Vol. 9, 79-84, Janua,y 1998 Cell Growth & Differentiation 79 mdm2 and bax, Downstream Mediators of the p53 Response, Are Degraded by the Ubiquitin-Proteasome Pathway1 Young-Chae Chang, Yun-Slk Lee, Tazuko Tejima, Keiji Tanaka, Satoshi Omura, Nicholas H. Hein, Youji Mitsui,2 and Junji Magae National Institute of Bioscience and Human-Technology, Tsukuba 305, Japan (V-C. C., V-S. L, T. T., V. M., J. M.]; The Tokyo Metropolitan Institute of Medal Science, Tokyo 113, Japan [K T.]; The Kitasato Institute, Tokyo 108, Japan [S. 0.]; and Departments of Pathology and Microbiology and Molecular Genetics, University of Vermont, Buriington, Vermont 05405 [N. H. H.] Abstract Upon activation in response to cellular stress or DNA damage, the p53 tumor suppressor induces the expression of gene products Involved in call cycle arrest and apoptosis. Using the proteasome-specffic inhibitors, MG132 (N-acetyl-L-Ieuclnyl-L-leucinal-L- leucinal) and lactacystin, here we show that the p53- response proteins, bax and mdm2 as well as p21 , are degraded by the ubiquitin-proteasome pathway In HeLa cells. MG132 also increased expression of the three proteins In cells that lack p53, showing that stabilization of the p53 response proteins Is not due to increased levels of p53 itself. Increases In mdm2 protein levels by MG132 was accompanied by increases in polyubiquitinated forms of the proteins. Our results indicate that ubiqultin-dependent protein degradation influences the turnover of downstream targets of p53, therefore suggesting that the proteasome plays a role In regulating apoptosis and cell cycle arrest In response to p53. Introduction Inactivation of the p53 tumor suppressor gene product is common event in the development of human neoplasia (1). Wild-type p53 has been shown to be a suppressor of tumor cell growth (2), and the activation of the p53 in response to DNA damage can affect cell fate, either through the induction of growth arrest at the G1-S or G2-M cell cycle check points or apoptotic cell death (3-9). Upon activation in response to DNA damage or cellular stress, wild-type p53 protein in- duces the expression of genes involved in growth arrest, DNA repair, and apoptosis, including p21/WAF1/Cipl , bax, GADD45, and mdm2 (4, 9-25). For example, p53 activates expression of p21 , which binds to and inhibits cdk3 corn- plexes (1 1-13). The inhibition of Gi cdks prevents phospho rylation of pRb, which in turn prevents activation of E2F, a family of transcription activators required for S-phase entry (25). mdm2 and bax are also regulated by p53. mdm2 is an oncogene product that associates with p53 and inhibits its transcriptional activation, thereby preventing G1 arrest and apoptosis mediated by p53 (1 7-1 9). mdm2 also regulates the expression level of p53 by influencing the sensitivity of p53 protein to the ubiquitin/proteasome system (26, 27). bax is a bcl-2-related cellular protein that heterodimerizes with bcl-2 and accelerates cell death (21-24). In the ubiquitin-proteasome pathway, proteins are marked for degradation by conjugation to multiple molecules of ubiq- uitin, which targets proteins for rapid hydrolysis by the 26 5 proteasome (28-30). In addition to catalyzing the rapid elim- ination of proteins with abnormal conformations, the protea- some also degrades a number of regulatory proteins, includ- ing regulators of the cell cycle (31-36) and several transcription factors, including p53 (34, 36-43). In certain settings, accelerated degradation of p53 may play a role in tumorigenesis. For example, the papillorna oncoprotein E6 accentuates p53 degradation by conjugating ubiquitin to p53 (40, 41). Recent evidence using specific inhibitors shows involvement of the ubiquitin-proteasome pathway in apop- tosis (44-47). These observations suggest that ubiquitina- tion and subsequent degradation of ubiquitinated proteins are major regulatory mechanisms for the activity of fac- tors involved in cell cycle progression, oncogenesis, and apoptosis. In this study, we examined the turnover of several gene products that are elevated by p53 during the cellular stress response, including p21 , bax, and mdm2. Stabilization of these proteins by the highly specific proteasome inhibitors, MGi 32 and lactacystin, in cells that lack p53 suggests that many p53 response proteins may be degraded by the ubiq- uitin-proteasome pathway. Results Received 7/28/97; revised 1 1/1 1/97; accepted 11/24/97. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement In accordance with 18 U.S.C. Section 1734 solely to mdi- cate this fact. I c. and V-S. L were supported by Grant Center of Excellence from the Science and Technology Agency. V. M. and J. M. were supported by the Proposal-Based Advanced Industrial Technology R&D Program of the New Energy and Industrial Technology Development Organization of Ja- pan. 2 To whom requests for reprints should be addressed, at National Institute of Bioscience and Human-Technology, 1-1 Higashi, Tsukuba 305, Japan. Phone: 81-298-54-6070; Fax: 81-298-54-6095; E-mail: yrnitsuiOnibh. go.jp. Effect of MG132 on the Expression of Proteins Regulat- ing Cell Proliferation. Recently, ubiquitination-dependent proteolysis of a number of less abundant cellular proteins has been demonstrated (34-36, 38, 39, 42, 43) by using MGi 32 or lactacystin, highly specific inhibitors of the 26 5 proteasome (48, 49). To study the role of the ubiquitin-pro- 3 The abbreviations used are: cdk, cyclmn-dependent kmnase; MG132, N- acetyl-L-leucinyl-L-leucinal-L-leucinal; ALLN, N-acetyl-L-leucinyl-L-leuci- nyl-L-norieucinal; HA, hemagglutinin.
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mdm2 and bax, Downstream Mediators of the p53 Response,Are Degraded by the Ubiquitin-Proteasome Pathway1
Young-Chae Chang, Yun-Slk Lee, Tazuko Tejima,Keiji Tanaka, Satoshi Omura, Nicholas H. Hein�,Youji Mitsui,2 and Junji MagaeNational Institute of Bioscience and Human-Technology, Tsukuba 305,Japan (V-C. C., V-S. L, T. T., V. M., J. M.]; The Tokyo MetropolitanInstitute of Med�al Science, Tokyo 113, Japan [K T.]; The KitasatoInstitute, Tokyo 108, Japan [S. 0.]; and Departments of Pathology andMicrobiology and Molecular Genetics, University of Vermont,Buriington, Vermont 05405 [N. H. H.]
Abstract
Upon activation in response to cellular stress or DNAdamage, the p53 tumor suppressor induces theexpression of gene products Involved in call cyclearrest and apoptosis. Using the proteasome-specfficinhibitors, MG132 (N-acetyl-L-Ieuclnyl-L-leucinal-L-leucinal) and lactacystin, here we show that the p53-response proteins, bax and mdm2 as well as p21 , aredegraded by the ubiquitin-proteasome pathway InHeLa cells. MG132 also increased expression of thethree proteins In cells that lack p53, showing thatstabilization of the p53 response proteins Is not due toincreased levels of p53 itself. Increases In mdm2protein levels by MG132 was accompanied byincreases in polyubiquitinated forms of the proteins.Our results indicate that ubiqultin-dependent proteindegradation influences the turnover of downstreamtargets of p53, therefore suggesting that theproteasome plays a role In regulating apoptosis andcell cycle arrest In response to p53.
Introduction
Inactivation of the p53 tumor suppressor gene product iscommon event in the development of human neoplasia (1).Wild-type p53 has been shown to be a suppressor of tumorcell growth (2), and the activation of the p53 in response toDNA damage can affect cell fate, either through the inductionof growth arrest at the G1-S or G2-M cell cycle check pointsor apoptotic cell death (3-9). Upon activation in response toDNA damage or cellular stress, wild-type p53 protein in-
duces the expression of genes involved in growth arrest,DNA repair, and apoptosis, including p21/WAF1/Cipl , bax,GADD45, and mdm2 (4, 9-25). For example, p53 activatesexpression of p21 , which binds to and inhibits cdk3 corn-plexes (1 1-13). The inhibition of Gi cdks prevents phospho�rylation of pRb, which in turn prevents activation of E2F, afamily of transcription activators required for S-phase entry(25). mdm2 and bax are also regulated by p53. mdm2 is anoncogene product that associates with p53 and inhibits its
transcriptional activation, thereby preventing G1 arrest andapoptosis mediated by p53 (1 7-1 9). mdm2 also regulates the
expression level of p53 by influencing the sensitivity of p53protein to the ubiquitin/proteasome system (26, 27). bax is a
bcl-2-related cellular protein that heterodimerizes with bcl-2and accelerates cell death (21-24).
In the ubiquitin-proteasome pathway, proteins are marked
for degradation by conjugation to multiple molecules of ubiq-uitin, which targets proteins for rapid hydrolysis by the 26 5proteasome (28-30). In addition to catalyzing the rapid elim-
ination of proteins with abnormal conformations, the protea-
some also degrades a number of regulatory proteins, includ-ing regulators of the cell cycle (31-36) and several
transcription factors, including p53 (34, 36-43). In certain
settings, accelerated degradation of p53 may play a role in
tumorigenesis. For example, the papillorna oncoprotein E6accentuates p53 degradation by conjugating ubiquitin to p53(40, 41). Recent evidence using specific inhibitors shows
involvement of the ubiquitin-proteasome pathway in apop-tosis (44-47). These observations suggest that ubiquitina-tion and subsequent degradation of ubiquitinated proteins
are major regulatory mechanisms for the activity of fac-tors involved in cell cycle progression, oncogenesis, andapoptosis.
In this study, we examined the turnover of several geneproducts that are elevated by p53 during the cellular stress
response, including p21 , bax, and mdm2. Stabilization ofthese proteins by the highly specific proteasome inhibitors,
MGi 32 and lactacystin, in cells that lack p53 suggests thatmany p53 response proteins may be degraded by the ubiq-uitin-proteasome pathway.
Results
Received 7/28/97; revised 1 1/1 1/97; accepted 11/24/97.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement In accordance with 18 U.S.C. Section 1734 solely to mdi-cate this fact.I � c. and V-S. L were supported by Grant Center of Excellence fromthe Science and Technology Agency. V. M. and J. M. were supported bythe Proposal-Based Advanced Industrial Technology R&D Program of theNew Energy and Industrial Technology Development Organization of Ja-pan.2 To whom requests for reprints should be addressed, at National Instituteof Bioscience and Human-Technology, 1-1 Higashi, Tsukuba 305, Japan.
Effect of MG132 on the Expression of Proteins Regulat-ing Cell Proliferation. Recently, ubiquitination-dependentproteolysis of a number of less abundant cellular proteinshas been demonstrated (34-36, 38, 39, 42, 43) by usingMGi 32 or lactacystin, highly specific inhibitors of the 26 5proteasome (48, 49). To study the role of the ubiquitin-pro-
3 The abbreviations used are: cdk, cyclmn-dependent kmnase; MG132, N-acetyl-L-leucinyl-L-leucinal-L-leucinal; ALLN, N-acetyl-L-leucinyl-L-leuci-nyl-L-norieucinal; HA, hemagglutinin.
MG132(�tM) 00.11 10 00.1110
mdm2 -#{248}�
p2l+� p �MII��
�+ cdk2
�+ cdk4
.� alP� �+p27+� -� -� _______
.-.�,.p16-0
cyclinA -0
c-myc
�;:z�
cyclinBi -o
.,+ c-fos
.,+ c-jun
80 Proteasome-mediated Degradation of mdm2 and bax
p53 +� � �:‘: �
Bax +� �
Bcl-2 +� �
+cyclinDl
�
�- �+ cyclinE
� �+ cdc2
Fig. 1. Effect of MG132 on rag-ulators of cell proliferation. HeLacells were treated with the mdi-cated concentration of MG132for 1 6 h, and the levels of endog-enous cellular proteins were an-alyzed by Western blothng.
teasome system in the expression of p53 response proteins
and several other regulators of cell proliferation, HeLa cells
were treated with MGi 32 for 1 6 h, and protein expression
was analyzed by Western blotting (Fig. 1). Expression of p53,
the cdk inhibitors p21 and p27, and cyclins B1, E, and Dl
was increased by MG132 treatment. MG132 did not affect
the amount of p16, cyclin A, cdc2, cdk2, and cdk4. Thus, in
HeLa cells the ubiquitin-proteasome system degrades some,
but not all, cyclins or cdk/cyclin kinase inhibitors. The tran-
scription factors c-jun, c-fos, and c-myc were also increased
by MG132. In addition to p21 , we found that two other
proteins, which are regulated by p53, bax, and mdm2, were
also increased by the treatment of MGi 32. In contrast, ex-
pression of bcl-2, a heterodimeric partner of bax (21 , 22),was not affected by MGi 32.
Because the expression and degradation of many cell
cycle regulators is required for passage through different
stages of the cell cycle, the DNA content of HeLa cells was
analyzed by flow cytometry. We found that MG132 did not
cause any significant change in flow cytometry profiles (data
not shown). The concentration of MGi 32 necessary for in-
creases in protein expression was slightly different among
proteins; stabilization of p53 and cyclin B1 were significantly
increased only at 1 0 �.LM, whereas c-fos and c-jun were
similarly increased at 1 and 1 0 �.LM. Most proteins showed
moderate increases at 1 MM and more substantial increases
in expression at 1 0 �LM (i.e., for bax, mdm2, p21 , p27, and
cyclin E).
Expression of p53-responsive gene products was also
examined in cells treated with lactacystin, the most specific
proteasome inhibitor known (49), and a less specific inhibitor,
ALLN, which also inhibits calpain (48). The antibody used
for Western blotting against p21 in this experiment de-
tected two bands around the appropriate molecular
weight of p21 (Fig. 2). The lower band, which did not
appear in Fig. 1,may be a nonspecific cross-reactive
band, because another monoclonal antibody for p21 did
not recognize this band. The addition of lactacystin (1 0 �.LM)
or MG132 (1 MM) in HeLa cells increased expression of
bax, p21 , and mdm2. The result is consistent with the K�s
of lactacystin and MG132 for the purified rat 20 S protea-
some, which are 7.5 and 0.9 �M, respectively, using suc-
cinyl-Leu-Leu-VaI-Tyr-4-methyl-coumaryl-7-amide as a
substrate (data not shown). ALLN at 10 �.tM slightly in-
creased p21 but not bax or mdm2.
Increased expression of p53-responsive gene prod-ucts is independent of p53. To test the possibility that
increases in expression of p21 ,bax, and mdm2 resulted from
increased levels of p53 that occur in the presence of MG132
and lactacystin, the effect of the inhibitors on protein expres-
sion was examined in Saos-2 cells (Fig. 2), an osteosarcoma
cell line that lacks p53 (50). As shown in Fig. 2, MGi 32
caused increased levels of bax, p21 , and mdm2 proteins in
Saos-2 cells. This result suggests that stabilization of p21,
bax, and mdm2 by inhibiting the ubiquitin-proteasome sys-
tem occurs in a p53-independent manner, consistent with
previous reports that expression of p21 is regulated in a
p53-independent manner (51-55).
Increases in bax, mdm2, and p21 protein levels were ob-
served as early as 2 h after the addition of MG132 (Fig. 3A).
.� ALLN lactacystin MG132 � MG132
� � �. � � �. � � �. � � � �. � (�tM)
-�-� m
�:�#{248}- p21
� . �+. mdm2
A
MG132 0 p.M
time(h) 0 2 4 6 8
p21 -�
mdm2 -*�
Bax -#{248}’�� -
B
Cell Growth & Differentiation 81
cells (data not shown).
Fig. 2. p53-independent accu-mulation of p21, mdm2, and baxby MG132. HeLa cells or Saos-2cells were treated with the mdi-cated concentration of protea-some inhibitors for 16 h, and cel-lular proteins were analyzed withWestern blotting.
- -�_
HeLa
��-Bax
SAOS-2
_________ 10 �.tM1 12468
-.�-�
MG132 Oi.tM 101AMI 1 I I
time(min) 0 30 60 90 120 0 30 60 90120
p2l-* �
mdm2 -*� � m � � � � �.
Bax -* � � �-R--�=-. � �
Fig. 3. MG132 stabilizes bax, p21 , and mdm2. In A, HeLa cells weretreated with MG132 for the indicated time, and cellular proteins wereanalyzed with Western blotting. In B, HeLa cells were incubated in thepresence or the absence of MG132 for 30 mm, and cycloheximide (10�g/ml) was added to the culture (0 mm). Cells were further incubated forthe indicated time, and cellular proteins were analyzed by Western blot-ting.
We therefore examined the stability of these proteins in the
presence and absence of MGi 32. HeLa cells were incubated
with or without MG132, and cycloheximide was added to
prevent new protein synthesis (Fig. 3B). In the absence of
MG1 32, the three p53-responsive gene products decreased
rapidly and were almost completely undetectable by 60 mm,
indicating the proteins have a half-life of about 30 mm. A
similar result was obtained in the experiment using Saos-2
Accumulation of Polyubiquitinated mdm2 by MG132.To confirm that p21 and mdm2 proteins are ubiquitinated, we
cotransfected Saos-2 cells with expression vectors for p21,
mdm2, and bax and pCMV-HA-ubiquitmn, which expressed
ubiquitin tagged with the HA epitope. Bax, p21 , and mdm2
were immunoprecipitated with specific antibodies, resolved
by electrophoresis, and blotted, and the HA epitope was
detected with monoclonal antibody 12CA5. We failed to
detect the ectopically expressed bax protein (data not
shown), presumably because expression of bax induced the
apoptosis (23). mdm2 was expressed as a faint M, 90,000
band, accompanied by the Mr 67,000 band. The expressions
of both bands were greatly increased in the presence of
MG132 (Fig. 4A, lower panel). p21 was expressed as a M,
21 000 singlet, which was accompanied by Mr 30,000 and Mr
34,000 bands. They were again increased by the treatment
with MG132 (Fig. 4B, lowerpanel). When these immunopre-
cipitates were blotted with anti-HA antibody, p21 and mdm2
immunoprecipitates from cells treated with MG132 displayed
increases in high molecular weight HA immunoreactive ma-
terial as compared with transfected cells not treated with
MG132 (Fig. 4, upper panels). No HA-reactive material was
observed when specific antibody was omitted from the im-
munoprecipitation reactions. It should be noted that faint
bands around Mr 90,000 and Mr 1 1 0,000 were observed in
an anti-HA blot of anti-mdm2 immunoprecipitates, although
we are not sure that the Mr 90,000 protein observed on the
anti-mdm2 blot corresponds to the Mr 90,000 band on the
anti-HA blot (Fig. 4A). M� 34,000 and Mr 30,000 bands of p21
can be assumed to be ubiquitinated forms of the protein, but
the anti-HA blot of p21 immunoprecipitate had no band that
corresponded to these high molecular weight bands (Fig.
4B), probably because polyubiquitination of these bands was
not sufficient to be detected by the anti-HA Western blotting.
DiscussionUbiquitin-dependent proteolysis mediated by the 26S pro-
teasome is involved in metabolism of many short-lived pro-
teins, including several regulators of cell cycle progression or
transcription. The detection of ubiquitin-conjugated proteins
in vivo is often difficult because of rapid protein turnover by
B
+II-+
+II-+
�. � �IgG
anti-HA blot
Aanti-mdm2
IIMG132 - +
lOlkDa-+
83kDa-#{248}�
5OkDa -*�
lOlkDa-*
83kDa-#{248}
5OkDa .-#{248}’�
anti-p21II
MG132 - +
101kDa-+�
83kDa-*
5OkDa -*
101kDa-#{248}�83kDa-*
5OkDa -+�
35kDa-+�
29kDa-+
anti-HA blot
Fig. 4. Polyubiquitination of ec-topically expressed p21 andrndrn2 proteins in the presence otMGi 32. Saos-2 cells were trans-tected with pCMV-HA-ubiquitinand pCMV-rndm2 (A) or pCMV-HA-ubiquitin and pCMV-p21 (B).Cells were incubated with plas-mid DNA for 10 h, washed, andthen incubated in fresh mediumwithout or with 10 � MG132 for24 h. Cell lysates were incubatedwith or without the indicatedantibody to irnrnunoprecipitatemdm2 (A) or p21 (B) and then withanti-lgG agarose. The immuno-precipitates were resolved bySOS-PAGE and irnmunoblottedwith antibodies against HA (upperpanels), rndm2 (lowerpanel, A), orp21 (lowerpanel, B). Positions ofmolecular weight markers (left)and IgG heavy chain from the an-tibodies used for the immunopre-cipitations as well as expressedp21 and rndm2 proteins (right) areindicated with arrows.
anti-mdm2 blot.�#{248}-p21
82 Proteasome-mediated Degradation of mdm2 and bax
abundant proteasome complexes. Highly specific inhibitors
have proven to be a useful tool to study the involvement of
ubiquitin-proteasome system in the degradation of such
short-lived proteins. Here we studied the stability of several
proteins that are induced by the activation of p53 and found
that the proteasome inhibitors MG132 and lactacystin in-
crease the expression of at least three p53-responsive gene
products, bax, mdm2, and p21 . Increases in the stability of
these three proteins was independent of p53, which at least
in part explains the p53-independent regulation of p21 ex-
pression reported previously (51-55). We also show that
MG132 increases polyubiquitinated forms of mdm2 and p21.
Together, our results demonstrate that bax, mdm2, and p21
are degraded by the ubiquitin/proteasome system.
The function of p53 is carefully regulated. Once activated
by cellular stress, p53 mediates growth arrest and apoptosis
by activating the expression of a number of cellular genes (4,
9, 1 0, 1 7, 24). Because recovery of cells from the p53 re-
sponse presumably requires inactivation of p53 target genes,
we reasoned that p53 response proteins may also be short-
lived, and therefore be subject to degradation by ubiquitin-
dependent proteolysis. For at least three p53-responsive
genes, this prediction appears to be correct. Thus, p53 mayimpinge on the normal cell cycle machinery by inducing
elevated expression of many proteins that are normally short
lived. Recently, it is reported that mdm2 protein associates
with p53 and promotes its degradation (26, 27). Our present
results, together with these observations, demonstrate that
p53 and mdm2 regulate the expression level of each other
through the ubiquitin/proteasome system.
The ubiquitin/proteasome system may play several impor-
tant roles in the p53 response pathway, leading to cell cycle
arrest or apoptosis. The system regulates the expression of
p53 itself, and it regulates several downstream targets in-
volved in growth arrest and apoptosis. In regard to the ap-
optotic response, we found that bcl-2 is not subject to deg-
radation by the proteasome, whereas bax is rapidly
degraded by the system. Bax is an apoptosis-inducing pro-
tein, whereas bcl-2 protects cells from apoptosis by het-
erodimerizing with bax (21 , 22). p53 induces apoptosisthrough the induction of bax (24). Thus, the ubiquitin-protea-
some may negatively regulate p53-dependent apoptosis by
Cell Growth & Differentiation 83
degrading the p53 downstream target, bax. This notion is
consistent with studies showing that proteasome inhibitors
induce apoptosis (44-46). Phosphorylation inactivates the
ability of bcl-2 to prevent apoptosis, because okadaic acid,
a phosphatase inhibitor, induces apoptosis and hyperphos-
phorylation of bcl-2 (56). Thus, cells may regulate the ability
of bax to influence cell death by two different but comple-
mentary mechanisms: ubiquitination of bax and phosphoryl-ation of bcl-2.
Clearly, the ubiquitin-proteasome system influences thebalance between many proteins that positively or negativelyregulate cell cycle progression and apoptosis. How alter-
ations in this proteolytic system influence cell transformation
is the focus of our present efforts.
Materials and MethodsCell Culture. HeLa, Saos-2, and CHOC 400 cells were cultured at 37#{176}Cunder 5% CO2 atmosphere, using DMEM supplemented with 5% fetalbovine serum and 50 �g/ml kanamycin. CHOC 400 cells are a Chinese
hamster cell line resistant to methotrexate (57).Western Blotting. Cells were scraped off from the culture dish,
washed with PBS (0.9% NaCI, pH 7.4), and suspended in 2x SOS samplebuffer [4% SDS, 20% glycerol, 120 mr�i Tris (pH 6.8), 0.01 % bromphenol
blue, and 100 mM OTT], sonicated for 10 s, and heated to 95#{176}Cfor 10 mm.Samples were resolved by SDS-PAGE and transferred to polyvmnylidenedifluoride membrane (Millipore, Bedford, MA) by eiectroblothng. Mem-
branes were blocked in Tris-buffered saline (�BS; 20 m� Tris, 0.8% NaCI,
pH 7.5)contamning 5% nonfat dry milk, and 0.1 % Tween 20 for 1 h at 50#{176}C,incubated with primary antibody diluted with blocking buffer for 1 h,washed three times with blocking buffer, and incubated with horseradishperoxidase-conjugated anti-mouse or anti-rabbit secondary antibody
(Arnersham Corp., Ariington Heights, IL) diluted 1:1000 with blockingbuffer for 1 h. The membranes were then washed tour times with blockingbuffer and two times with TBS containing 0.1 % Tween 20, and signalswere visualized with the ECL detection system (Arnersham).
Transfection and Immunoprecipitatlon. Cells (1 x 106) were platedin 85-mm culture dishes in 10 ml of medium, incubated overnight, andchanged into fresh medium; 6 h later, they were transtected with 24 �g ofDNA per plate using the calcium phosphate coprecipitation method asdescribed previously (58). Transfections typically contained 8 �g of each
expression vector and salmon sperm DNA as carrier. Cells were incubated
with DNA for 10 h, washed three times with PBS, and further incubated
with or without MG132 for 24 h. For immunoprecipitations, transtectedcells werelysed with PBS containing 2% SOS. The lysates were sonicated
for 10 s, incubated at 95#{176}Cfor 10 mm, cleared by centritugation at 14,000rpm for 10 mm, and diluted with PBS. Diluted lysate was incubated withspecific primary antibody for 60 mm and then with anti-rabbit or mouse-lgG agarose (Sigma Chemical Co., St. Louis, MO) for 60 mm at roomtemperature. The beads were washed with TBS containing 0.1 % Tween
20 six times, and bound protein was eluted by heating the beads at 95#{176}Cfor 10 mm in SDS sample buffer.
AntIbodIes and Plasmlds. Anti-p21 (C-i 9), anti-bax (N-20), anti-p27(M-197), anti-cyclin A (BF683), anti-cyclin Bi (GNS1), anti-cyclin Di
(H-295), anti-cyclin E (HE1 1 1), anti-odc2 p34 (17), anti-cdk2 (M2), anti-cdk4 (C-22), anti-c-myc (C-33), anti-c-fos (4), and anti-c-jun (KM-i) werepurchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-p53 (Pabi8Oi), anti-mdm2 (IF-2), and anti-bcl-2 (0P60) were purchasedfrom Oncogene Science, Inc. (Uniondale, NV). Anti-p16 were from PharM-ingen (San Diego, CA). Anti-HA monoclonal antibody (12CA5) was kindly
provided by Dr. C-L Wu (Massachusetts General Hospital, Boston, MA).Expression vectors of human mdm2, p21 , HA-tagged ubiquitin, and
mouse bax proteins were kind gifts from Drs. J. Xiao (Boston University
School of Medicine, Boston, MA), A. Noda (Kobe University Medical
School, Kobe, Japan), P. M. Howley (Harvard Medical School, Boston,MA), and V. Tsujimoto (Osaka University Medical School, Osaka, Japan),respectively.
Chemicals. MG132 was kindly donated by Peptide Institute, Inc.(Osaka, Japan). Lactacystin was purified as described previously (59).ALLN was purchased from Sigma.
AcknowledgmentsWe are grateful to Drs. C-L Wu (Boston, MA), J. Xiao (Boston, MA), A.Noda (Odawara, Japan), P. M. Howley (Boston, MA), and V. Tsujimoto(Osaka, Japan) for anti-HA antibody and expression vectors for mdm2,
p21 HA-tagged ubiquitin, and mouse bax proteins.
References1. Hollstein, M., Sidransky, 0., Vogelstemn, B., and Hams, C. C. p53
mutations in human cancers. Science (Washington DC), 253: 49-53,1991.
2. Levine, A, J., Momand, J., and Fmnlay, C. A. Thep53 tumour suppressorgene. Nature (Lond.), 351: 453-456, 1991.
3. Kuerbitz, S. J., Plunkett, B. S., Walsh, W. V., and Kastan, M. B.Wild-type p53 is a cell cycle checkpoint determinant following irradiation.Proc. NatI. Acad. Sci. USA, 89: 7491-7495, 1992.
4. Kastan, M. B., and Zhan, Q., El-Dairy, W. S., Carrier, F., Jacks, T.,Walsh, W. V., Plunkett, B. S., Vogeistemn, B., and Fomace, A. J., Jr. A
mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 isdetective in ataxia-telangiectasia. Cell, 71: 587-597, 1992.
5. Lu, X., and Lane, 0. P. Differential induction of transcriptionally activep53 following UV or ionizing radiation: detects in chromosome instability
syndromes? Cell, 75: 765-778, 1993.
6. Debbas, M., and White, E. Wild-type p53 mediates apoptosis by E1A,which is inhibited by El B. Genes 0ev., 7: 546-554, 1993.
7. Wu, X., and Levine, A. J. p53 and E2F-l cooperate to mediate apop-tosis. Proc. NatI. Acad. Sci. USA, 91: 3602-3606, 1994.
8. Wagner, A. J., Kokontis, J. M., and Hay, N. Myc-mediated apoptosisrequires wild-type p53 in a manner independent of cell cycle arrest andthe ability of p53 to induce p2iwal�IPl. Genes 0ev., 8: 2817-2830, 1994.
9. Ko, L J., and Prives, C. p53: puzzle and paradigm. Genes 0ev., 10:1054-1072, 1996.
10. El-Diery, W. 5, Tokino, T., Velculescu, V. E., Levy, 0. B., Parsons, A.,Trent, J. M., Un, 0., Mercer, W. E., Kinzler, K. W., and Vogeistemn, B.WAF1, a potential mediation of p53 tumorsuppression. Cell, 75: 81 7-825,1993.
1 1.Gu, V., Turck, C. W., and Morgan, 0. 0. Inhibition of cdk2 activity invivo by an associated 20K regulatory subunit. Nature (Lond.), 366: 707-710, 1993.
12. Harper, J. W., Adami, G. A., Wei, N., Keyomaarsi, K., and Elledge,S. J. The p21 cdk-interacting protein Cipi is a potent inhibitor of G1cyclmn-dependent kinases. Cell, 75: 805-816, 1993.
13. Xiong, V., Hannon, G. J., Zhang, H. Casso, 0., Kobayashi, R., and
Beach, 0. p21 is a universal inhibitorot cyclmn kinases. Nature(Lond.), 366:70i-704, 1993.
14. Wu, X., Bayle, H., Olson, 0., and Levine, A. L The p53-mdm-2autoregulatory feedback loop. Genes 0ev., 7: 1 126-i 132, 1993.
15. Barak, V., Juven, T., Hoffner, A., and Oren, M. mdm2 expression isinduced by wild type p53 activity. EMBO J., 12: 461-468, 1993.
16. Perry, M. E., Piette, J., Zawadzki, J. A., Harvey, 0., and Levine A. J.
The mdm-2 gene is induced in response to UV light in a p53-dependentmanner. Proc. NatI. Acad. Sci. USA, 90: 1 1623-i 1627, 1993.
17. Momand, J., Zambetti, G. P., Olson, 0. C., George, 0., and Levine,A. L The mdm-2 oncogene product forms a complex with the p53 protein
and inhibits p53-mediated transactivation. Cell, 69: 1237-1245, 1992.
18. Chen, J., Wu, X., Un, J., and Levine, A. J. mdm-2 inhibits the Gi arrestand apoptosis functions of the p53 tumor suppressor protein. Mol. Cell.
Biol., 16: 2445-2452, 1996.
19. Haupt, V., Barak, V., and Oren, M. Cell type-specific inhibition ofp53-mediated apoptosis by mdm2. EMBO J., 15: 1596-1606, 1996.
20. Miyashita, T., and Reed, J. C. Tumor suppressor p53 is a directtranscriptional activator of the human bax gene. Cell, 80: 293-299, 199g.
84 Proteasome-mediated Degradation of mdm2 and bax
21 . Yin, X-M., Oltvai, Z. N., and Korsmeyer, S. J. BH1 and BH2 domainsof Bcl-2 are required for inhibition of apoptosis and heterodimerizationwith Bax. Nature (Lond.), 369: 321-323, 1994.
22. Oltvai, Z. N., Milliman, C. L, and Korsmeyer, S. J. Bcl-2 heterodimer-izes in vivo with a conserved homolog, Bax, that accelerates programmedcell death. Cell, 74: 609-619, 1993.
23. Xiang, J., Chao, 0. T., and Korsmeyer, S. J. Bax-induced cell deathmay not require interleukin 1 p-converting enzyme-like proteases. Proc.Nati. Acad. Sci. USA, 93: 14559-14563, 1996.
24. Han, J., Sabbatini, P., Perez, 0., Aao, L, Modha, 0., and White, E. The
El B 19K protein blocks apoptosis by interacting with and inhibiting thep53-inducible and death-promoting Bax protein. Genes Day., 10: 461-477, 1996.
25. Weinberg, A. A. The retinoblastoma protein and cell cycle control.
Cell, 81: 323-330, 1995.
26. Haupt, V., Maya, A., Kazaz, A., and Oren, M. mdm2 promotes therapid degradation of p53. Nature (Lond.), 387: 296-299, 1997.
27. Kubbatat, M. H. G., Jones, S. N., and Vousden, K. H. Regulation ofp53 stability by mdm2. Nature (Lond.), 387: 299-303, 1997.
28. Goldberg, A. L. The mechanism and function of AlP-dependentproteases in bacterial and animal cells. Eur. J. Biochem., 203: 9-23, 1992.
29. Hershko, A., and Ciechanover, A. The ubiquitin system for proteindegradation. Annu. Aev. Biochem., 61: 761-807, 1992.
30. Coux, 0., Tanaka, K, and Goldberg, A. L Structure and functions ofthe 205 and 265 proteasomes. Annu. Rev. Biochem., 65: 801-847, 1996.
31 .Goltzer, M., Murray, A. W., and Kirschner, M. W. Cyclin is degraded
by the ubiquitmn pathway. Nature (Lond.), 349: 132-138, 1991.
32. Clurman, B. E., Sheaff, A. J., Thress, K., Groudine, M., and Roberts,J. M. Turnover of cyclin E by the ubiquitmn-proteasome pathway is regu-lated by cdk2 binding and cyclin phosphorylation. Genes Day., 10: 1979-1990, 1996.
33. Seufert, W., Futcher, B., and Jentsch, S. Role of a ubiquitin-conju-gating enzyme in degradation of 5- and M-phase cyclins. Nature (Lond.),373: 78-83, 1995.
34. Maki, C. G., Huibregtse, J. M., and Howley, P. M. In vivo ubiquitmnationand proteasome-mediated degradation of p53. Cancer Res., 56: 2649-2654. 1996.
35. Blagosklonny, M. V., Wu, G-S., Omura, S., and El-Diery, W. S. Pro-teasome-dependent regulation of p21 WAFI/CIPI expression. Biochem.Biophys. Aes. Commun., 227: 564-569, 1996.
36. Pagano, M., Tam, S. W., Theodoras, A. M., Beer-Romero, P., Sal,G. 0., Chau, V., Yew, P. A., Draetta, G. F., and Rolte, M. Role of theubiquitin-proteasome pathway in regulating abundance of the cyclmn-dependent kinase inhibitor p27. Science (Washington DC), 269:682-685,
1995.
37. Ciechanover, A., DiGiuseppe, A. J., Bercovich, B., Orian, A., Richter,J. 0., Schwartz, A. L, and Brodeur, G. M. Degradation ot nuclear onco-proteins by the ubiquitin system in vitro. Proc. NatI. Acad. Sal. USA, 88:139-143, 1991.
38. Treier, M., Staszewski, L M., and Bohmann, D. Ubiquitin-dependentc-jun degradation in vivo is mediated by the & domain. Cell, 78: 787-798,1994.
39. Palombella, V. J., Aando, 0. J., Goldberg, A. L, and Maniatis, T. The
ubiquitmn-proteasome pathway is required for processing the NF-icBlprecursor protein and the activation of NF-scB. Cell, 78: 773-785, 1994.
40. Aolfe, M., Beer-Aomero, P., Glass, S., Eckstemn, J., Berdo, I., The-odoras, M., Pagano, M., and Draetta, G. Reconstitution of p53-ubiquiti-nylation reactions from purified components: the role of human ubiquitmn-
conjugating enzyme UBC4 and E6-associated protein (E6AP). Proc. NatI.
Acad. Sci. USA, 92: 3264-3268, 1995.
41 . Scheffner, M., Huibregtse, J. M., and Howley, P. M. Identification of ahuman ubiquitin-conjugating enzyme that mediates the E6-AP-dependent
ubiquitmnation of p53. Proc. NatI. Acad. Sci. USA, 91: 8797-8801, 1991.
42. Hateboer, G., Kerkhoven, A. M., Shvarts, A., Bemards, A., andBeijersbergen, A. L Degradation of E2F by the ubiquitin-proteasomepathway: regulation by retinoblastoma family proteins and adenovirustransforming proteins. Genes 0ev., 10: 2960-2970, 1996.
43. Hotmann, F., Martelli, F., Livingston, D. M., and Wang, Z. The retino-blastoma gene product protects E2F-i from degradation by the ubiquitin-
44. Shinohara, K., Tokioka, M., Nakano, H., Tone, S., Ito, H., andKawashima, S. Apoptosis induction resulting from proteasome inhibition.Biochem. J., 317: 385-388, 1996.
45. Drexler, H. C. A. Activation of the cell death program by inhibition ofproteasome function. Proc. NatI. Acad. Sci. USA, 94: 855-860, 1997.
46. lmajoh-Ohmi, S., Kawaguchi, T., Sugiyama, S., Tanaka, K., Omura, S.,and Kikuchi, H. Lactacystin, a specific inhibitor of the proteasome, in-
duces apoptosis in human monoblast U937 cells. Biochem. Biophys. Aes.Commun., 217: 1070-1077, 1995.
47. Grimm, L M., Goldberg, A. L, Poirier, G. G., Schwartz, L M., and
Osborne, B. A. Proteasomes play an essential role in thymocytes apop-tosis. EMBO J., 15: 3835-3844, 1996.
48. Rock, K. L, Gramm, C., Aothstein, L, Clark, K., Stein, A., Dick, L,Hwang, 0., and Goldberg, A. L Inhibition of the proteasome block thedegradation of most cell proteins and the generation of peptides pro-sented on MHC class I molecules. Cell, 78: 761-771 , 1994.
49. Fenteany, G., Standaert, A. F., Lane, W. S., Choi, S., Corey, E. J.,and Schreiber, S. L Inhibition of proteasome activities and subunit-
specific amino-terminal threonine modification by lactacystmn. Science(Washington DC), 268: 726-731 , 1995.
50. DilIer, L, Kassel, J., Nelson, C. E, Gryka, M. A., Utwak, G., Gebhardt,M., Bressac, B., Ozturk, M., Baker, S. J., Vogelsteln, B., and Friend, S. H.p53 functions as a cell cycle control protein in osteosarcomas. Mol. Cell.Biol., 10: 5772-5781 , 1990.
51 .Michieli, P., Chedid, M., Lin, D., Peirce, J. H., Mercer, W. E., and Givol,0. Induction of WAF1/CIP1 by a p53-independent pathway. Cancer Aes.,54: 3391-3395, 1994.
52. Macleod, K. F., Sherry, N., Hannon, G., Beach, 0., Tokino, I., Kinzler,K., Vogelstemn, B., and Jacks, T. p53-dependent and independent expres-sion of p21 during cell growth, differentiation, and DNA damage. GenesDay., 9: 935-944, 1995.
53. Halevy, 0., Novitch, B. G., Spicer, 0. B., Skapek, S. X., Ahee, J.,Hannon, G. J., Beach, D., and Lassar, A. B. Correlation of terminal cellcycle arrest of skeletal muscle with induction of p21 by MyoD. Science(Washington DC). 267: 1018-1021 , 1995.
54. Parker, S. B., Eichele, G., Zhang, P., Rawls, A., Sands, A. T., Bradley,
A., Olson, E. N., Harper, J. W., and Elledge, S. J. p53-independentexpression of p2lciPi in muscle and other terminally differentiating cells.
Science (Washington DC), 267: 1024-1027, 1995.
55. Canman, C. E, Gilmer, I. M., Coutts, S. B., and Kastan, M. B. Growthfactor modulation of p53-mediated growth arrest versus apoptosis. GenesDay., 9: 600-611, 1995.
56. Haldar, S., Jena, N., and Croce, C. M. Inactivation of Bcl-2 by phos-phorylation. Proc. NatI. Acad. Sci. USA, 92: 4507-451 1 , 1995.
57. Milbrandt, J. D., Heintz, N. H., White, W. C., Aothman, S. M., andHamlin, J. L Methotrexate-resistant Chinese hamster ovary cells have
amplified a 135-kilobase-pair region that includes the dihydrofolate re-ductase gene. Proc. NatI. Acad. Sd. USA, 78: 6042-6047, 1981.
58. Magae, J., Wu, C-L, Illenye, S., Hariow, E., and Heintz, N. H. Nuclearlocalization of OP and E2F transcription factors by heterodimeric partners
and retinoblastoma protein family members. J. Cell Sci., 109: 1717-1726,
1996.
59. Omura, S., Fujimoto, T., Otoguro, K., Matsuzaki, K., Moriguchi, A.,Tanaka, V., and Sasaki, V. Lactacystin, a novel microbial metabolite,induces neurite-genesis of neuroblastoma cells. J. Antibiot., 44:113-116,1991.