Antiproliferative proteins of the BTG/Tob family are degraded by the ubiquitin-proteasome system Hitoshi Sasajima, Koji Nakagawa and Hideyoshi Yokosawa Department of Biochemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan BTG/Tob family proteins, which are characterized by similarities in their N-terminal BTG/Tob homology domains, control cell growth negatively. Among the BTG/ Tob family members, BTG2/TIS21/PC3 proteins have beenreportedtohaveshortlivesandtobedegradedby the proteasome. However, the mechanisms regulating the stabilities of other BTG/Tob family proteins have not yet been clarified. Here, we report that BTG1, Tob, and Tob2 proteins, as well as BTG2 protein, are degraded by the ubiquitin–proteasome system; the degradation of Tob protein in HeLa cells and the degradation of BTG1, BTG2, Tob and Tob2 proteins transiently expressed in HEK293 cells were inhibited by treatments with protea- some-specific inhibitors. Co-expression of BTG1, BTG2, Tob, or Tob2 protein with ubiquitin in HEK293 cells revealed specific multiubiquitination of each of the four proteins. Although the full-length and N-terminal trun- cated forms of BTG1, BTG2, Tob, and Tob2 proteins were unstable, the respective C-terminal truncated forms were found to be almost stable, suggesting that the C-terminal regions control the stabilities of BTG1, BTG2, Tob, and Tob2 proteins. In addition, it was found that the respective C-terminal regions confer instability on green fluorescent protein, a normally stable protein. Thus, it can be concluded that the C-terminal regions are necessary and sufficient to control the stabilities of BTG1, BTG2, Tob, and Tob2 proteins. Keywords: BTG; Tob; ubiquitin; proteasome; degradation signal. The BTG/Tob family is composed of at least six distinct members in vertebrates, namely BTG1, BTG2/TIS21/PC3, BTG3/ANA, PC3B, Tob and Tob2 [1]. The main charac- teristic of this family is the presence of a highly conserved 110-amino-acid N-terminal region, designated the BTG/ Tob homology domain. The BTG/Tob family members are involved in cell growth control (antiproliferation) and differentiation. PC3 and TIS21 were isolated as immediate early genes that were induced by stimulation of nerve growth factor in a rat PC12 cell line and by stimulation of phorbol ester in a mouse 3T3 cell line, respectively [2,3]. BTG2, a molecule showing high similarity to BTG1, was isolated as a human homolog of rodent PC3/TIS21 [4]. BTG1 was cloned as a gene involved in a t(8;12)(q24;q22) chromosomal translocation in B-cell chronic lymphocytic leukemia [5]. On the other hand, Tob was isolated as a protein associating with the ErbB2 growth factor receptor [6] and, subsequently, Tob2 was isolated on the basis of its similarity to Tob [7,8]. In addition, other family members were isolated using different cloning strategies [9–12]. The BTG/Tob homology domain contains two highly homologous regions, designated the A and B boxes, and the A box has been suggested to play an antiproliferative role [9,13]. It has been shown that the expression of BTG2, a transcriptional target gene of p53, is upregulated by a DNA- damaging reagent and that the expressed BTG2 protein down-regulates the transcription of cyclin D1, leading to inhibition of progression of the cell cycle at the G1 phase through declining phosphorylated Rb [4]. In addition, the BTG/Tob family has been reported to be involved not only in antiproliferative function but also in differentiation [14,15]. Variations in functions of the BTG/Tob family proteins seem to be due to their interactions with other proteins. For example, BTG1 and BTG2/TIS21/PC3 interact with type 1 protein arginine methyltransferase, and their associations may be important in neuronal differentiation [16–19]. Several BTG/Tob family members associate with transcriptional factors: Tob binds Smad1, Smad5, and Smad8 and nega- tively regulates BMP2-dependent bone formation by inhibit- ing transcriptional activity of Smad [20]. BTG1 and BTG2/ TIS21/PC3 interact with Hoxb9 [21], while BTG1, BTG2/ TIS21/PC3, BTG3/ANA, Tob, and Tob2 interact with CAF1, a component of the CCR4 transcriptional regulatory complex [8,22–25]. In these cases, it has been proposed that the respective BTG/Tob family proteins function as cofac- tors of the transcriptional factors [26]. A balance between the expression of proliferative genes (proto-oncogenes) and that of antiproliferative genes (tumor suppressor genes) regulates cell cycle progression, cell growth control, differentiation, and apoptosis. Both synthesis and degradation of these gene products are important for Correspondence to H.Yokosawa,DepartmentofBiochemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan. Fax: + 81 11 706 4900, Tel.: + 81 11 706 3754, E-mail: yoko@pharm.hokudai.ac.jp Abbreviations: BTG, B-cell translocation gene; Tob, transducing molecule of ErbB2; TIS, TPA-induced sequence 21; PC3, pheo- chromocytoma cell-3; ANA, abundant in neuroepithelium area; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin ligase; GFP, green fluorescent protein. (Received 21 March 2002, revised 22 May 2002, accepted 17 June 2002) Eur. J. Biochem. 269, 3596–3604 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.3052.x determining their functioning. It has been shown that the ubiquitin–proteasome system mediates the degradation of various proliferative and antiproliferative gene products, such as c-Jun [27], c-Fos [28], c-Myc [29], p53 [30], and b-catenin [31]. This system plays an important role in intracellular degradation of short-lived regulatory proteins and abnormal proteins [32–35]. In this system, target proteins are first tagged with multiubiquitin chains via isopeptide bonds, catalyzed by the sequential actions of E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3 (ubiquitin ligase). The multiubiquitinated target proteins thus produced are degraded by the 26S proteasome. On the other hand, some proteins, such as ornithine decarboxylase, are degraded by the proteasome without ubiquitination [36]. Although the degradation of BTG2 is inhibited by lactacystin, a proteasome-specific inhibitor [37], it remains to be clarified whether the degra- dation of BTG2 is dependent on ubiquitination or whether other members of the BTG/Tob family are subjected to degradation by the proteasome through ubiquitination. In this paper, we present evidence that the ubiquitin– proteasome system plays a key role in downregulation of BTG1, BTG2, Tob, and Tob2 among the BTG/Tob family members. We found that these four proteins are multiubiq- uitinated and then degraded by the 26S proteasome. In addition, analyses of the stabilities of truncated mutants of BTG1, BTG2, Tob, and Tob2 revealed that their C-terminal regions control the stabilities of the respective BTG/Tob family proteins. MATERIALS AND METHODS Materials Protease inhibitors, MG115, MG132, and E64d, were pur- chased from Peptide Institute, Inc. (Osaka, Japan). Cycloh- eximide, an inhibitor of protein synthesis, was purchased from Wako Pure Chemicals (Osaka, Japan). M-PER TM mammalian protein extraction reagent was purchased from Pierce. Monoclonal mouse anti-(T7-tag) Ig and anti-(T7- tag) Ig-immobilized agarose were purchased from Novagen. Polyclonal rabbit anti-(hemagglutinin epitope) (HA) Ig and polyclonal anti-actin Ig were purchased from Santa Cruz Biotechnology and Sigma, respectively. Monoclonal anti- (green fluorescent protein) (GFP) Ig and anti-Tob Ig (4B1) were obtained from Clontech and Immuno-Biological Laboratories (Gunma, Japan), respectively. Horseradish peroxidase-conjugated anti-(rabbit IgG) Ig and anti-(mouse IgG) Ig were from Amersham Pharmacia Biotech. Cell culture and transfection HEK293 and HeLa cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum at 37 °C under 5% CO 2 atmosphere. Transfection was performed using Effectene transfection reagent (Qia- gen) or LipofectAmine 2000 reagent (Life Technologies, Inc.), according to the manufacturer’s protocol. Plasmid constructions Human BTG1, Tob, and Tob2 cDNAs were obtained by RT-PCR with total RNA from K562 cells using forward and reverse primers, BTG1F and BTG1R, TobF and TobR, and Tob2F and Tob2R, respectively (Table 1). Human BTG2 cDNA was obtained by PCR with human universal Quick-Clone cDNA (Clontech) using forward and reverse primers, BTG2F and BTG2R (Table 1). To generate BTG1, BTG2, Tob, and Tob2 expression plasmids, pCI-neo-T7-BTG1, pCI-neo-T7-BTG2, pCI- neo-T7-Tob, and pCI-neo-T7-Tob2, respectively, the PCR products subcloned in the pGEM-T-vector (Pro- mega) were digested with EcoRI and SalI and then inserted into the EcoRI and SalI sites of the pCI-neo-T7- vector (38). The terminal-truncated mutants of BTG1 were constructed by PCR with pCI-neo-T7-BTG1 as a template using primers shown in Table 1. The PCR products were subcloned and inserted into the EcoRI and SalI sites of the pCI-neo-T7-vector. Other truncated mutant expression vectors of BTG2, Tob, and Tob2 were constructed in the same way as described above using primers shown in Table 1. pEGFP-C2-BTG1 (111–171), pEGFP-C2-BTG2 (98–158), pEGFP-C2-Tob (285–345), and pEGFP-C2-Tob2 (284–344) were constructed by PCR with the expression vectors stated above as templates using primers shown in Table 1. The PCR products subcloned in the pGEM-T-vector were digested with EcoRI and SalIandtheninsertedintotheEcoRI and SalI sites of pEGFP-C2 (Clontech). To generate the ubiquitin expression plasmid, pAS2-1-ubiquitin plasmid (a gift from M. Fujimuro of our laboratory) was digested with EcoRI and SalIandtheninsertedintotheEcoRI and SalI sites of the pCI-neo-HA vector that had been generated by inserting the oligonucleotides encoding the HA epitope (YPDYDVPDYA) into the NheI and EcoRI sites of pCI-neo. All of the constructs were verified by DNA sequence analysis. Immunoblotting Proteins were separated by SDS/PAGE on 12.5 or 15% gel and transferred to a nitrocellulose membrane (Advan- tec, Tokyo, Japan). The membrane was blocked with 5% nonfat milk in NaCl/P i containing 0.1% Tween 20 for 1 h at room temperature, incubated with the primary antibody at room temperature for 1 h and then with a horseradish peroxidase-conjugated anti-(rabbit IgG) Ig or anti-(mouse IgG) Ig at room temperature for 30 min, and developed by an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech). Analysis of protein stability To analyze the stability of Tob, HeLa cells were treated with 50 l M MG115, MG132 and E64d, each dissolved in dimethyl sulfoxide, for 2 h. The cells were then washed with NaCl/P i and harvested. For Western blotting, the cells were disrupted by M-PER TM reagent containing 50 l M MG132 and a protease inhibitor cocktail (Roche) for 5 min, and the lysate was centrifuged at 13 000 g.The resulting supernatant was subjected to SDS/PAGE and then to Western blotting with anti-Tob Ig and anti-actin Ig as a control. Alternatively, to analyze the effect of MG132 on degradation of endogenous Tob, HeLa cells were treated with 50 l M MG132 for 1 h and then with 25 lgÆmL )1 cycloheximide for the indicated periods. The cell lysate was Ó FEBS 2002 Ubiquitin-dependent degradation of BTG/Tob family proteins (Eur. J. Biochem. 269) 3597 prepared and subjected to SDS/PAGE and then to Western blotting as described above. To analyze the stability of BTG1, BTG2, Tob, or Tob2 transiently expressed in HEK293 cells, the cells were transfected with 0.5 lg each of pCI-neo-T7-BTG1, pCI- neo-T7-BTG2, pCI-neo-T7-Tob, or pCI-neo-T7-Tob2 using Effectene transfection reagent in 35-mm dishes. After a 24-h incubation period, the transfected cells were treated with 50 l M MG132, dissolved in dimethylsulfox- ide, for 2 h. The cell lysates were prepared as described above, and the protein levels of BTG1, BTG2, Tob, and Tob2 were detected by Western blotting with anti-(T7-tag) Ig. Alternatively, to analyze the effect of MG132 on degradation of BTG1, BTG2, Tob, or Tob2 transiently expressed in HEK293 cells, the cells were transfected with 2.0 lg each of pCI-neo-T7-BTG1, pCI-neo-T7-BTG2, pCI-neo-T7-Tob, or pCI-neo-T7-Tob2 using Lipofect- Amine 2000 in 35-mm dishes. After a 24-h incubation period, the transfected cells were treated with 50 l M MG132 for 1 h and then with of 25 lgÆmL )1 cyclohex- imide for the indicated periods. The cell lysates were prepared and subjected to SDS/PAGE and Western blotting, as described above. The stabilities of deletion mutants were analyzed in the presence of cycloheximide as described above. Ubiquitination of BGT/Tob family proteins HEK293 cells were transfected with several combinations of 4.5 lg of pCI-neo-HA-ubiquitin and 0.5 lgofpCI-neo-T7- (a BTG/Tob family member) using LipofectAmine 2000 in 100-mm dishes (the total amount of plasmid DNA being adjusted to 5 lg with the empty vector pCI-neo-T7), incubated for 24 h, and then treated with 50 l M MG132 for 12 h. The cells were disrupted with M-PER TM reagent containing 0.5% SDS, 50 l M MG132 and a protease inhibitor cocktail, sonicated for 10 s, and then cleared by centrifugation at 13 000 g for 15 min The resulting super- natant was diluted 10-fold with M-PER TM reagent contain- ing MG132 and a protease inhibitor cocktail and subjected to immunoprecipitation. The diluted supernatant was incubated with 20 lg of anti-(T7-tag) Ig-immobilized aga- rose at 4 °C for 1 h. The beads were collected and washed four times with washing buffer containing 4.29 m M Na 2 HPO 4 ,1.47m M KH 2 PO 4, 2.7 m M KCl, 137 m M NaCl, 0.1% Tween 20, and a protease inhibitor cocktail, and the proteins adsorbed were eluted with elution buffer containing 100 m M citric acid, pH 2.2, and 1% SDS. The eluate was neutralized with 2 M Tris base, pH 10.4, and subjected to SDS/PAGE and Western blotting with anti-HA Ig or anti-(T7-tag) Ig. Bands were detected with a Vectastain Table 1. Forward and reverse primers for PCR. Construct Forward primer Reverse primer BTG1 (full length) 5¢-GAATTCATGCATCCCTTCTACACC-3¢ (BTG1F) 5¢-GTCGACTTAACCTGATACAGTCAT-3¢ (BTG1R) (36–171) 5¢-GAATTCCAGCTGCAGACCTTCAGC-3¢ BTG1R (71–171) 5¢-GAATTCCGCATCAACCATAAAATG-3¢ BTG1R (96–171) 5¢-GAATTCAGGCTTCTCCCAAGTGAA-3¢ BTG1R (1–141) BTG1F 5¢-GTCGACTTATTGCACGTTGGTGCTGTT-3¢ GFP–BTG1 (111–171) 5¢-GAATTCTCCTACAGAATTGGAGAGG-3¢ BTG1R BTG2 (full length) 5¢-GAATTCATGAGCCACGGGAAG-3¢ (BTG2F) 5¢-GTCGACCTAGCTGGAGACTGCCA-3¢ (BTG2R) (34–158) 5¢-GAATTCAGGCTTAAGGTCTTCAGC-3¢ BTG2R (69–158) 5¢-GAATTCCGCATCAACCACAAGATG-3¢ BTG2R (1–129) BTG2F 5¢-GTCGACTTAGGCCAGTGGGGCC-3¢ GFP–BTG2 (98–158) 5¢-GAATTCGAGCTGACCCTGTGGG-3¢ BTG2R Tob (full length) 5¢-GAATTCATGCATCCCTTCTACACC-3¢ (TobF) 5¢-GTCGACTTAGTTAGCCATAACAGGC-3¢ (TobR) (59–345) 5¢-GAATTCCACATAGGGGAGAAAGTG-3¢ TobR (84–345) 5¢-GAATTCGGCAATCTGCCACAGGAT-3¢ TobR (114–345) 5¢-GAATTCGTGGATGATAATAATGAA-3¢ TobR (1–315) TobF 5¢-GTCGACTTAGCCTCCATAGGCTGC-3¢ (1–277) TobF 5¢-GTCGACTTAAGGAAAAATAAATTCCTT-3¢ GFP–Tob (285–345) 5¢-GAATTCACCAATGGAATGTTCCCA-3¢ TobR Tob2 (full length) 5¢-GAATTCATGCAGCTAGAGATCAAAGT-3¢ (Tob2F) 5¢-GTCGACTCAGTTGGCCAGCACCA-3¢ (Tob2R) (59–344) 5¢-GAATTCCACATTGGGGAGATGGTG-3¢ Tob2R (84–344) 5¢-GAATTCGCCAATGTGCCTGAGGAG-3¢ Tob2R (114–344) 5¢-GAATTCCTGGATGACAGTGAGGGT-3¢ Tob2R (1–313) Tob2F 5¢-GTCGACTTAGAAGAGGCTGTTGGC-3¢ (1–273) Tob2F 5¢-GTCGACTTAATCAAAGAAGAGGCTGGG-3¢ GFP–Tob2 (284–344) 5¢-GAATTCCCGTTTGGAGGCAGTG-3¢ Tob2R 3598 H. Sasajima et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Universal Elite ABC kit (Vector Laboratories, Inc.) using biotinylated anti-(rabbit IgG) Ig as a secondary antibody. RESULTS Tob is degraded by the 26S proteasome BTG2/TIS21/PC3 is a short-lived protein with a half-life of less than 15 min [39]. Various labile proteins are degraded by the ubiquitin–proteasome system [32–35]. Recently, it has been demonstrated that lactacystin, a proteasome-specific inhibitor, inhibits the degradation of BTG2 protein in prostate cells [37]. To determine whether the ubiquitin–proteasome system plays an important role in the degradation of BTG/Tob family members, we first examined the effects of proteasome inhibitors on the steady-state level of Tob protein in HeLa cells. HeLa cells were treated with several protease inhibitors, and then the level of Tob protein was analyzed by Western blotting with an antibody against Tob (Fig. 1A). Treatment of HeLa cells with the proteasome inhibitors MG115 and MG132 resulted in accumulation of Tob protein, com- pared with that in the case of treatment with E64d (a cysteine protease inhibitor that inhibits calpain and lysosomal protease). Next, to determine whether the proteasome inhibitor directly affects degradation of Tob protein, we measured its effect on the stability of Tob protein under conditions in which protein synthesis had been blocked by cycloheximide. HeLa cells were subjected to MG132 inhibition followed by treatment with cycloheximide. Western blot analysis with an anti-Tob Ig (Fig. 1B) revealed that Tob protein was stabilized in the presence of MG132, indicating that the proteasome inhibitor directly inhibited degradation of the Tob protein. Thus, Tob protein is degraded by the 26S proteasome. BTG1, BTG2, and Tob2 are also degraded by the 26S proteasome As endogenous Tob protein is degraded by the 26S proteasome, we examined the effects of the proteasome inhibitor MG132 on the levels of BTG1, BTG2, Tob, and Tob2 proteins transiently expressed in HEK293 cells. The HEK293 cells, in which the respective four proteins tagged with T7 epitope were transiently expressed, were treated with MG132, and the levels of the four proteins were analyzed by Western blotting with an antibody against T7- tag (Fig. 2). In either case, the treatment of HEK293 cells with MG132 resulted in remarkable accumulation of the respective protein. Next, we analyzed the effects of MG132 on the degra- dation of BTG1, BTG2, and Tob2 proteins under condi- tions in which protein synthesis had been blocked by cycloheximide: The HEK293 cells, transiently expressing T7-tagged BTG1, BTG2, and Tob2, were subjected to MG132 inhibition and then to treatment with cyclohexi- mide. Western blot analysis with anti-(T7-tag) Ig (Fig. 3) showed that BTG1, BTG2, and Tob2 proteins were Fig. 1. Effects of proteasome inhibitors on the level of Tob protein. (A) HeLa cells were treated with the protease inhibitors MG115 (b), MG132 (c), and E64d (d) at concentrations of 50 l M for 2 h and 0.5% dimethyl sulfoxide (a) was used as a control. The cell lysates were prepared, and the protein levels of Tob and b-actin were analyzed by Western blotting with antibodies against Tob and actin, respectively. (B) HeLa cells were treated with 50 l M MG132 or 0.5% dimethyl- sulfoxide (DMSO) for 1 h and then incubated with 25 lgÆmL )1 cycloheximide for the indicated periods. The cell lysates were prepared at the indicated times, and the protein levels of Tob and b-actin were analyzed as described in (A). Fig. 2. Effects of the proteasome inhibitor on the levels of BTG/Tob family proteins. T7-tagged BTG/Tob family proteins, T7-BTG1 (a), T7-BTG2 (b), T7-Tob (c) and T7-Tob2 (d), were transiently expressedinHEK293cells,andthecellsweretreatedwith50l M MG132 (+) or 0.5% dimethylsulfoxide (–) for 2 h. The cell lysates were prepared, and the protein levels of BTG/Tob proteins and b-actin were analyzed by Western blotting with antibodies against T7-tag and actin, respectively. Nonspecific bands are indicated with an asterisk. Ó FEBS 2002 Ubiquitin-dependent degradation of BTG/Tob family proteins (Eur. J. Biochem. 269) 3599 stabilized in the presence of MG132, indicating that the proteasome inhibitor directly inhibited the degradation of BTG1, BTG2, and Tob2 proteins. The result in the case of transiently expressed T7-tagged Tob was the same as that in the case of endogenous Tob (data not shown). Taken together, the results suggest that the BTG/Tob family members BTG1, BTG2, Tob, and Tob2 are degraded by the 26S proteasome. BTG/Tob family proteins are multiubiquitinated To determine whether BTG1, BTG2, Tob, and Tob2 proteins are multiubiquitinated prior to degradation by the 26S proteasome, we transiently expressed both T7-tagged BTG/Tob family proteins and HA-tagged ubiquitin in HEK293 cells simultaneously. After the transiently expressed cells had been treated with MG132 for 12 h, cell extracts were subjected to immunoprecipitation with anti- (T7-tag) Ig-immobilized agarose, and the immunoprecipi- tates produced were subjected to SDS/PAGE and then to Western blotting with an anti-HA Ig (Fig. 4). High- molecular-mass materials accumulated only in the case of cotransfection with expression plasmids containing T7- tagged BTG/Tob family proteins and HA-tagged ubiquitin. It should be noted that a long incubation time (12 h) in the presence of MG132 was a prerequisite for detecting multiubiquitination. Expression of T7-tagged BTG/Tob family proteins was confirmed by immunoblotting with anti-(T7-tag) Ig (data not shown). These results strongly suggest that BTG1, BTG2, Tob, and Tob2 are multiubiq- uitinated prior to their degradation by the 26S proteasome. The C-terminal regions of BTG/Tob family proteins act as protein degradation signals To determine the region required for degradation of BTG1, BTG2, Tob, and Tob2 proteins by the ubiquitin–protea- some system, we constructed several truncated BTG1, BTG2, Tob, and Tob2 expression plasmids. We first assumed that the regions controlling the stabilities of BTG/Tob family proteins are situated on the conserved N-terminal BTG/Tob homology domains, because BTG1, BTG2, Tob, and Tob2 proteins are all degraded by the ubiquitin–proteasome system and the C-terminal regions of BTG1/BTG2 show little similarity to those of Tob/Tob2. We constructed N-terminal truncated BTG1, BTG2, Tob, and Tob2 expression plasmids (Fig. 5A,C,E,G) and tran- siently expressed them in HEK293 cells. The stabilities of the N-terminally truncated mutants were analyzed by Western blotting with anti-(T7-tag) Ig under conditions in Fig. 3. Effects of the proteasome inhibitor on the stabilities of BTG/Tob family proteins. T7-tagged BTG/Tob family proteins, T7-BTG1 (A), T7-BTG2 (B) and T7-Tob2 (C), were transiently expressed in HEK293 cells, and the cells were treated with 50 l M MG132 or 0.5% dimethyl- sulfoxide (DMSO) for 1 h and then incubated with 25 lgÆmL )1 cycloheximide for the indicated periods. The cell lysates were prepared at the indicated times, and the protein levels of BTG/Tob proteins and b-actin were analyzed as described in Fig. 2. Fig. 4. Ubiquitination of BTG/Tob family proteins. HEK293 cells were transiently transfected with the indicated combinations of HA-tagged ubiquitin and T7-tagged BTG/Tob family protein expression plasmids, mock (a), T7-BTG1 (b), T7-BTG2 (c), T7-Tob (d) and T7-Tob2 (e), and at 24 h after transfection, the cells were treated with 50 l M MG132 for 12 h. (A) The cell lysates were subjected to immunoprecipitation with anti-(T7-tag) Ig-immobilized agarose, and the immunoprecipi- tates produced were then subjected to Western blotting with anti-HA Ig. The high molecular bands indicate multiubiquitinated T7-tagged BTG/Tob family proteins. IP, immunoprecipitation; Ub, ubiquitin. (B) Parts of the same cell lysates were directly subjected to Western blotting with anti-HA Ig to check the expression level of HA-tagged ubiquitin. 3600 H. Sasajima et al. (Eur. J. Biochem. 269) Ó FEBS 2002 which protein synthesis had been blocked by cycloheximide (Fig. 5B,D,F,H). In contrast to our assumption, none of the N-terminally truncated mutants displayed resistance to degradation, suggesting that the BTG/Tob homology domain is not required for degradation by the ubiquitin- proteasome system. Next, we constructed C-terminal trun- cated expression plasmids (Fig. 5A,C,E,G) and transiently expressed them in HEK293 cells. The stabilities of the C-terminally truncated mutants were analyzed in the same way as that in the above-described experiments using the N-terminally truncated mutants (Fig. 5B,D,F,H). Unex- pectedly, BTG1, BTG2, Tob, and Tob2 mutants with truncation of C-terminal amino acids displayed almost complete resistance to degradation, suggesting that the C-terminal region controls the stability of the BTG/Tob family proteins. To confirm that the C-terminal regions of BTG1, BTG2, Tob, and Tob2 act as degradation signals, we constructed GFP fusion protein expression plasmids in which the sequences of BTG1 (111–171), BTG2 (98–158), Tob (285–345), and Tob2 (284–344) were fused to the C-terminus of GFP to generate chimeric proteins, GFP– BTG1 (111–171), GFP–BTG2 (98–158), GFP–Tob (285–345) and GFP–Tob2 (284–344) fusion proteins, respectively (Fig. 6A). We then transiently expressed the chimeric proteins, together with intact GFP, in HEK293 cells. The stabilities of the respective GFP fusion proteins were analyzed by Western blotting with an anti-GFP Ig under conditions in which protein synthesis had been blocked by cycloheximide (Fig. 6B). Although intact GFP was stable, the chimeric proteins containing the C-terminal 60 amino acids of BTG1, BTG2, Tob, and Tob2 were remarkably unstable, indicating that the C-terminal regions of BTG1, BTG2, Tob, and Tob2 confer instability on GFP. In addition, it was found that treatment with MG132 blocked the degradation of GFP fusion proteins. Taken together, the results suggest that the C-terminal regions of BTG1, BTG2, Tob, and Tob2 act as protein degradation signals. DISCUSSION In this study, we found that BTG1, BTG2, Tob, and Tob2 proteins of the antiproliferative BTG/Tob family are multiubiquitinated and are degraded by the 26S protea- some. These findings are consistent with results of previous Fig. 5. Stabilities of N-terminally and C-ter- minally truncated mutants of BTG1, BTG2, Tob, and Tob2. (A,C,E,G)Schematicrep- resentation of BTG1, BTG2, Tob, and Tob2, and their mutants. BTHD, BTG/Tob homol- ogy domain. (B, D, F, H) HEK293 cells were transiently transfected with the respective mutant expression plasmids, and at 24 h after transfection, the cells were treated with 50 l M MG132 or 0.5% dimethylsulfoxide (DMSO) for 1 h and then incubated with 25 lgÆmL )1 cycloheximide for the indicated periods. The cell lysates were prepared at the indicated times, and the protein levels of mutants were analyzed by Western blotting with anti-(T7- tag) Ig. Note that Western blotting with anti- actin antibody showed a constant level of b-actin in any case (data not shown). Ó FEBS 2002 Ubiquitin-dependent degradation of BTG/Tob family proteins (Eur. J. Biochem. 269) 3601 studies showing that various short-lived oncogenic proteins and tumor suppressor proteins are degraded by the ubiquitin–proteasome system [32–35]. The proteasome inhibitor MG132 had dramatic stabilizing effects on BTG/ Tob family proteins. Although MG132 also inhibits calpain, E64d (a calpain inhibitor) had little effect on the degrada- tion of BTG/Tob family proteins. Thus, we conclude that inhibition of the proteasome activity results in the accumu- lation of BTG/Tob family proteins. In addition, we demonstrated that BTG/Tob family members are multi- ubiquitinated before degradation, i.e. in the presence of the proteasome inhibitor. We found that the C-terminal regions of BTG1, BTG2, Tob, and Tob2 act as signals for their rapid degradation by the ubiquitin–proteasome system. The life spans of the C-terminal truncated mutants were much longer than those of the full-length and the N-terminal mutants with truncation of the BTG/Tob homology domain. The BTG/Tob family members are short-lived proteins, but algorithm analysis using a PESTFIND program predicts that they lack the PEST sequence, a protein degradation signal [40]. Based on the results of our analyses of four members of the BTG/Tob family, we propose that the C-terminal region of this family controls its stability. The C-terminal sequences containing 60 amino acids in BTG1 and BTG2 show high homology (55%) with each other, while those in Tob and Tb2 also show high homology (42%). However, the C-terminal sequences of the former BTG1/BTG2 show a very low homology to those of the latter Tob/Tob2 (for example, 15% in comparison between BTG1 and Tob); neither of the regions show high degree of similarity in hydropathy and secondary structure plots (data not shown). In addition, both C-terminal regions lack known degradation signals. Although it is not clear whether the C-terminal degrada- tion signals of BTG/Tob family members recognize common and/or different targets, it can be inferred that the C-terminal regions are necessary for recognition by E3 or interaction with the proteasome. This possibility will be verified by investigating proteins interacting with the respective C-terminal regions. Another possibility is that the lysine residues within the C-terminal regions (two residues in either BTG1 or BTG2 and one residue in Tob or Tob2) are sites for ubiquitination. Determination of ubiquitination sites in BTG/Tob family proteins will clarify this point. It has been shown that expression levels of BTG1 and BTG2 mRNAs increase in the early G1 phase of the cell cycle [4,5] and that BTG/Tob family proteins are involved in G1 arrest [7,11,13]; BTG/Tob family proteins accumulate at the G1 phase and inhibit the progression to the S phase. Therefore, it can be inferred that rapid degradation of BTG/ Tob family proteins is necessary for release from G1 arrest and that this degradation is induced in response to growth factors. Although it is not clear what kind of signaling mechanism works to induce the cell cycle-dependent degradation of BTG/Tob family proteins, it is possible that phosphorylation is a signal for this degradation, because it has been reported that Tob is phosphorylated by a Tob- associating kinase [41] and that cyclin-dependent kinase inhibitors, functioning at the G1 and G1/S phases, are degraded by the ubiquitin-proteasome system in a phos- phorylation-dependent manner [34]. 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