (CHX, 100 µg/mL) was added to the medium containing DMSO (lanes 1–5) or MG132 (20 µM) (lanes 6–10), and the culture was incubated for 10 min to 4 h. The cells were lysed and 10 µg of total lysate was subjected to SDS-PAGE, followed by immunoblotting (WB) using an anti-TdT or anti-actin antibody. BPOZ-2 expression in the total lysate (40 µg; middle panel) was also analyzed by immunoblotting (WB) using an anti-BPOZ-2 antibody. (D) BPOZ-2 mRNA expression in J.EcoR cells. J.EcoR cells were infected with pMXs-IG (empty vector) (lane 1) or pMXs-IG-BPOZ-2 (lane 2). BPOZ-2 mRNA was detected by RT-PCR analysis. The integrity of each RNA template was controlled by GAPDH amplification.

We have elucidated that TdT degradation by the 26S proteasome is promoted by BPOZ-2 in vivo. However, BPOZ-2 and TdT have been observed to localize within the cytoplasm and nucleus, respectively (Unoki & Nakamura 2001; Thai & Kearney 2004). Because BPOZ-2 should associate with TdT to be ubiquitinated within the cytoplasm or nucleus, we determined where TdT is ubiquitinated. We initially observed the localization of TdT or BPOZ-2 by expressing DsRed-TdT and EGFP-BPOZ-2 in the 293T cells, respectively. As shown in Fig. 9B, when only DsRed-TdT or EGFP-BPOZ-2 was expressed, DsRed-TdT was observed within the nucleus and, although EGFP-BPOZ-2 was mainly expressed within the cytoplasm, EGFP-BPOZ-2 was also observed within the nucleus. We then determined the site of TdT ubiquitination by fractionating the cells into the cytoplasmic and nuclear fractions (CF and NF, respectively). After co-expressing Myc-TdT, Flag-BPOZ-2, Flag-CUL3, Flag-Rbx1, and His-Ub in the 293T cells in the presence of MG132, the cells were lysed in NP-40 and then the cell lysate was fractionated by centrifugation. CF and NF were then applied to a Ni²⁺-agarose column. After the SDS-PAGE of the purified proteins followed by Western blotting, ubiquitinated Myc-TdT was cross-reacted with an anti-Myc antibody. As shown in Fig. 8 (lanes 7–9), ubiquitinated Myc-TdT was detected in only NF, whereas no ubiquitinated Myc-TdT was detected in CF (lanes 4-6), indicating that TdT is ubiquitinated within the nucleus. These findings also strongly suggest that ubiquitinated TdT is degraded only within the nucleus and is not recruited to the cytoplasm for degradation.

View larger version (30K): [in this window] [in a new window]   Figure 8  TdT is ubiquitinated within the nucleus. Ubiquitinated Myc-TdT was detected in the nuclear extract of 293T cells over-expressing BPOZ-2. The cells were transfected with plasmids encoding 6xHis-ubiquitin (lanes 1–9), Myc-TdT (lanes 1–9), Flag-BPOZ-2 (lanes 2, 3, 5, 6, 8, and 9), Flag-CUL3 (lanes 3, 6, and 9), and Flag-Rbx1 (lanes 3, 6, and 9) in the indicated combinations. After incubation for 36 h, 20 µM MG132 was added to the medium and the culture was incubated for another 7 h. The cells were fractionated in NF and CF. NF and CF were suspended in a denaturing buffer, and ubiquitin-containing protein complexes were affinity-purified and subjected to SDS-PAGE. Ubiquitinated TdT and total ubiquitinated proteins were detected by immunoblotting using anti-Myc and anti-Ub antibodies, respectively. Myc-TdT, Flag-BPOZ-2, Flag-CUL3, and Flag-Rbx1 expressed in the transfected cells were detected by immunoblotting using anti-Myc or anti-Flag antibodies. β-Tubulin and HP1 in NF and CF, respectively, were used as controls.

Next, to determine where TdT co-localizes with BPOZ-2, we co-expressed EGFP-BPOZ-2 and DsRed-TdT in 293T cells and observed them under fluorescence microscopy. As shown in Fig. 9D(5), EGFP-BPOZ-2 co-localized with DsRed-TdT adjacent to the nucleolus within the nucleus in the presence of MG132. Notably, a large speckle of TdT was observed adjacent to the nucleolus by co-expressing BPOZ-2, when compared with TdT alone (Fig. 9D(4)). We further determined the localization of TdT, BPOZ-2, and CUL3 by co-expressing them in 293T cells. As shown in Fig. 9E (4) and (6), they co-localized as large speckles within the nucleus in the presence of MG132, supporting our finding that TdT, BPOZ-2, and CUL3 form a ternary complex in vivo.

	Discussion

Top Abstract Introduction Results Discussion Experimental procedures References

 
We have demonstrated that BPOZ-2 is ubiquitinated through CUL3-based E3 ligase and degraded by the 26S proteasome and that TdT is ubiquinated within the nucleus and degraded by the 26S proteasome as promoted by BPOZ-2. BPOZ-2 is considered to be a human counterpart of yeast Btb3p and a substrate of CUL3 ligase.

BPOZ-2 consists of putative functional domains of ankyrin repeats, two BTB/POZ domains and a C-terminal region and binds to CUL3 through the two BTB/POZ domains. Compared with BPOZ-2 that has two BTB/POZ domains as well as the tumor suppressor RhoBTB2, Keap1 and SPOP have only one BTB/POZ domain. From the detailed structural analysis of Keap1 by constructing truncated or mis-sense mutants, Keap1 has been found to form a homodimer and bind to CUL3 through the BTB/POZ domain (McMahon et al. 2006). At least two BTB/POZ domains may generally be essential for binding to one CUL3 molecule. Therefore, Keap1 with only one BTB/POZ domain is necessary to form a homodimer for binding to one CUL3 molecule, whereas it is not necessary for RhoBTB2 and BPOZ-2 to form a homodimer for binding to one CUL3 molecule because they have two BTB/POZ domains. CUL3 is enzymatically active when it forms a dimer (Wimuttisuk & Singer 2007). Since we found that BPOZ-2 forms a homodimer (data not shown), a hetero tetramer consisting of two BPOZ-2 molecules and two CUL3 molecules should be formed. We thus expect that SPOP with one BTB/POZ domain form a homodimer. From the finding showing that the C-terminal region of BPOZ-2 binds to TdT, it is considered that this region functions as a substrate binding region. The function of ankyrin repeats in the N-terminal region has not yet been clarified.

Five substrate proteins are degraded through the CUL3-based ubiquitin–proteasome system; cyclin E, which promotes the cell cycle transition from the G1 phase to S phase (Singer et al. 1999), RhoBTB2 (Wilkins et al. 2004), the transcription factor Nrf2 (Kobayashi et al. 2004), the oncogenic collaborator BMI1 (Hernandez-Munoz et al. 2005), and the anti-apoptotic protein Daxx (Kwon et al. 2006). Although the protein that binds to cyclin E has not yet been identified, RhoBTB2, Nrf2, and BMI1 have been identified as substrates of RhoBTB2, Keap1, and SPOP, respectively. Daxx has also been reported to be a substrate of SPOP (Kwon et al. 2006). We have shown that TdT is degraded through the CUL3-based ubiquitin–proteasome system. Although no notable enhancement of TdT ubiquitination by only BPOZ-2 was detected, TdT ubiquitination was markedly promoted when both BPOZ-2 and CUL3 were co-expressed in cells, strongly suggesting that TdT is a substrate of BPOZ-2. TdT was ubiquitinated even when BPOZ-2 and CUL3 were not expressed. Although an endogenous BPOZ-2/CUL3 complex might ubiquitinate TdT, it is also possible that an adaptor-dependent E3 ligase for TdT other than BPOZ-2-dependent CUL3 ligase is present to ubiqutinate TdT or that the BPOZ-2/CUL3 complex promotes further ubiquitination of ubiquitinated-TdT.

TdT expression is strictly restricted at the N-region synthesis during the V(D)J recombination of Ig heavy chains in pro-B-cells or the TcR and β genes in pro- and pre-T cells (Fowler & Suo 2006). After the stage of pro-B- or pre-T cells during lymphocyte development, TdT is rapidly degraded. Therefore, in pre-B cells, no N-region is synthesized at the junction between the V and J DNA segments in the Ig light-chain genes. The specific degradation of target proteins is generally carried out through the 26S proteasome system after the polyubiquitination of the proteins. Our results suggest that TdT is degraded through the ubiquitin–proteasome system within the nucleus in pre-B cells, or CD4⁺CD8^– or CD4^–CD8⁺ T cells, which are developed from pre-T cells. RAG2, which regulates RAG1 by forming a complex to carry out V(D)J recombination, is degraded through the CUL1-based ubiquitin–proteasome system (Mizuta et al. 2002; Jiang et al. 2005). Considering that RAG1 and RAG2 function in pro- and pre-B cells during lymphocyte development, the stage-specific expressions of RAG1 and RAG2 may be regulated differently from that of TdT, which is expressed only in pro-B cells. This difference in expressional regulation could reflect the use of the CUL1- or CUL3-based ubiquitin–proteasome system for the degradation of RAG2 and TdT, respectively. Although we could expect that RAG1 is degraded through the CUL1-based ubiquitin–proteasome system as well as RAG2, RAG1 exhibits E3 ligase activity by itself (Jones & Gellert 2003). At present, it is too early to determine which E3 ligases, that is, CUL1, CUL2, CUL3, CUL4A, CUL4B, CUL5, and CUL7 ligases, are used for the ubiquitination of substrates on the basis of precise standards such as the specific amino acids sequence, structure and function. Proteins such as DDB2 and XPC, which are involved in DNA repair, are ubiquitinated by the CUL4A E3 ligase (Sugasawa et al. 2005; El-Mahdy et al. 2006). On the other hand, the DNA polymerase µ, which belongs to the DNA polymerase X family and functions in DNA repair, binds to BPOZ-2 (Maezawa et al. data not shown) to be ubiquitinated through the same CUL3-based ubiquitin–proteasome system as that for TdT. Further data accumulation is necessary to elucidate of the general rule showing the relationship between E3 ligase and its substrate.

When we observed the localization of BPOZ-2 expressed in 293T cells, BPOZ-2 was mainly expressed within the cytoplasm and hardly detected within the nucleus. However, when we observed BPOZ-2 expressed in the presence of MG132, it notably accumulated within the nucleus, strongly suggesting that BPOZ-2 within the cytoplasm is recruited into the nucleus. The fact that no export signal sequence is found in TdT or BPOZ-2 and that no accumulation of BPOZ-2 within the nucleus is detected in the presence of leptomycin B (data not shown), which is a specific inhibitor of CRM1/exportin 1, supports our speculation. We are now elucidating the molecular mechanism, underlying the recruit of BPOZ-2 from the cytoplasm to the nucleus. The possible role of BPOZ-2 within the nucleus after being recruited from the cytoplasm is to degrade TdT by serving as an adaptor for CUL3. The signal from mature TcR or Ig produced by functional V(D)J recombination should be relayed from the cytoplasm to the nucleus to degrade TdT. In addition to the possible function as an adaptor for CUL3, we speculate that BPOZ-2 also serves as a transmitter for relaying the signal from mature TcR or Ig to the nucleus.

BPOZ-2 over-expression inhibits cell cycle progression during the G1/S transition (Unoki & Nakamura 2001). Given that BPOZ-2 is the adaptor for CUL3 in the ubiquitin–proteasome system, we can speculate that cell cycle arrest is caused by the degradation of proteins involved in cell cycle progression through the BPOZ-2-dependent ubiquitin–proteasome system. Likewise, the growth suppressive effects of BPOZ-2 may be caused by the degradation of proteins, which positively regulate cell growth, through the BPOZ-2-dependent ubiquitin–proteasome system. Note that we have isolated the Nek2A and Rassf1c genes whose products are directly involved in the cell cycle progression among the fifty clones isolated using a yeast two-hybrid system. Nek2A, which functions in centrosome separation and spindle formation, is regulated in a cell-cycle-dependent manner by proteasomal destruction in mitosis (Fry & Yamano 2006; Hayes et al. 2006). Rassf1c, which promotes β-catenin accumulation by interacting with β-TrCP, stimulates human cancer cell proliferation (Amaar et al. 2006; Estrabaud et al. 2007).

In conclusion, BPOZ-2 is ubiquitinated by CUL3-based E3 ligase and degraded by the 26S proteasome. BPOZ-2 is considered to be a human counterpart of yeast Btb3p and substrate for CUL3 ligase. TdT is ubiquitinated within the nucleus and degraded by the 26S proteasome as promoted by BPOZ-2. Adaptor proteins with BTB/POZ domains have been proposed to function as adaptors for E3 ligase CUL3 (Furukawa et al. 2003). To finally determine whether BPOZ-2 is a specific adaptor for CUL3 or TdT is a specific substrate of BPOZ-2, we are attempting to establish the in vitro BPOZ-2-dependent ubiquitination system. We also consider it essential to study the in vivo mechanism of the ubiqtintation and degradation of TdT in B- or T cells.

	Experimental procedures

Top Abstract Introduction Results Discussion Experimental procedures References

 
Plasmids

The cDNA clone for BPOZ-2 was isolated by screening the human thymus cDNA library using a yeast two-hybrid system with TdT interacting factor 1 (TdIF1) as the bait. cDNA for Rbx-1 was synthesized by PCR using the human thymus cDNA library as the template. TdT cDNA isolation was described previously (Koiwai & Morita 1988). cDNA for CUL3 was obtained from Dr T. Nagase (Kazusa DNA Reserch Institute, Chiba, Japan). The expression vectors for BPOZ-2 tagged with 6xHis, GST, Flag epitope, Myc epitope or EGFP at the N-terminus were constructed by inserting their genes into pET28 (Novagen, Madison, WI), pGEX4T (Amersham Biosciences, Uppsala, Sweden), pME18s-2xFlag (a kind gift from Dr T. Masai), pME18s-Myc (a kind gift from Dr. T. Masai), and pEGFP-C3 (Clontech, Palo Alto, CA), respectively. The expression vectors for CUL3 tagged with GST, Flag epitope or EGFP at the N-terminus were constructed by inserting their genes into pGEX5X (Amersham Biosciences), pME18s-2xFlag and pEGFP-C1 (Clontech), respectively. The expression vectors for TdT tagged with 6xHis, GST, Myc epitope, EGFP, or DsRed-monomer at the N-terminus were constructed by inserting their genes into pET28, pGEX5X, pCMVmyc (Invitrogen, Carlsbad, CA), pEGFP-C3 and pDsRed-monomer-C1 (Clontech), respectively. The expression vectors for Rbx1 tagged with the Flag epitope at the N-terminus were constructed by inserting the gene into pME18s-2xFlag. Deleted DNA fragments of the BPOZ-2 or TdT gene were subcloned into pAS2-1 (Clontech), and the BPOZ-2 and CUL3 genes were subcloned into pACT2 (Clontech). Histidine 129 or 286 of BPOZ-2 was replaced with leucine using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) to prevent its interaction with CUL3. Mutagenesis was performed using pUC19-BPOZ-2 as the template and the following mutagenic primers: H129L, 5'-CCATTCCGGGTGCTTCGCTGCGT-CCTG-3' (forward) and 5'-CAGGACGCAGCGAAGCACCCGGAATGG-3' (reverse); H286L, 5'-GCAGCTTCCTCTGCCTCAAGGCCTTTT TCTG-3' (forward) and 5'-CAGAAAAAGGCCTTGAGGCA GAGGAAGCTGC-3' (reverse). The His-tagged Ub expression vector pMT107 is a kind gift from Dr D. Bohmann (Treier et al. 1994).

Antibodies and chemical reagents

A rabbit polyclonal antibody (pAb) against BPOZ-2 was generated using a purified recombinant His-tagged BPOZ-2 (amino acids 61–478) as the antigen. His-tagged BPOZ-2 was expressed in E. coli and purified with nickel resin. To detect endogenous BPOZ-2 in the bovine thymocytes, the rabbit pAb against BPOZ-2 was obtained from Proteintech Group, Inc (Chicago, IL). The goat pAb against CUL3 (C-18) was obtained from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). The rabbit pAb against TdT was raised using purified calf TdT. The monoclonal mouse anti-β-tubulin antibody used was kindly provided by Dr T. Arai (Tokyo University of Science, Chiba, Japan). Mouse monoclonal antibodies (mAbs) against the Flag epitope tag (M2), actin (AC40), and ubiquitin (6C1) were obtained from Sigma (St Louis, MO). The mouse mAbs against the Myc epitope tag (4A6) and HP1 (15.19s2) were from Upstate (Lake Placid, NY). The mouse mAb against GFP (GF200) was from Nacalai tesque (Kyoto, Japan). The rabbit pAb against the GST epitope tag was from Affinity BioReagents (Golden, CO). The secondary anti-mouse IgG antibodies conjugated with Alexa Fluor 350 and 546 were from Molecular Probes (Eugene, OR). The secondary anti-mouse and anti-rabbit IgG antibodies conjugated with HRP were from New England Biolabs (Ipswitch, MA). MG132 and 3-amino-1,2,4-trizole (3AT) were purchased from Sigma. Cycloheximide (CHX) was from Nacalai Tesque.

Cell culture and transient transfection

COS7 and 293T cells were cultured in Dulbecco's modified Eagle medium (Gibco-BRL) supplemented with 10% fetal bovine serum and 100 µg/mL kanamycin. Jurkat cells expressing the ecotropic receptor (J.EcoR cells) were kindly provided by Dr. T. Saito (RIKEN, Kanagawa, Japan) (Yamasaki et al. 2001). J.EcoR cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum and 100 µg/mL kanamycin. Transfection was performed using Lipofectamine 2000 (Invitrogen) or HilyMax (Dojindo, Gaithersburg, MD) transfection reagent according to the manufacturer's instructions.

Yeast two-hybrid screening and interaction assays

For the yeast two-hybrid screening, the human thymus Matchmaker library in pACT2 (Clontech) was used to isolate the target clones using the manufacturer's recommended conditions. In brief, S. cerevisiae strain Y190 was sequentially transfected with the bait vector pAS2-1-BPOZ-2 (full) and the cDNA library by the lithium acetate method. Transformants were selected on plates without histidine, tryptophan, and leucine in the presence of 45 mM 3AT. Positive clones were then isolated after incubation for 4–6 days at 30 °C, and β-galactosidase activity was assayed by the filter method. Extrachromosomal DNA was isolated by the glass bead method from β-galactosidase-positive clones that grew in the absence of histidine. Prey plasmids were transformed in E. coli DH5 cells and purified. Then, the DNA sequences of the genes inserted in the plasmids were determined. To determine the binding regions in a protein using deletion mutants, the interaction between a protein (prey) and a mutant (bait) was also detected from β-galactosidase activity using a yeast two-hybrid system.

GST pull-down assay, immunoprecipitation, and Western immunobloting

Recombinant BPOZ-2, TdT, and CUL3 proteins were expressed in E. coli strain BL21 (DE3) as GST-fusion proteins. BL21 cells were grown at 37 °C until 0.6 OD600 and then the expression of the proteins was induced by adding 100 µM isopropyl-1-thio-β-D-galactopyranoside and incubating the mixture for an additional 20 h at 22 °C. The cells were harvested by centrifugation at 1560 g for 5 min, and the pellet was resuspended in the lysis buffer with 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% TritonX-100, 10% glycerol, 1 mM DTT, 1 mM EDTA, 1 mM PMSF, 1 mg/mL pepstatin A, 1 mg/mL leupeptin, and 5 mM benzamidine. After sonication, the cell lysate was centrifuged at 21 880 g for 20 min. GST-fused proteins were coupled to glutathione Sepharose 4B beads (Amersham Bioscience) by incubation for 1 h at 4 °C. The beads were washed 5 times in 1 mL of lysis buffer and incubated with His-tagged proteins. After an additional incubation for 1 h, the beads were washed 5 times in 1 mL of lysis buffer, and bound proteins were analyzed by SDS-PAGE followed by immunoblotting using an anti-GST, anti-TdT, or anti-BPOZ-2 antibody.

For immunoprecipitation, COS7 cells, 293T cells and the bovine thymocytes were lysed in Nonidet P-40 lysis buffer (50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Nonidet P-40, 1 mM EDTA, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 µg/mL leupeptin, 10 µg/mL aprotinin, 1 mM benzamidine, and 2 µg/mL pepstatin A). The cell lysate was incubated with the appropriate antibodies for 2 h at 4 °C and then with 20 µL of 50% slurry of protein A-Sepharose or protein G-Sepharose for another 1 h. After washing 5 times in 1 mL of wash buffer (50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 10% glycerol, and 0.01% Nonidet P-40), bound proteins were eluted by boiling and subjected to SDS-PAGE followed by immunobloting.

Retrovirus production and infection

Full-length cDNA coding for BPOZ-2 was inserted into the pMXs-IG vector (kindly provided by Dr T. Kitamura, University of Tokyo, Tokyo, Japan). Plat-E packaging cells (kindly provided by Dr T. Kitamura), the potent retrovirus-packaging cell line derived from 293T cells (Morita et al. 2000), were plated on a 6-well plate, incubated for 24 h, and then transfected with 1 µg of pMXs-IG-BPOZ-2 in conjunction with 3 µL of FuGENE6 (Roche) according to the manufacturer's instructions. 24 h after transfection, the culture medium was replaced with 2 mL of fresh medium (10% FBS/RPMI 1640), the cells were cultured for another 24 h, and the virus-containing medium was collected and filtered. J.EcoR cells were infected twice with a viral supernatant containing 1% of HilyMax on subsequent days.

In vivo ubiquitination assay

293T cells were transfected with several combinations of plasmids together with His-tagged Ub vector as described in Figure legends (Treier et al. 1994). 36 h after the transfection, the cells were treated with DMSO or MG132 (final concentration, 20 µM) for 7 h to abrogate proteasome activity. Whole cell extracts were prepared in the lysis buffer U (20 mM Tris–HCl (pH 7.4), 0.5 M NaCl, 0.02% Nonidet P-40, 8 M urea, and 5 mM imidazole) and incubated overnight on a HIS-Select Nickel Affinity Gel (Sigma). After washing 4 times in the lysis buffer U, the beads were boiled in the sample buffer and the eluate was subjected to immunoblotting using an anti-Myc antibody.

Cell fractionation

Nuclear and cytosolic protein extracts were prepared according to Nguyen et al.'s method with minor modifications (Nguyen et al. 2005). 293T cells cultured on 35-mm dishes were transfected and treated with MG132 or DMSO, as described above. After washing, the cells were harvested by scraping in ice-cold phosphate-buffered saline and collected by centrifugation. The cells were lysed with 200 µL of CF lysis buffer (50 mM Tris–HCl (pH 7.4), 10 mM NaCl, 5 mM MgCl₂, and 0.5% Nonidet P-40) for 10 min on ice. After centrifugation, the supernatant (or cytosolic extract) was collected and centrifuged at 15 000 g for 30 min to remove cellular debris and then diluted with four volumes of buffer containing 12.5 mM Tris–HCl (pH 7.4), 600 mM NaCl, 10 M urea, and 6.25 mM imidazole. The nuclear pellet was washed 3 times with the CF buffer and resuspended by vortexing in a lysis buffer (20 mM Tris–HCl (pH 7.4), 0.5 M NaCl, 0.1% Nonidet P-40, 8 M urea, and 5 mM imidazole). The nuclear extract was isolated from the debris by centrifugation, as described above. Protein concentration was estimated using protein assay (Bio-Rad) with bovine serum albumin as standard. A small aliquot of the extracts was used for direct immunoblotting, and the remaining extracts were subjected to ubiquitination assay, as described above.

Immunocytochemistry (ICC)

293T cells were grown on coverslips and transfected with plasmids. The cells were then treated with 10 µM MG132 for 7 h and fixed in PBS with 4% paraformaldehyde (PFA) for 20 min. Next, the cells were permeabilized in PBS containing 0.2% Triton X-100 for 10 min, incubated in PBS with 1% BSA for 30 min and further incubated with an anti-Ub or anti-Flag antibody for 1 h at room temperature. After extensive washing, the cells were incubated with Alexa Fluor 350- or 546-conjugated goat anti-mouse IgG for 40 min, washed and mounted on a glass slide with PBS containing 4,6-diamido-2-phenylindole (DAPI). Fluorescence images were obtained under fluorescence microscopy (Axiovert 200, Carl Zeiss) or laser scanning confocal microscopy (FV1000-D, Olympus or TSC-SP2, Leica).

	Acknowledgements

 
We are indebted to Dr Bohmann for his kind gift of plasmids encoding ubiquitin, Dr Nagase for the cDNA encoding CUL3, Dr Saitou for the J.EcoR cells, and Dr Kitamura for the Plat-E cells and pMXs-IG vector. We are also grateful to Dr M. Watanabe, M. Kinoshita, and T. Yanai for valuable suggestions and technical support.

	Footnotes

 
Communicated by: Fumio Hanaoka

^* Correspondence: Email: j6407705{at}ed.noda.tus.ac.jp

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Received: 1 October 2007
Accepted: 27 January 2008

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