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.

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:
Abbreviations: BTG, B-cell translocation gene; Tob, transducing
molecule of ErbB2; TIS, TPA-induced sequence 21; PC3, pheo-

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-

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

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

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¢

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

(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.

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

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/

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-

MG132 or 0.5% dimethylsulfoxide (DMSO) for
1 h and then incubated with 25 lgÆmL
)1
cycloheximide for the indi-
cated periods. The cell lysates were prepared at the indicated times, and
the protein levels of GFP and GFP fusion proteins were analyzed by
Western blotting with anti-GFP Ig.
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