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Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Noda, Chiba, Japan
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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One hundred eighty-three human proteins with a BTB/POZ domain (BTB proteins) including BPOZ-2 have been reported, and these BTB proteins function in various physiological transactions including transcriptional repression, regulation of cytoskeleton or gating of ion channels (Collins et al. 2001). Recently, human RhoBTB2 and Caenorhabditis elegans MEL-26 have been identified as adaptors for E3 ubiquitin ligase (Ub ligase) scaffold proteins (Pintard et al. 2003; Wilkins et al. 2004). They mediate the recruitment of substrates to Cullin 3 (CUL3), which is a component of the Skp1-Cullin 1-F-box (SCF)-like E3 complex. RhoBTB2 is an atypical RhoGTPase of 83 kDa. It has a GTPase domain at the N-terminal followed by two BTB/POZ domains. RhoBTB2 directly binds to CUL3 through its first BTB/POZ domain and is degraded through the CUL3-dependent ubiquitin–proteasome system (UPS) (Wilkins et al. 2004). MEL-26, which contains MATH and BTB/POZ domains, has been identified as a substrate-specific adaptor for CUL3-based Ub ligase. The substrate of MEL-26 is down-regulated by UPS (Pintard et al. 2003). In addition to RhoBTB2 and MEL-26, yeast Btb3p, which contains an ankyrin repeat and two BTB/POZ domains at N- (residues 51–114) and C-terminals (residues 165–262 and 297–405), respectively, has also been reported as a putative substrate-specific adaptor for Pcu3p-based Ub ligase and Btb3p itself is degraded by the Pcu3p-dependent UPS (Geyer et al. 2003).
BPOZ-2 is a human counterpart of yeast Btb3p and functions as an adaptor for E3 Ub ligase to degrade a ubiquitylated substrate through the 26S proteasome system (Maezawa et al. 2008). BPOZ-2 itself is ubiquitylated through the CUL3-based E3 Ub ligase as well as yeast Btb3p. We thus attempted to isolate the genes whose products directly bind to BPOZ-2 using a yeast two-hybrid system and found that BPOZ-2 binds to eukaryotic elongation factor 1A1 (eEF1A1) and terminal deoxynucleotidyltransferase (TdT), which is a DNA polymerase synthesizing extra nucleotides (N nucleotides) at the V–J, V–D and D–J junctions during the V(D)J recombination of immunoglobulin (Ig) or T-cell receptor genes (Komori et al. 1993).
eEF1A1 is a GTP binding protein of 50 kDa, which transfers the aminoacyl-tRNA to an acceptor site in ribosome during elongation in translation. eEF1A1 also binds to F-actin or microtubles to function as an activator of phosphoinositol 4-kinase and is involved in apoptosis or cell cycle control (Ejiri 2002). In addition to its function in translation, eEF1A1 is essential for the ubiquitin-dependent degradation of N-
-acetylated proteins such as histone H2A (Gonen et al. 1991, 1994). eEF1A1 also functions in the degradation of misfolded or unfolded proteins through the UPS (Chuang et al. 2005). eEF1A1 directly binds to the 26S proteasome regulatory particle triple A-ATPase subunit T1 (Rpt1), strongly suggesting that eEF1A1 is a mediator between damaged proteins and the 26S proteasome. We show that BPOZ-2 binds to eEF1A1 to promote eEF1A1 ubiquitylation and degradation, inhibit GTP binding to eEF1A1, and finally prevent translation in vitro.
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Results |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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BPOZ-2 is a human counterpart of yeast Btb3p, which is a putative substrate-specific adaptor for E3 Ub ligase Pcu3p (Maezawa et al. 2008). We then attempted to identify the substrate proteins of BPOZ-2 by isolating genes whose products directly bind to BPOZ-2 using a yeast two-hybrid system. After screening a human thymus cDNA library using BPOZ-2 as the bait, 52 candidate clones were isolated and their DNA sequences were determined. The amino acid sequences of 11 of 52 candidate clones were identical to that of eEF1A1. The longest cDNA clone for eEF1A1 was 1750 bp long and its DNA sequence shared a 99.9% identity with that of eEF1A1. G1743 in the 3'-noncoding region was replaced by A. The three clones isolated from the cDNA library were TdT genes. TdT is degraded through the BPOZ-2/CUL3-based UPS (Maezawa et al. 2008).
Binding between BPOZ-2 and eEF1A1
To confirm the direct binding between BPOZ-2 and eEF1A1, we initially performed GST pull-down assay in vitro using GST-eEF1A1 as the bait. Escherichia coli cell lysate expressing MBP-BPOZ-2 was reacted with the GST-eEF1A1-bound beads. After thoroughly washing the beads, we attempted to determine whether MBP-BPOZ-2 bound to the beads by Western blotting using an anti-BPOZ-2 antibody. As shown in Fig. 1A, MBP-BPOZ-2 was precipitated with the GST-eEF1A1-bound beads (lane 3), but not with the GST-bound beads (lane 4). To further confirm the association between BPOZ-2 and eEF1A1 in the mammalian cells, we performed co-immunoprecipitation assay. We co-transfected pEGFP-C3-BPOZ-2 and pME18s-flag (+2)-eEF1A1 into COS7 cells using Lipofectamine and determined whether EGFP-BPOZ-2 is co-immunoprecipitated together with FLAG-eEF1A1 using an anti-FLAG antibody as the bait. As shown in Fig. 1B, EGFP-BPOZ-2 was co-precipitated together with FLAG-eEF1A1 (lane 4), indicating that BPOZ-2 associates with eEF1A1 in mammalian cells. We also determined the binding between eEF1A1 and BPOZ-2 in the tissue cells. Although we initially attempted to determine their binding using rat liver, the expression level of BPOZ-2 is too low to detect the binding by immunoprecipitation assay. We then attempted to perform immunoprecipitation using a rat heart, because BPOZ-2 mRNA was strongly expressed in the heart when the mRNA expression was checked by RT-PCR. However, the expression level was too low to detect BPOZ-2 as well as liver. We then performed immunoprecipitation with bovine thymus. Bovine thymus was homogenized and fractionated to nuclear, mitochondrial, lysosomal, microsomal and cytoplasmic fractions. Immunoprecipitation was performed with the cytoplasmic fraction using a rabbit anti-BPOZ-2 antibody as the bait. As shown in Fig. 1C, eEF1A1 and BPOZ-2 were detected in the immunoprecipitant (lane 2), indicating that BPOZ-2 associates with eEF1A1 in the thymocytes. To further observe their localization in COS7 cells, we constructed the expression vectors pEGFP-C3-BPOZ-2 and pDsRed-monomer C1 (+2)-eEF1A1, co-transfected the vectors into the cells and observed their localizations under fluorescence microscopy. EGFP-BPOZ-2 and DsRed-eEF1A1 expressions in the absence of MG132 were observed as three patterns. As shown in Fig. 1D(ii–iv), EGFP-BPOZ-2 and DsRed-eEF1A1 co-localized within the entire cytoplasm (ii), within the entire cytoplasm and also as speckles (iii) and only as speckles (iv), respectively.
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We next attempted to identify the BPOZ-2 binding region in eEF1A1 by pull-down assay in vitro. In accordance with the three-dimensional structure of eEF1A1, eEF1A1 is composed of three domains, namely, Domains I, II and III (Fig. 2A(i); Gaucher et al. 2002). We thus constructed three eEF1A1 mutants (Fig. 2A(i)), which express the GST-fused N-terminal half region (Del 1), C-terminal half region (Del 2), and Domain II (Dom II). The eEF1A1 mutants and MBP-BPOZ-2 were used as the bait and prey, respectively. Both Del 1 and Del 2 bound to BPOZ-2, but not Dom II (data not shown), suggesting that eEF1A1 binds to BPOZ-2 through Domains I and III. We then determined whether Domains I and III of eEF1A1 actually bind to BPOZ-2 by pull-down assay after constructing the plasmids expressing GST-fused Domains I and III (Dom I and Dom III). As shown in Fig. 2A(ii), Dom I and Dom III bound to BPOZ-2 but not Dom II, indicating that eEF1A1 binds to BPOZ-2 through Domains I and III.
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eEF1A1 binding region in BPOZ-2
We also attempted to determine the eEF1A1 binding region in BPOZ-2 by GST pull-down assay in vitro. BPOZ-2 has two ARs, two BTB/POZ domains (BTBs 1 and 2) and a predicted coiled-coil (CC) (Fig. 2B(i)). Four deletion mutants were constructed and expressed in E. coli (Fig. 2B(i); Del 1–Del 4). Each cell lysate expressing Del 1–Del 4 was reacted with GST-eEF1A1-bound beads and mutant proteins bound to the beads were analyzed by Western blotting using an anti-BPOZ-2, anti-MBP or anti-GST antibody. As shown in Fig. 2B(ii), Del 1 (lane 8) and Del 2 (lane 9) bound to eEF1A1 but Del 3 (lane 10) and Del 4 (lane 11) did not, indicating that BPOZ-2 binds to eEF1A1 through the region containing ARs and two BTB/POZ domains, BTBs 1 and 2. Note that both BTB/POZ domains in BPOZ-2 are required to bind to eEF1A1. We further confirmed the eEF1A1 binding region in BPOZ-2 by constructing the mis-sense mutants H129L and H286L. Residues H129 and H286 in BTBs 1 and 2 in BPOZ-2, respectively, were replaced by leucine (H129L and H286L, respectively). Because BPOZ-2 binds to eEF1A1 through both BTB/POZ domains but does not to only BTB 1 or BTB 2 (Del 3 and Del 4), we expected that H129L and H286L do not bind to eEF1A1. We over-expressed H129L and H286L in E. coli and reacted their cell lysates with GST-eEF1A1-bound glutathione Sepharose beads. H129L and H286L bound to the beads were analyzed by Western blotting using an anti-BPOZ-2 antibody. As shown in Fig. 2C(ii), H129L and H286L did not bind to eEF1A1 as expected (lanes 8 and 9). These results indicate that both BTB 1 and BTB 2 are necessary for BPOZ-2 to bind to eEF1A1. This is the same result as the binding between BPOZ-2 and CUL3, which is a major subunit of E3 Ub ligase for ubiquitylation. BPOZ-2 also binds to CUL3 through both BTB/POZ domains (Maezawa et al. 2008).
eEF1A1 ubiquitylation
BPOZ-2 binds to CUL3 both in vitro and in vivo and promotes TdT ubiquitylation (Maezawa et al. 2008). Because BPOZ-2 binds to eEF1A1 in vivo, we expected that eEF1A1 is ubiquitylated through the BPOZ-2/CUL3-based ubiquitylation system. We then determined whether eEF1A1 is actually ubiquitylated using in vivo ubiquitylation assay by expressing FLAG-eEF1A1 and His-Ub in 293T cells. We first fixed ubiquitylated proteins to nickel beads and determined whether ubiquitylated FLAG-eEF1A1 is detected in the ubiquitylated proteins by Western blotting using an anti-FLAG antibody. As shown in Fig. 3A, ubiquitylated eEF1A1 bands with higher molecular weights than eEF1A1 were detected in the form of a ladder (lane 2).
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Because BPOZ-2 binds to both eEF1A1 (Fig. 1) and CUL3 (Maezawa et al. 2008), we attempted to determine whether BPOZ-2, eEF1A1 and CUL3 form a ternary complex by in vitro GST pull-down and immunoprecipitation assays. As shown in supplementary Fig. S1, eEF1A1 bound to BPOZ-2 but not to CUL3, indicating that eEF1A1, BPOZ-2 and CUL3 do not form a ternary complex. Note that although eEF1A1 binds to BPOZ-2, it does not bind to the BPOZ-2/CUL3 complex. BPOZ-2 binds to eEF1A1 or CUL3 through its ankyrin repeat, the first and second BTB/POZ domains, or through the first and second BTB/POZ domains in BPOZ-2, respectively. Therefore, eEF1A1 and CUL3 are considered to competitively bind to the region containing the first and second BTB/POZ domains in BPOZ-2. These results strongly suggest that eEF1A1 is not ubiquitylated by CUL3-based Ub ligase, and consequently eEF1A1 is not degraded through the CUL3-dependent UPS.
Considering the above results, although we initially speculated that BPOZ-2 functions as a specific adaptor for CUL3, eEF1A1 may be ubiquitylated by BPOZ-2 using E3 Ub ligase other than CUL3. The in vitro ubiquitylation assay using BPOZ-2 and CUL3 also showed that eEF1A1 is not ubiquitylated through the CUL3-based ubiquitylation system (data not shown).
BPOZ-2 promotes eEF1A1 degradation
Since we clarified that BPOZ-2 promotes eEF1A1 ubiquitylation, we next attempted to determine whether eEF1A1 degradation is also promoted by BPOZ-2, when protein synthesis is inhibited by cycloheximide (CHX). The amount of FLAG-eEF1A1 expressed in 293T cells was monitored for 180 min in the presence or absence of MG132, when BPOZ-2 was or was not co-expressed. As shown in Fig. 4A and B, when BPOZ-2 was expressed in the cells, eEF1A1 degradation was detected soon after CHX treatment and no eEF1A1 was detected after 180 min in the absence of MG132, whereas no marked eEF1A1 degradation was detected in the presence of MG132. Thus, eEF1A1 ubiquitylation and degradation were promoted by BPOZ-2.
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Since eEF1A1 and BPOZ-2 were degraded by the 26S proteasome, we observed EGFP-BPOZ-2 and DsRed-eEF1A1 expressed in COS7 cells in the presence of MG132. As shown in Fig. 1E(i), when EGFP-BPOZ-2 or DsRed-eEF1A1 was solely expressed, EGFP-BPOZ-2 was detected within the cytoplasm and as speckles within the nucleus, and DsRed-eEF1A1 was detected with in the cytoplasm. When EGFP-BPOZ-2 and DsRed-eEF1A1 were co-expressed, BPOZ-2 localized as speckles within the cytoplasm in almost all the observed cells and eEF1A1 was localized within the entire cytoplasm and as speckles (Fig. 1E(ii)). These contrast with our previous observations, in which EGFP-BPOZ-2 and DsRed-eEF1A1 localized in the cells as three patterns when they were observed in the absence of MG132 (Fig. 1D). Considering that MG132 is a specific inhibitor for the 26S proteasome, BPOZ-2 and eEF1A1 may accumulate as speckles at the place of the 26S proteasome. We then observed the localization of BPOZ-2, eEF1A1 and the 26S proteasome in the presence or absence of MG132. EGFP-BPOZ-2 and EGFP-eEF1A1 were co-expressed in COS7 cells, and their localizations were observed together with the endogenous 26S proteasome by immunostaining using an anti-P32 (20S) antibody. As shown in Fig. 1F(i), when EGFP-BPOZ-2 or EGFP-eEF1A1 was solely expressed, they co-localized with the 26S proteasome within the entire cytoplasm. Note that EGFP-eEF1A1 did not always co-localize with the 26S proteasome (Fig. 1F(i), shown by **). These localization patterns were greatly changed when EGFP-eEF1A1 and FLAG-BPOZ-2 or FLAG-eEF1A1 and EGFP-BPOZ-2 were co-expressed. As shown in Fig. 1F(ii), EGFP-eEF1A1 and the endogenous 26S proteasome co-localized within the entire cytoplasm and also as speckles or only as speckles both in the presence or absence of MG132. The co-localization pattern detected only as speckles was mainly observed in the cells. When FLAG-eEF1A1 and EGFP-BPOZ-2 were co-expressed, EGFP-BPOZ-2 and the endogeneous 26S proteasome were also observed within the entire cytoplasm and as speckles or only as speckles. Note that the nuclear 26S proteasome does not co-localize with EGFP-eEF1A1 or EGFP-BPOZ-2. These results support that eEF1A1 and BPOZ-2 are degraded by the 26S proteasome.
BPOZ-2 inhibits GTP binding to eEF1A1
eEF1A1, aminoacyl-tRNA and GTP form a ternary complex in the elongation step in translation. eEF1A1 GTP-dependently delivers the aminoacyl-tRNA to the acceptor site in a ribosome (Jefferies et al. 1994), which is then released from the ribosome as a GDP/eEF1A1 complex after hydrolyzing GTP (reviewed by Browne & Proud 2002). GTP binds to eEF1A1 through N156 in Domain I (Ejiri 2002). In addition, RhoBTB2, which is a putative adaptor for CUL3-based Ub ligase, was found to GTP-dependently bind to CUL3 through the first BTB/POZ domain (R. Kawahito, M. Tada, K. Kontani and T. Katada, personal communication; Wilkins et al. 2004). We thus attempted to determine whether GTP affects the BPOZ-2 binding to eEF1A1 in vitro by constructing a mis-sense mutant of eEF1A1, N156D, which has a much higher Km for GTP than normal eEF1A1 (Cavallius & Merrick 1998). The binding between BPOZ-2 and N156D was examined by GST pull-down assay using GST-eEF1A1 and MBP-BPOZ-2 as the bait and prey, respectively, in the presence of various concentrations of GTP. As shown in Fig. 5A, BPOZ-2 GTP-independently bound to eEF1A1 (lanes 1–6) and N156D (lane 7), indicating that GTP does not affect the binding between eEF1A1 and BPOZ-2. That is, eEF1A1 ubiquitylation and degradation may be GTP-independently promoted by BPOZ-2, and GTP binding to eEF1A1 is considered to be crucial only for the elongation step in translation. However, note that GTP or BPOZ-2 binds to Domain I (Andersen et al. 2001), and Domains I and III in eEF1A1, respectively. We therefore suspected that BPOZ-2 releases GTP from the GTP/eEF1A1 complex or inhibits GTP binding to eEF1A1 and consequently prevents translation. To determine whether BPOZ-2 actually inhibits GTP binding to eEF1A1, the effect of BPOZ-2 on GTP binding to eEF1A1 was initially examined. 32P-labeled--GTP (GTP*) in various amounts was first bound to GST-eEF1A1 coupled with glutathione Sepharose 4B beads, and then purified MBP-BPOZ-2 was added to the GTP*/GST-eEF1A1 containing mixture. After GST pull-down using GST-eEF1A1 as the bait, the radioactivity of GTP*/eEF1A1 was determined with a scintillation counter. As shown in Fig. 5B, GTP*/GST-eEF1A1 decreased to 15.7%, indicating that GTP was released from GTP/eEF1A1 by BPOZ-2. Furthermore, GTP* was first bound to GST-eEF1A1 and then MBP-BPOZ-2 in various amounts was added to GTP*/eEF1A1. After incubation and centrifugation, the radioactivity of GTP*/eEF1A1 in the precipitant was determined. As shown in Fig. 5C, the radioactivity of GTP*/eEF1A1 decreased with increasing amount of MBP-BPOZ-2, indicating that almost all GTP* was released from GTP*/eEF1A1. These results also show that BPOZ-2 does not have the ability to bind to GTP. If GTP binds to BPOZ-2 to form a GTP/BPOZ-2 complex, a GTP/BPOZ-2/eEF1A1 ternary complex should be precipitated and no decrease in radioactivity should be detected.
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On the basis of these results, we expected that BPOZ-2 prevents translation by binding to eEF1A1. To determine whether BPOZ-2 really prevents translation, purified BPOZ-2 was added to rabbit reticulocytes in an in vitro translation system. A plasmid encoding glutathione S-transferase (GST) gene was initially transcribed using a T7 promoter system and then GST was synthesized using the in vitro translation system containing indicated amounts of MBP-BPOZ-2. As shown in Fig. 6 (A and B), GST translation was expectedly prevented by up to 24% of the original rate of protein synthesis. Because no cDNA clone encoding the translation-related protein other than eEF1A1 was isolated by screening a cDNA library with BPOZ-2 as the bait, our results strongly suggest that BPOZ-2 directly binds to eEF1A1 to inhibit GTP binding to eEF1A1 and consequently prevents translation. However, to obtain conclusive results, we should construct an eEF1A1 mutant that maintains its elongation activity and simultaneously abolishes the ability to bind to BPOZ-2, or a BPOZ-2 mutant that only abolishes the ability to bind to eEF1A1.
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Discussion |
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The proteins with BTB/POZ domain(s) (BTB proteins) such as actinfilin, Btb3p, Keap1, MEL-26, RhoBTB2 and SPOP have been reported to directly bind to E3 Ub ligase CUL3 through the BTB/POZ domain(s) (Pintard et al. 2004; Wilkins et al. 2004; Kwon et al. 2006; Lo & Hannink 2006). We recently elucidated that BPOZ-2 also directly binds to CUL3 to ubiquitylate TdT (Maezawa et al. 2008). Both the first and second BTB/POZ domains in BPOZ-2 are required to bind to CUL3, and the C-terminal region without the second BTB/POZ domain is required to bind to TdT. In contrast with the bindings to CUL3 and TdT, the ARs or two BTB/POZ domains in BPOZ-2 are required to bind to eEF1A1. Although BPOZ-2 is one of the adaptors of CUL3-based Ub ligase, we have shown that BPOZ-2 CUL3-independently promotes eEF1A1 ubiquitylation, indicating that BPOZ-2 is not a specific adaptor for CUL3-based Ub ligase and eEF1A1 is ubiquitylated by E3 Ub ligase other than CUL3-based Ub ligase. Our results also indicate that adaptor proteins containing BTB/POZ domain do not always function as adaptors for CUL3.
Given that, what kind of E3 Ub ligase is used to ubiquitylate eEF1A1? Two hundred eleven yeast membrane-associated proteins have been identified as the ubiquitylated forms and eighty-three among them including eEF1A1 were classified as potential substrates of NPL4- or UBC7-dependent ERAD by a genetic/proteomic technique, which takes advantage of the differential ubiquitylation of proteins in the npl4 and ubc7 yeast mutants (Hitchcock et al. 2003). Interferon (IFN) induces the accumulation of ubiquitylated eEF1A1 in the lung cancer cell line H1355 in the presence of the 26S proteasome inhibitor lactacystin (Lamberti et al. 2007). BPOZ-2 is transcriptionally activated by PTEN in endometrial cells and mediates the growth-suppressive effect on PTEN (Unoki et al. 2001). From these facts, eEF1A1 may be ubiquitylated through the E3 ligase involved in the ERAD, IFN or PTEN signaling pathway. Since E3 Ub ligases such as ring-finger protein 125 (RNF125) in the IFN pathway, and Doa10, gp78, Hrd1 and Rfp2 in the ERAD pathway have been reported to ubiquitylate the substrates (Fang et al. 2001; Kikkert et al. 2004; Ravid et al. 2006; Arimoto et al. 2007; Lerner et al. 2007), eEF1A1 might also be ubiquitylated through such E3 Ub ligases.
After ubiquitylation in 293T cells, eEF1A1 is degraded by the 26S proteasome (Fig. 4). In normal living cells, eEF1A1 is up-regulated in growing cells as stimulated by epidermal growth factor (EGF), heregulin-β1 (HRG) or serum, whereas eEF1A1 is down-regulated in resting cells to reduce the expression level of proteins (Jefferies et al. 1994; Talukder et al. 2001). The eEF1A1 degradation by the 26S proteasome would be induced and promoted by BPOZ-2 during the transition from growing cells to resting cells. Research to determine whether BPOZ-2 really promotes the eEF1A1 ubiquitylation and degradation during the transition in vivo is now underway at our laboratory.
eEF1A1 is a GTP binding protein, which transfers the aminoacyl-tRNA as a GTP–eEF1A1–aminoacyl-tRNA complex to the ribosome, and GTP binding to eEF1A1 is necessary for the elongation in translation. In translational regulation, we here propose that BPOZ-2 releases GTP from the GTP/eEF1A1 complex by directly binding to eEF1A1 to negatively regulate translation. That is, by inhibiting GTP binding to eEF1A1, BPOZ-2 inhibits the functional formation of a GTP/eEF1A1/aminoacyl-tRNA complex. Similarly to BPOZ-2, translationally controlled tumor protein (TCTP), which is proposed to function in the growth-related activities, associates with eEF1A1 and eEF1Bβ (Cans et al. 2003). TCTP preferentially binds to GDP/eEF1A1 to impair the GDP-GTP exchange reaction performed by eEF1Bβ. Although both BPOZ-2 and TCTP can bind to eEF1A1, the BPOZ-2 binding to eEF1A1 is considered to have a different physiological significance from the TCTP binding to eEF1A1. That is, after inhibiting the binding between GTP and eEF1A1 by direct binding to eEF1A1, BPOZ-2 further promotes the eEF1A1 ubiquitylation and degradation. In contrast to BPOZ-2, TCTP may temporarily prevent translation without eEF1A1 ubiquitylation and degradation. Note that two proteins negatively regulate translation at the stage of GTP binding to eEF1A1, indicating that the formation of a GTP/eEF1A1 complex is one of the most critical stages for translation.
Recent research has revealed that eEF1A1 plays multifunctional roles such as apoptosis and protein degradation (Ejiri 2002) in addition to translation. eEF1A1 is crucial for the ubiquitin-dependent degradation of N--acetylated proteins such as histone H2A (Gonen et al. 1991, 1994). When cells synthesize misfolded or unfolded proteins, eEF1A1 binds to the unusual proteins to transfer them to the 26S proteasome. eEF1A1 plays a role as a mediator by directly binding to Rpt1 in the 19S regulatory particle of the 26S proteasome (Chuang et al. 2005). In addition to its possible function as an adaptor for E3 Ub ligase, we also suspected that BPOZ-2 promotes the function of eEF1A1 as a mediator between misfolded or unfolded nascent proteins and the 26S proteasome or, from the view point of eEF1A1, eEF1A1 mediates the transfer of excessively expressed BPOZ-2 to the 26S proteasome for its degradation. Thus, BPOZ-2, eEF1A1 and Rpt1 should form a BPOZ-2/eEF1A1/Rpt1 ternary complex. We thus examined whether eEF1A1, BPOZ-2 and Rpt1 form a ternary complex in vitro. As shown in supplementary Fig. S3, BPOZ-2 did not bind to Rpt1 and no BPOZ-2/eEF1A1/Rpt1 ternary complex was formed, suggesting that BPOZ-2 is not degraded by the 26S proteasome through eEF1A1 as a mediator. These results indicate that BPOZ-2 does not promote the function of eEF1A1 as a mediator and that eEF1A1 does not mediate the transfer of BPOZ-2 to the 26S proteasome for its degradation.
BPOZ-2 is transcriptionally activated by over-expressing PTEN in endometrial cancer cells. BPOZ-2 also mediates the growth-suppressive effect of PTEN (Unoki et al. 2001). PTEN antagonizes the phosphatidylinositol 3-kinase (PI3K)–Akt signaling pathway by dephosphorylating phosphatidylinositol 3,4,5-triphosphate (PIP3) (Choi et al. 2002). The PI3 K–Akt signaling pathway activates the mammalian target of rapamycin (mTOR) through the inactivation of tuberous sclerosis complex (TSC) (Tee et al. 2003). mTOR triggers the phosphorylation of key regulators of the translation machinery including ribosomal p70 S6 kinase (p70 S6K) and eukaryote initiation factor 4E binding protein 1 (4EBP1) to activate protein synthesis (Burnet et al. 1998). That is, PTEN inactivates protein synthesis by antagonizing the PI3K–Akt signaling pathway. Therefore, we can expect that PTEN induces BPOZ-2 expression and the expressed BPOZ-2 degrades eEF1A1 to prevent translation. Transient PTEN expression in PTEN-null glioma cell lines causes cell cycle arrest at the G1 phase (Furnari et al. 1998; Myers et al. 1998). We also speculate that transcriptional control by PTEN through the PI3K–Akt signaling pathway, which is mediated by BPOZ-2, results in cell cycle arrest.
In conclusion, BPOZ-2 inhibits GTP binding to eEF1A1 and promotes eEF1A1 degradation by the 26S proteasome to prevent translation. eEF1A1 is considered to be a target protein of the adaptor protein BPOZ-2 and is ubiquitylated by other E3 Ub ligase than CUL3. Thus, we are attempting to determine the E3 Ub ligase specifically using BPOZ-2 and eEF1A1 as its adaptor and substrate, respectively.
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Experimental procedures |
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For the yeast two-hybrid screening, the human thymus Matchmaker library in pACT2 (Clontech, Palo Alto, CA) was used to isolate the target clones under the manufacturer's recommended conditions. In brief, Saccharomyces cerevisiae strain Y190 was sequentially transfected with the bait vector pAS2–1-BPOZ-2 (full) and cDNA library by the lithium acetate method. Transformants were selected on plates lacking 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 into E. coli DH5 cells and purified. Then DNA sequences of the genes inserted in the plasmids were determined. To determine the binding regions in a protein by constructing deletion mutants, the interaction between a protein (prey) and mutant (bait) was also detected by β-galactosidase activity using a yeast two-hybrid system.
Site-directed mutagenesis
A site-directed mutagenesis of eEF1A1 was performed by a PCR technique using a Quick Change site directed mutagenesis kit (Stratagene Inc., La Jolla, CA). A pair of mutagenic primers were used for eEF1A1 N156D: 5'-TCGGTGTTAACAAAATGAAT TCCACTGAGCCACCC-3' and 5'-GGGTGGCTCAGT GGAATTCATTTTGTTAACACCGA-3'. The substituted bases are underlined. The nucleotide sequences were determined by the dideoxy termination method. cDNA fragments containing each mutation were subcloned into pGEX 4T-1 (+2).
Over-expression in Escherichia coli
GST-eEF1A1, GST-CUL3, GST-Rpt1, GST, His-eEF1A1, His-CUL3, His-BPOZ-2, MBP-BPOZ-2 and MBP were over-expressed in E. coli BL21 (DE3) in LB broth (50 mL) supplemented with ampicillin or kanamycin (50 µg/mL) at 37 °C until an OD600 of 0.6. cDNA of CUL3 was obtained from Dr T. Nagase (Kazusa DNA Research Institute, Chiba, Japan). The expression of each protein was induced with IPTG (final 0.01 mM) for 14 h at 20 °C. Cells were harvested by centrifugation at 6000 g for 7 min and suspended in lysis buffer T (20 mM Tris-HCl (pH 7.4), 1 mM EDTA, 200 mM NaCl, 1% Triton X-100, 5% glycerol, 1 µg/mL pepstatin A, 0.5 µg/mL leupeptin, 5 mM benzamijine, 5 mM DTT, and 0.1 mM PMSF). The cells were then transferred to a new 1.5-mL tube, centrifuged at 13 000 g for 2 min and suspended in 800 µL of lysis buffer T. The cells were then thoroughly disrupted with a sonicator. The samples were centrifuged at 16 000 g at 4 °C for 20 min.
Purification of MBP and MBP-BPOZ-2
An amylose affinity column was prepared as follows. One gram of potato starch (Wako, Osaka, Japan) and 4 mL of dH2O were mixed at 50 °C. Six milliliters of 5 N NaOH and 3 mL of epichlorohydrin were gently added to the mixture at 50 °C. After cooling down to room temperature (R.T.), the gel was triturated in 80 mL of distilled H2O using a syringe. After washing in the wash buffer (20 mM Tris–HCl and 200 mM NaCl), 10 mL of the gel solution was packed in a glass column (
= 1 cm).
MBP or MBP-BPOZ-2 expression was induced in E. coli BL21 (DE3; pMAL c2 or pMAL c2 (+2)-BPOZ-2) at 20 °C for 20 h in 150 mL of culture medium. After harvesting them, the cells were suspended in 20 mL of lysis buffer T and sonicated. The soluble lysate was centrifuged at 7500 rpm for 20 min, and the supernatant was loaded onto the amylose affinity column. After thoroughly washing with 50 mL of the wash buffer, the proteins bound to the column were eluted in 20 mL of elution buffer (250 mM maltose, 20 mM Tris–HCl, and 200 mM NaCl) at a 100 µL/min of eluent flow rate, and 1.5-mL fractions were collected. Fractions 5 and 6 were mixed, dialyzed against the dialysis buffer (20 mM Tris–HCl, and 200 mM NaCl), and concentrated using Amicon Ultra 50 000 MWCO (Amicon, Beverly, MA).
GST and His pull-down assay
The E. coli soluble lysate expressing the bait proteins was mixed with glutathione SepharoseTM 4B (GE Healthcare. Little Chalfont, UK) or HIS-Select Nickel affinity beads (Sigma, St Louis, MO) for 1.5 h at 4 °C. After washing 4 times, the beads were incubated with the soluble lysate of E. coli expressing the prey proteins for 2 h at 4 °C. After washing 4 times, the proteins bound to the beads were eluted in the sample buffer and the resulting mixture was heated in boiling water. The proteins in each sample were subjected to SDS-PAGE (20 mA/gel), transferred to a nitrocellulose membrane (150 mA/gel, 1.5 h), and analyzed by Western blotting using the indicated antibodies.
Cell culture, transfection and expression
COS7 and HEK293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C under 5% of CO2. Lipofectamine (Invitrogen, Carlsbad, CA) and HilyMax (Dojindo, Kumamoto, Japan) were utilized in the transfection methods.
Fluorescence microscopy
COS7 cells expressing EGFP-BPOZ-2 and/or DsRed-eEF1A1 were fixed with 8% paraform aldehyde for 30 min and observed under confocal microscope, Leica TCS SC2. The fixed cells were permeabilized in PBS containing 5% BSA and 0.1% Triton X-100. Proteasome was immunoreacted using anti-P32 (20S) and Alexa 546 anti-mouse IgG antibodies as the first and second antibodies, respectively.
Immunoprecipitation
After harvesting the cells, the cells were lysed in 800 µL of lysis buffer N (20 mM Tris–HCl (pH 7.4), 1 mM EDTA, 150 mM NaCl, 1% Nonidet P-40, 5% glycerol, 1 µg/mL pepstatin A, 0.5 µg/mL leupeptin, 5 mM benzamidine, 5 mM DTT, and 0.1 mM PMSF) for 1 h at 4 °C and sonicated. Soluble lysate was obtained by centrifugation at 13 000 rpm for 20 min at 4 °C. Five hundred micrograms of soluble lysate was mixed with an anti-FLAG or an anti-HA antibody (2 µg) and incubated for 2 h at 4 °C. Protein A SepharoseTM 4 Fast Flow (GE Healthcare) was added to the mixture and incubated for another 2 h at 4 °C. After washing 4 times, proteins were eluted with the sample buffer and boiled. The proteins in each sample were subjected to SDS-PAGE (20 mA/gel), transferred to a nitrocellulose membrane (150 mA/gel, 1.5 h), and analyzed by Western blotting using indicated antibodies.
Cell fractionation
Eight hundred micrograms of bovine thymus were homogenized in 5 mL of 0.25 M sucrose buffer (50 mM Tris–HCl (pH 7.4) and 0.25 M sucrose) (homogenate). After filtration through gauze, homogenate was fractionated by centrifugation at 600 g for 10 min to nuclear fraction (NF, ppt) and cytosolic fraction 1 (CSF1, sup). CFS1 was fractionated by centrifugation at 5000 g for 10 min to mitochondrial fraction (MitF, ppt) and cytosolic fraction 2 (CSF2, sup). CFS2 was fractionated by centrifugation at 8000 g for 10 min to lysosomal fraction (LF, ppt) and cytosolic fraction 3 (CSF3, sup). CFS3 was fractionated by centrifugation at 105 000 g for 60 min to microsomal fraction (MicF, ppt) and cytoplasmic fraction (CF, sup). NF, MitF, LF and MicF were suspended in 1 mL, 0.3 mL, 0.3 mL and 0.3 mL of 0.25 M sucrose buffer, respectively.
In vivo ubiquitylation assay
HEK 293T cells were transfected with pMT107, pME18s-flag (+2)-eEF1A1 and/or pEGFP-C3-BPOZ-2 using HilyMax. After changing the medium to DMEM with 10% FBS, proteins were expressed for 48 h. DMSO or MG132 at a final concentration of 10 µM was added to the medium and incubated for 12 h. Cells were lysed in 600 µL of lysis buffer U (20 mM Tris–HCl (pH 7.5), 500 mM NaCl, 8 M urea, 0.02% NP-40, and 5 mM imidazole) and sonicated. The soluble fraction was centrifuged at 16 000 g for 15 min. Five hundred micrograms of the soluble fraction was mixed with 15 µL of HIS Select Affinity Gel for 12 h at 4 °C. After washing with 500 µL of Lysis buffer U 3 times, the proteins bound to the gel were eluted in 30 µL of SDS sample buffer. The proteins were subjected to SDS-PAGE and analyzed by Western blotting using the indicated antibodies.
CHX and MG132 treatment
HEK 293T cells were transfected with pEGFP-C3-BPOZ-2 and pME18s-flag (+2)-eEF1A1 using HilyMax and incubated for 48 h. The medium was changed to DMEM containing 10 µg/mL CHX and/or 10 µM MG132. After incubation for the indicated time, the cells were harvested in the suspension buffer. The cells were lysed in 70 µL of lysis buffer N by sonication (50%, level 1, three times). The soluble fraction was centrifuged at 16 000 g for 10 min. The soluble fraction was subjected to SDS-PAGE and analyzed by Western blotting using the indicated antibodies.
Binding assay between GTP and eEF1A1
GST-eEF1A1 (wt)- or GST-bound glutathione SepharoseTM 4B beads were mixed with MBP-BPOZ-2 in the absence or presence of GTP. The proteins bound to the beads were mixed with MBP-BPOZ-2 in the presence of GTP (100 µM). MBP-BPOZ bound or unbound to eEF1A1 was detected by Western blotting using an anti-BPOZ-2 antibody. The effect of BPOZ-2 on GTP binding to eEF1A1 was analyzed as follows. 32P-labeled--GTP (GTP*) was added to the reaction mixture containing GST-eEF1A1 or GST. After the pre-incubation of GST-eEF1A1 and GTP*, MBP-BPOZ-2 was added to each reaction mixture. After centrifugation, the radioactivity of the GS-4B-bound GST-eEF1A1/GTP* in the pellet was determined using a liquid scintillation spectrometer (Beckman, Fullerton, CA). GST-eEF1A1 and GST fixed on GST-Sepharose 4B beads were incubated with 100 µM GTP*, and MBP-BPOZ-2 or MBP was added to each reaction mixture. After centrifugation, the radioactivity of GST-eEF1A1/GTP* in the pellet was determined.
In vitro translation
The rabbit reticulocyte in vitro translation system (Merk Ltd., Nottingham, UK) was used to analyze the effect of BPOZ-2 on the translation of glutatione S-transferase (GST). Single Tube ProteinTM System 3, T7 was purchased from Novagen. The GST inserted into pET42a was used as a template for its expression. After transcription of GST, the purified MBP or MBP-BPOZ-2 was added to the reaction mixture and incubated at 37 °C for 1 h. The translational products were subjected to SDS-PAGE, transferred to a nitrocellulose membrane, and analyzed by Western blotting using an anti-GST antibody.
Cell counting
HEK 293T cells transfected pEGFP or pEGFP-C3-BPOZ-2 were cultured for 2 days. Transfectants seeded and cultured on 24-well plate for 0–144 h were counted with haemacytometer.
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* Correspondence: Email: koiwai{at}rs.noda.tus.ac.jp; projecterkkk{at}hotmail.com
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Received: 17 December 2007
Accepted: 4 March 2008
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) or GST (
). After pre-incubation of GST-eEF1A1 and GTP*, 10 µg of MBP-BPOZ-2 was added to each reaction mixture (
). After centrifugation, the radioactivity of the glutathione SepharoseTM 4B-bound GST-eEF1A1/GTP* in the pellet was determined using a scintillation counter. (C) GST-eEF1A1 (50 µg) and GST (50 µg) fixed to glutathione SepharoseTM 4B were incubated with 100 µM GTP* and the indicated amounts (0, 20, 40 and 100 µg/mL) of MBP-BPOZ-2 (