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 degradat
Trang 1Antiproliferative 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
been reported to have short lives and to be degraded by
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 faccofac-tors [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, Department of Biochemistry,
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)
Trang 2determining 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
M A T E R I A L S A N D M E T H O D S
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-PERTM
mammalian protein extraction reagent was purchased from
Pierce Monoclonal mouse 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 37C under 5% CO2 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, neo-T7-BTG1, 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 EcoRIand SalIand then inserted into the EcoRIand SalIsites 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 EcoRIand SalIsites 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 EcoRIand SalI and then inserted into the EcoRI and SalIsites 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 EcoRIand SalI and then inserted into the EcoRI and SalIsites of the pCI-neo-HA vector that had been generated by inserting the oligonucleotides encoding the
HA epitope (YPDYDVPDYA) into the NheIand 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/Pi 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 lM MG115, MG132 and E64d, each dissolved in dimethyl sulfoxide, for 2 h The cells were then washed with NaCl/Piand harvested For Western blotting, the cells were disrupted by M-PERTM reagent containing 50 lM 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 lM MG132 for 1 h and then with 25 lgÆmL)1 cycloheximide for the indicated periods The cell lysate was
Trang 3prepared 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 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 lM 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 lM
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 lg of pCI -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 lM MG132 for 12 h The cells were disrupted with M-PERTMreagent containing 0.5% SDS, 50 lM 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-PERTMreagent 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 4C for 1 h The beads were collected and washed four times with washing buffer containing 4.29 mM
Na2HPO4, 1.47 mMKH2PO4,2.7 mMKCl, 137 mMNaCl, 0.1% Tween 20, and a protease inhibitor cocktail, and the proteins adsorbed were eluted with elution buffer containing
100 mMcitric acid, pH 2.2, and 1% SDS The eluate was neutralized with 2MTris 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.
BTG1 (full length) 5¢-GAATTCATGCATCCCTTCTACACC-3¢
(BTG1F)
5¢-GTCGACTTAACCTGATACAGTCAT-3¢ (BTG1R)
GFP–BTG1 (111–171) 5¢-GAATTCTCCTACAGAATTGGAGAGG-3¢ BTG1R
BTG2 (full length) 5¢-GAATTCATGAGCCACGGGAAG-3¢
(BTG2F)
5¢-GTCGACCTAGCTGGAGACTGCCA-3¢ (BTG2R)
GFP–BTG2 (98–158) 5¢-GAATTCGAGCTGACCCTGTGGG-3¢ BTG2R
Tob (full length) 5¢-GAATTCATGCATCCCTTCTACACC-3¢
(TobF)
5¢-GTCGACTTAGTTAGCCATAACAGGC-3¢ (TobR)
GFP–Tob (285–345) 5¢-GAATTCACCAATGGAATGTTCCCA-3¢ TobR
Tob2 (full length) 5¢-GAATTCATGCAGCTAGAGATCAAAGT-3¢
(Tob2F)
5¢-GTCGACTCAGTTGGCCAGCACCA-3¢ (Tob2R)
GFP–Tob2 (284–344) 5¢-GAATTCCCGTTTGGAGGCAGTG-3¢ Tob2R
Trang 4Universal Elite ABC kit (Vector Laboratories, Inc.) using
biotinylated anti-(rabbit IgG) Ig as a secondary antibody
R E S U L T S
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 expressed in HEK293 cells, and the cells were treated with 50 l 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.
Trang 5stabilized 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-T7-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.
Trang 6which 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
D I S C U S S I O N
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) Schematic
rep-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
(T7-tag) Ig Note that Western blotting with
anti-actin antibody showed a constant level of
b-actin in any case (data not shown).
Trang 7studies 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 aPESTFINDprogram 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] In connection with this, it should be noted that double bands were detected in cases of Tob and Tob2 (see Figs 1–3,5) Whether these double bands are caused by phosphorylation is a future issue for us to resolve Thus, the actions of BTG/Tob family proteins in cell cycle progression are controlled through degradation by the ubiquitin-proteasome system
A C K N O W L E D G E M E N T S
This work was supported in part by grants-in-aid from the Ministry
of Education, Science, Sports, and Culture of Japan.
R E F E R E N C E S
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Fig 6 Stabilities of GFP fusion proteins containing the C-terminal
regions of BTG/Tob family proteins (A) Schematic representation of
chimeric proteins, GFP–BTG1 (111–171), GFP–BTG2 (98–158),
GFP–Tob (285–345), and GFP–Tob2 (284–344) (B) HEK293 cells
were transiently cotransfected with GFP and GFP fusion protein
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)1cycloheximide for the
indi-cated periods The cell lysates were prepared at the indiindi-cated times, and
the protein levels of GFP and GFP fusion proteins were analyzed by
Western blotting with anti-GFP Ig.
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