From the Laboratory of Molecular Cell Biology,
Institute of Biochemistry and Cell Biology, Shanghai Institutes for
Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Road,
Shanghai 200031, People's Republic of China and the ¶ National
Laboratory of Medical Neurobiology, Fudan University Medical Center,
Shanghai 200032, People's Republic of China
Received for publication, October 9, 2002, and in revised form, December 2, 2002
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ABSTRACT |
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Oncoprotein Mdm2 is a master negative regulator
of the tumor suppressor p53 and has been recently shown to regulate the
ubiquitination of The tumor suppressor protein p53 is a potent inhibitor of cell
growth that can induce growth arrest and apoptosis (1-3). Oncoprotein
Mdm2 is a key negative regulator of p53 function, which has been
demonstrated by many studies including allelic knockout mice. Mice
possessing homozygous deletions of Mdm2 die at around day 5 of
embryogenesis; whereas mice possessing a homozygous deletion of both
Mdm2 and p53 are viable and develop normally (4, 5). On the other hand,
overexpression of Mdm2 leads to the loss of p53 and tumor development
(6). Transcription of the Mdm2 gene is strongly stimulated by the
activated p53. The Mdm2 protein, in turn, binds to p53 and conceals its
transactivation domain. Moreover, Mdm2 functions as a RING
finger-dependent E3 ubiquitin ligase and promotes the
ubiquitination and degradation of p53 by the proteasome (6, 7). Thus,
Mdm2 and p53 form an autoregulatory feedback loop between the
expression and function of p53 and Mdm2. It has been well established
that the feedback loop connected Mdm2 and p53 is significantly
activated in response to stress such as DNA damage or activation of
oncogenes (6, 8). However, other important signaling may also
potentially influence the Mdm2/p53 pathway.
Mdm2 interacts with several cellular proteins in addition to p53 and
some of them can regulate the Mdm2/p53 feedback loop by a change in the
cellular distribution of Mdm2, alternation of the stability of Mdm2, or
inhibition the E3 ligase activity of Mdm2 (6-9). The distribution and
shuttling of Mdm2 between the nucleus and the cytoplasm (10-12) are
greatly modulated by its association with the regulators, such as ARF,
which assists the sequestering of Mdm2 in nucleoli and prevents its
export to the cytoplasm (13, 14). Moreover, Mdm2 regulates its own
stability by autoubiquitination via the interaction with its partner.
For example, the direct binding of Tsg101 or MdmX to Mdm2 leads to the
inhibition of the degradation and autoubiquitination of Mdm2 (6, 9,
15). Recently, G-protein-coupled receptors
(GPCRs),1 a large superfamily
of seven transmembrane cell-surface receptors, are responsible for many
physiological processes. Stimulation with agonist causes the
phosphorylation of the receptor by G-protein-coupled receptor kinases
(GRKs) (17). Then Yeast Two-hybrid System--
The ProQuest two-hybrid system and
the mouse fetal brain cDNA library were purchased from Invitrogen.
The bait for library screening was the full-length of human
Cell Culture and Transfection--
Human embryonic kidney
(HEK)293 cells were cultured in minimal essential medium supplemented
with 10% heat-inactivated fetal bovine serum. Normal liver L02 and
HepG2 cells were cultured in 1640 supplemented with 10% fetal bovine
serum. p53-null human osteosarcoma Saos2 and lung adenocarcinoma H1299
cells were maintained in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum. All cells were transfected
using the calcium phosphate-DNA co-precipitation method as described
previously (24). For all transfection experiments, CMV- Plasmids--
Full-length Mdm2, MdmX, and p53 were cloned from
mouse brain into modified pcDNA3 vector in-frame with HA or FLAG
tag at the N terminus. GFP-Mdm2 and the Mdm2 mutants were subcloned by
PCR from HA-Mdm2 into pcDNA3 with the GFP tag at the N terminus.
Adenovirus--
Recombinant adenoviruses encoding
Immunoprecipitation and Western Blotting--
The cells were
lysed in Lysis Buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl, 20 mM NaF,
0.5% Nonidet P-40, 10% glycerol, 1 mM
phenylmethylsulfonyl fluoride) for 1.5 h at 4 °C 48 h
after transfection. Cell extracts were cleared, and the supernatant was
incubated at 4 °C with anti-HA (1 µg; Roche Molecular
Biochemicals), anti-FLAG (1 µg, Santa Cruz Biotechnology) antibody or
anti- Immunofluorescence Microscopy--
Cells were grown on glass
coverslips and transfected with indicated plasmids. 24 h after
transfection, the cells were fixed with 4% polyformaldehyde for 20 min
and incubated with phosphate-buffered saline containing 0.1% Triton
X-100 for 5 min. HA-tagged fusion proteins were detected with anti-HA
antibody and a fluorescein isothiocyanate-conjugated secondary antibody
(green) (Jackson ImmunoResearch). Endogenous Mdm2 were detected with
anti-Mdm2 SIMP14 antibody and a Texas Red-conjugated IgG (red).
Coverslips were analyzed on Leica TCSNT laser confocal scanning microscope.
Ubiquitination Assay--
44 h after transfection, the cells
were treated with the cell-permeable proteasomal inhibitor, MG132 (10 µM, Sigma), for 4 h and then lysed in Lysis Buffer.
The Mdm2-ubiquitin and p53-ubiquitin complexes were immunoprecipitated
using anti-GFP antibody and anti-FLAG antibody and immunoblotted with
anti-HA antibody to detect ubiquitinated proteins. Endogenous Mdm2 were
immunoprecipitated with anti-Mdm2 antibody (H-221, Santa Cruz
Biotechnology) and p53 with anti-p53 antibody (FL-393, Santa Cruz
Biotechnology). Immunoprecipitates were then subjected to Western
blotting analysis with ubiquitin-specific antibody (Ub P4D1, Santa Cruz Biotechnology).
Apoptosis and Cell Death--
Saos2 cells were cultured in
6-well plates and transfected with indicated plasmids along with the
plasmid of CMV-EGFP. After 48 h, the cultures were analyzed under
a fluorescence microscope, and the transfected cells were marked by the
presence of green fluorescence. Apoptotic cells were identified and
accounted by their rounded morphology, in contrast to the spreaded-out
morphology of nonapoptotic cells (27). The assay for cell death was
performed by trypan blue staining (28). DNA fragmentation analysis was performed as described (29). Briefly, 48 h after infection with indicated virus, the cells were treated with UV (10 J/m2)
and cultured for additional 24 h. Both floating and adherent cells
were harvested and lysed with buffer containing 5 mM
Tris-HCl, pH 8.0, 10 mM EDTA, and 0.5% Triton X-100, and
left on ice overnight at 4 °C. After centrifugation at 13,000 × g for 20 min at 4 °C, the fragmented DNA in the
supernatant fraction was extracted twice with
phenol/chloroform/isopropyl alcohol (25:24:1, v/v) and once with
chloroform and then precipitated overnight at Activation of GPCRs Enhanced the Binding of Mdm2 to
Agonist stimulation of the
Then we tested the effect of endogenous GPCR activation on the
interaction between the endogenous
We further examined whether Mdm2 could colocalize with the activated
GPCRs under more physiological conditions. When DOR was expressed in
HEK293 cells, endogenous Mdm2 was predominantly distributed in nucleus
and scarcely colocalized with DOR at the plasma membrane. After the
cells were exposed to DPDPE, a proportion of Mdm2 was found to
translocate to the plasma membrane where it colocalized with DOR (Fig.
3C). Similar results were also obtained when the The N Terminus of
Because the shortest Mdm2 clone identified in our yeast two-hybrid
system-encoded fragment comprised amino acids from 358 to 489 in the C
terminus of Mmd2, we further tested the importance of this region to
the Mdm2 binding to Binding of
In all cases, the expression levels of the control protein GFP
cotransfected were not affected by cotransfection of other plasmids,
Then we used double-stranded RNA-mediated interference (RNAi), which
has recently emerged as a powerful reverse genetic tool to silence gene
expression in multiple organisms, to test the effect of endogenous
The effect of
To further test the effect of Accumulating evidence indicates that residues 220-437 of Mdm2 are
critical for its function since the mutant of Mdm2 lacking this region
is incapable of mediating p53 degradation (36). Many partners of Mdm2
are known to bind to this region of Mdm2 and thus exert their indirect
effects on p53 (6). For instance, Rb and ARF proteins, both of which
bind to Mdm2 in this region, are able to inhibit the ability of Mdm2 to
target p53 for degradation though different mechanisms may exist (6, 9,
37, 38). A recent study (39) shows that ATM kinase phosphorylates Mdm2 at serine 395 in this region in response to DNA damage and reduces the
capability of Mdm2 to promote p53 degradation. Our current results
demonstrate that the MdmX is structurally homologous to Mdm2 and has been demonstrated to
function as another critical negative regulator of p53 function
in vivo. Loss of MdmX results in p53-dependent
embryonic lethality in mice (43, 44). MdmX can directly bind to the RING finger domain of Mdm2 and thus stabilizes both Mdm2 and p53 via
inhibition of the E3 ubiquitin ligase activity of Mdm2 (40). In
addition, MdmX can directly interact with p53 and inhibit the acetylation of p53 induced by CBP/p300 (45) and inhibit the p53-mediated transcription. Recent evidence shows that MdmX is also a
ubiquitin ligase and mediates the ubiquitination of p53 (46). Although
MdmX has similar structure and function with Mdm2, our results showed
that the interaction between MdmX and Several lines of evidence from this study clearly demonstrated that the
association of -arrestin 2, an important adapter and scaffold in
signaling of G-protein-coupled receptors (GPCRs). However, whether
-arrestin 2 has any effect on the function of Mdm2 is still unclear.
Our current results demonstrated that the binding of Mdm2 to
-arrestin 2 was significantly enhanced by stimulation of GPCRs.
Activation of GPCRs led to formation of a ternary complex of Mdm2,
-arrestin 2, and GPCRs and thus recruited Mdm2 to GPCRs at plasma
membrane. Moreover, the binding of
-arrestin 2 to Mdm2
suppressed the self-ubiquitination of Mdm2 and consequently reduced the
Mdm2-mediated p53 degradation and ubiquitination. Further experiments
revealed that overexpression of
-arrestin 2 enhanced the
p53-mediated apoptosis while suppression of endogenous
-arrestin 2 expression by RNA interference technology considerably attenuated the
p53-mediated apoptosis. Our study thus suggests that
-arrestin 2 may serve as a cross-talk linker between GPCR and p53
signaling pathways.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-arrestin 2 has been identified as a novel partner of
Mdm2, which targets
-arrestin 2 for ubiquitination (16). But whether
-arrestin 2 has any effect on the function of Mdm2 is still to be investigated.
-arrestin translocates from cytoplasm to cell
membrane and binds to the phosphorylated GPCRs. Binding of
-arrestin
to GPCRs causes receptor desensitization and internalization and thus
regulates signaling of GPCRs (18, 19). Recent discoveries indicate that
the
-arrestin also plays important roles as scaffold-linking GPCRs
to mitogen-activated protein kinase (MAPK) cascades, such as JNK3 (20).
-Arrestin 2 recruits ASK1 and JNK3 together and enhances the
GPCR-mediated activation of JNK3, which has been reported to be tightly
associated with cell apoptosis (21). More interestingly, it is
demonstrated that the stable recruitment of visual arrestin to the
activated rhodopsin in the fly leads to retinal cell apoptosis (22,
23). These experiments suggest that arrestin may function as a
potential linker of GPCR signaling to apoptotic pathways, which,
however, remains to be further explored. In the current study, we
demonstrate that activation of GPCRs stimulates the association of
-arrestin 2 with oncoprotein Mdm2 and thus regulates the function of
Mdm2. Our results suggest that
-arrestin 2 play a potential role in
mediating GPCR signaling to p53 pathway.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-arrestin 2. Two-hybrid screening was carried out according to the
protocol provided by the manufacturer.
-gal was used
to compensate the total DNA input.
-Arrestin 2 and its mutant V54D clones were generated as described
(24). Ubiquitin plasmid was obtained from Dr. Dirk Bohmann (University of Rochester Medical Center). The authenticity of the DNA sequences was
confirmed by sequencing. Construction of RNAi plasmid for
-arrestin
2 was performed as described (25). The BS/U6 vector was a generous gift
from Dr. Yang Shi (Department of Pathology, Harvard Medical School,
Boston, MA). A 22-nt oligonucleotide (oligo1) corresponding to
nucleotides 216-237 of the
-arrestin 2 coding region was first
inserted into the BS/U6 vector with ApaI and XhoI. The inverted motif that contains the 6-nt spacer and 5 Ts (oligo2) was then subcloned into the XhoI and
EcoRI sites of the intermediate plasmid to generate
BS/U6/
-arrestin 2.
-galactosidase or
-arrestin 2 were prepared as described
previously (26). Briefly,
- galactosidase or
-arrestin 2 was
cloned into shuttle vector pAdTrack-CMV using standard cloning
protocol, and the shuttle vectors were recombined with pAdEasy-1, which
harbors a CMV-driven GFP, to form the viral constructs. The adenoviral
plasmids were transfected into HEK293 cells to generate recombinant
adenovirus. The L02 or Saos2 cells were infected with the adenovirus
for 12 h, and then the virus was removed to let cells recover. The
infection efficiency was determined by observing GFP fluorescence, and
the quantity of virus that afforded at least 80% transfection
efficiency was selected for the experiments.
-arrestin egg yolk IgY produced with recombinant
-arrestin 2 from chicken (1:1000) for 2 h. Immune complexes were immobilized
on protein A (Amersham Biosciences) or anti-chicken IgY Sepharose beads
(Promega) for 3 h, washed three times with Lysis Buffer, heated in
SDS sample buffer in a 50 °C water bath for 20 min. Western analysis
was performed as previously described (24). HA-tagged proteins were detected with anti-HA antibody and GFP-tagged protein with anti-GFP antibody. Endogenous Mdm2 was detected with antibody specific to Mdm2
(Ab3, Oncogene Research Products) or SIMP14 antibody (Santa Cruz Biotechnology).
20 °C. The DNA
fragments were separated by 1.4% agarose gel electrophoresis and
visualized by ethidium bromide staining.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Arrestin 2--
The full-length human
-arrestin 2 was fused to
the GAL4 DNA-binding domain (DBD) as a bait in the yeast two-hybrid
system to screen a mouse fetal brain complementary DNA library, and
Mdm2 was thus identified as a binding partner of
-arrestin 2. Their interaction in mammalian cells was confirmed by coimmunoprecipitation (Fig. 1). These results were consistent
with the recent report by Shenoy et al. (16). The
interaction between
-arrestin 2 and MdmX, a Mdm2-related protein,
was also examined in expressed HEK293 cells. As shown in Fig. 1,
-arrestin 2 interacted well with Mdm2 whereas the interaction
between MdmX and
-arrestin 2 was hardly detected under the same
conditions. Moreover, the presence of MdmX did not interfere with the
interaction between Mdm2 and
-arrestin 2. When the presence Mdm2,
MdmX could be coimmunoprecipitated with
-arrestin 2, indicating
-arrestin 2, Mdm2, and MdmX can form a ternary complex.
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Fig. 1.
-Arrestin 2 interacted with
Mdm2 but not MdmX. HEK293 cells were cotransfected with GFP-Mdm2,
GFP-MdmX, or HA-
-arrestin 2 as indicated, and then the cells were
subjected to immunoprecipitation with anti-HA antibody.
Coimmunoprecipitated Mdm2 or MdmX was detected with anti-GFP antibody.
arr,
-arrestin; IP, immunoprecipitate;
IB, immunoblot.
2-adrenergic receptor could
increase the association of
-arrestin 1 and its partner Src (30). Therefore, the effect of GPCR activation on the interaction between
-arrestin 2 and Mdm2 was examined. The HEK293 cells coexpressing
-arrestin 2, Mdm2, and
opioid receptor (DOR), which has been shown to interact with
-arrestin 2 well (31), were stimulated with
DOR-specific agonist DPDPE, and then
-arrestin 2 was
immunoprecipited. As shown in Fig.
2A, Mdm2 was present in the
-arrestin 2 immunoprecipitates, and an increase in
-arrestin
2-bound Mdm2 was clearly detected after 2 min of agonist exposure and
peaked at 5 min. Stimulation of another GPCR, the
2
adrenergic receptor, could also lead to the increase in the association
of these two proteins (data not shown).
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Fig. 2.
Activation of GPCRs enhanced the interaction
of -arrestin 2 with Mdm2. A, HEK293 cells cotransfected
with DOR, HA-
-arrestin 2, and GFP-Mdm2, were challenged with 1 µM DPDPE for the indicated time, and then the cells were
subjected to immunoprecipitation with anti-HA antibody.
Coimmunoprecipitated Mdm2 was detected with anti-GFP antibody.
Quantitative data are represented as fold values of the basal
coimmunoprecipitated Mdm2 and means ± S.E. of three separate
experiments. B, SK-N-SH cells were treated with or without
100 µM naloxone for 20 min prior to stimulation with 1 µM DPDPE for 10 min, and then the cells were subjected to
immunoprecipitation with anti-
-arrestin antibody. C,
HepG2 cells were treated with 1 µM bradykinin, and cells
were subjected to coimmunoprecipitation analysis.
-arrestin 2 and Mdm2. As shown in
Fig. 2B, DPDPE treatment apparently increased the
association of endogenous Mdm2 and
-arrestin in neuroblastoma
SK-N-SH cells and pretreatment with DOR antagonist naloxone blocked the
effect of DPDPE. Stimulation with bradykinin, which stimulates the
endogenous bradykinin receptors, for 30 min also enhanced the
interaction of endogenous Mdm2 and
-arrestin in HepG2 cells (Fig.
2C). These results provide clear evidence that stimulation
of GPCRs can regulate the interaction between
-arrestin 2 and Mdm2.
-Arrestin 2 Recruited Mdm2 to the Activated
GPCRs--
Then we asked whether
-arrestin 2 could bring Mdm2 to
the activated receptors through its binding to Mdm2. DOR was
coexpressed with Mdm2 in the presence or absence of
-arrestin 2 in
HEK293 cells. As shown in Fig. 3,
A and B, in the absence of coexpressed
-arrestin 2, DOR could not coprecipitate Mdm2 (this may be due to a
low level of endogenous
-arrestin 2 in HEK293 cells). In the
presence of
-arrestin 2 but without stimulation of DOR, only weak
binding of Mdm2 to DOR was observed. This binding was significantly enhanced after a 10-min exposure to DOR agonist. These data demonstrate that
-arrestin 2 can recruit Mdm2 to DOR, and suggest that DOR,
-arrestin 2, and Mdm2 form a ternary complex in an
agonist-dependent manner.
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Fig. 3.
Mdm2 was recruited to the activated GPCR.
A, recruitment of Mdm2 to the activated DOR in a
-arrestin 2-dependent manner. HEK293 cells were
cotransfected with HA-tagged DOR and GFP-Mdm2 in the presence or
absence of
-arrestin 2. After treated with 1 µM DPDPE
for 10 min, the cells were lysed and immunoprecipitated with anti-HA
antibody. The presence of Mdm2 was detected using anti-GFP antibody.
B, quantitative data are represented as fold values of the
basal coimmunoprecipitated Mdm2. C, colocalization of
endogenous Mdm2 with the activated DOR at plasma membrane. HEK293 cells
transfected with HA-DOR were treated with or without 100 µM naloxone for 20 min prior to stimulation with 1 µM DPDPE for 10 min. Receptors were detected with anti-HA
antibody (green), and Mdm2 was detected with its specific
antibody (red).
2 adrenergic receptor was stimulated (data not shown).
Moreover, pretreatment with naloxone completely blocked the
translocation of Mdm2 induced by DPDPE (Fig. 3C). Thus, our
data suggest that the subcellular distribution of endogenous Mdm2 can
be modulated by GPCR activation.
-Arrestin 2 Interacted with the Central
Region of Mdm2--
To map the Mdm2 interaction region in
-arrestin
2, we used
-arrestin 2 deletion mutants in coimmunoprecipitation
experiments. The wild type or mutant
-arrestin 2 was expressed in
HEK293 cells along with Mdm2, and coimmunoprecipitation of Mdm2 with
-arrestin 2 was monitored by immunoblotting. Deletion of amino acids
1-185 from the N terminus of
-arrestin 2 resulted in the complete
loss of Mdm2 binding. Conversely, a
-arrestin 2 fragment containing amino acids 1-185 associated with Mdm2 as efficiently as the
full-length
-arrestin 2, strongly indicating that Mdm2 binds to the
-arrestin 2 N terminus (Fig.
4A). Interestingly, an
N-terminal point mutation of
-arrestin 2, Val54
Asp
(V54D), which inhibits the internalization of GPCRs (32), also failed
to interact with Mdm2 (Fig. 4B). This further supports that
the N terminus of
-arrestin 2 contains the Mdm2 binding sites.
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Fig. 4.
Mapping the interaction region between
-arrestin 2 and Mdm2. A, HEK293 cells
were cotransfected with GFP-Mdm2 and full-length or truncated HA-tagged
-arrestin 2. Full-length and truncated
-arrestin 2 were
immunoprecipitated with anti-HA antibody, and the presence of Mdm2 in
the immunoprecipitate was detected using anti-GFP antibody.
B, HEK293 cells were cotransfected with GFP-Mdm2 and
HA-tagged wild type or V54D mutated
-arrestin 2 (
2V54D).
-Arrestin 2 was immunoprecipitated with anti-HA antibody. The
immunoblot was detected with anti-GFP antibody for Mdm2 and anti-HA
antibody for
-arrestin 2. C, schematic representation of
wild type and mutants of Mdm2. NLS, nuclear localization
signal; NES, nuclear export signal; RING, RING
finger domain; p53, p53 binding domain;
Arr2,
-arrestin 2 binding domain. D, HEK293 cells were
cotransfected with GFP-
-arrestin2 and full-length or truncated
HA-tagged Mdm2. Full-length and truncated Mdm2 were immunoprecipitated
with anti-HA antibody, and the presence of
-arrestin 2 in the
immunoprecipitate was detected using anti-
-arrestin antibody.
Immunoprecipitated Mdm2 was detected with an anti-HA antibody.
-arrestin 2. A series of Mdm2 deletion mutants
(Fig. 4C) were used in coimmunoprecipitation assays, and the
results showed that among them the mutants of Mdm2-(1-358) and
Mdm2-(1-383) lost their ability to bind to
-arrestin 2 (Fig.
4D). In contrast, Mdm2-(1-410) and Mdm2-(1-435) still associated with
-arrestin 2 to an extent comparable to the
full-length Mdm2 (Fig. 4D). These data indicate that the
region from 383 to 410 of Mdm2 is essential for
-arrestin 2 binding.
-Arrestin 2 with Mdm2 Negatively Regulated the
Function of Mdm2--
Mdm2 is a RING finger-dependent E3
ubiquitin ligase for p53 and targets p53 for ubiquitination and
degradation in a ubiquitin-dependent pathway. It has been
reported that degradation of p53 mediated by Mdm2 is regulated by
several binding partners of Mdm2 (13, 15). We therefore questioned
whether binding of
-arrestin 2 to Mdm2 could interfere with
Mdm2-directed p53 degradation. In a standard assay that tests the
potential effect of Mdm2 partner on Mdm2/p53 feedback loop,
coexpression of Mdm2 resulted in a significant reduction of p53 protein
level in Saos2 cells, which lack p53 (Fig.
5, A and B).
Further coexpression of increasing amounts of
-arrestin 2 could
effectively restore the protein level of p53 (Fig. 5, A and
B), indicating the reduction of the Mdm2-mediated p53
degradation by
-arrestin 2. Furthermore, the coexpression of
-arrestin 2-(1-185), which binds to Mdm2, could restore
p53 level while
-arrestin 2-(186-409), which does not bind to Mdm2, failed to do so (Fig. 5D). The above data
demonstrate that binding of
-arrestin 2 with Mdm2 functionally
regulates Mdm2-mediated p53 degradation. In support of this notion, our results showed that coexpression of
-arrestin 2 or
-arrestin 2-(1-185) but not
-arrestin 2-(186-409) also increased the protein level of Mdm2 since the binding of
-arrestin 2 with Mdm2 regulates the stability of Mdm2 (Fig. 5, A and C).
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Fig. 5.
-Arrestin 2 reduced the degradation of
Mdm2 and p53. A, stabilization of p53 and Mdm2 by
-arrestin 2. Saos2 cells were transfected with 100 ng of FLAG-p53
expression vector alone or together with 2 µg of HA-Mdm2 expression
vector in the absence or presence of 2, 4, or 6 µg of full-length
-arrestin 2 expression vector. 100 ng of CMV-GFP expression vector
was cotransfected to examine transfection efficiency, and CMV-
-gal
vector was used to compensate total DNA input. Cell lysates were
analyzed by Western blotting with anti-FLAG, anti-HA, or anti-GFP
antibody. B, quantitative data of the protein level of p53.
Data are means of three independent experiments. C,
quantitative data of the protein level of Mdm2. Data are represented as
the fold values of the control. D, stabilization of Mdm2 and
p53 by
-arrestin 2-(1-185) fragment. Wild type and mutant
-arrestin 2 were transfected into Saos2 cells with GFP-Mdm2 at a 3:1
ratio.
-arrestin 2, Mdm2, and p53 (Fig. 5A), indicating that coexpression of
-arrestin 2 unlikely interferes with the protein synthesis of Mdm2 or p53. To further investigate
-arrestin
2-mediated stabilization of the Mdm2 and p53, the effect of
-arrestin 2 on ubiquitination of Mdm2 and p53 was analyzed. As shown
in Fig. 6, A and B,
coexpression of
-arrestin 2 significantly reduced the
self-ubiquitination of Mdm2 and the Mdm2-mediated p53 ubiquitination. In contrast, coexpression of V54D
-arrestin 2 mutant, which does not
bind to Mdm2, had no effect on Mdm2-mediated p53 ubiquitination (Fig.
6B). In L02 cells, a normal liver cell line that expresses wild type p53 (32), infection with adenovirus expressing
-arrestin 2 could similarly reduce the ubiquitination of both endogenous Mdm2 and
p53 as compared with that with the control virus (Fig. 6, C
and D). Taken together, these data indicate that
-arrestin 2 negatively regulates self-ubiquitination of Mdm2 and
Mdm2-mediated ubiquitination of p53 via its binding with Mdm2.
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Fig. 6.
-Arrestin 2 reduced the
ubiquitination of Mdm2 and p53. A, reduction of Mdm2
self-ubiquitination. Saos2 cells were transfected with GFP-Mdm2,
HA-ubiquitin, and full-length
-arrestin 2 in various combinations.
Cells were pretreated with 10 µM MG132 for 4 h, and
Mdm2 was immunoprecipitated with anti-GFP antibody. The Mdm2-ubiquitin
complex was detected with anti-HA antibody. B, reduction of
Mdm2-mediated p53 ubiquitination by
-arrestin 2. Saos2 cells were
transfected with plasmids in combination as indicated. p53 was
immunoprecipitated with anti-FLAG antibody, and the p53-ubiquitin
complex was examined with anti-HA antibody. C, reduction of
the ubiquitination of endogenous Mdm2 by
-arrestin 2. L02 cells
infected with control adenovirus or
-arrestin 2 adenovirus were
treated with 10 µM MG132 for 4 h, and Mdm2 was
immunoprecipited with anti-Mdm2 antibody. The ubiquitinated Mdm2 was
detected with ubiquitin-specific antibody. D, reduction of
the ubiquitination of endogenous p53 by
-arrestin 2. L02 cells
infected with control adenovirus or
-arrestin2 adenovirus were
treated with 10 µM MG132 for 4 h and p53 was
immunoprecipitated with p53-specific antibody. The ubiquitinated p53
was detected with ubiquitin-specific antibody.
-Arrestin 2 Enhanced p53-mediated Apoptosis--
Since
overexpression of
-arrestin 2 could effectively reduce the
Mdm2-mediated p53 degradation, its consequence on the p53 function was
next examined in Saos2 cells. As shown in Fig.
7A, introduction of p53 into
Saos2 cells resulted in apoptosis in 60% of the transfected cells and
coexpression of Mdm2 significantly inhibited the apoptotic function of
p53. Further coexpression of
-arrestin 2 strongly restored
p53-mediated apoptosis (Fig. 7A), consistent with its
reduction of p53 degradation. Similar results were also observed in
another p53-null cell line H1299 (data not shown).
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Fig. 7.
-Arrestin 2 enhanced p53-mediated
apoptosis. A, reduction of the anti-apoptotic function of
Mdm2 on p53-mediated apoptosis by
-arrestin 2. Saos2 cells were
transfected with plasmids in various combinations. A GFP expression
vector was included in all the combinations to mark the transfected
cells. The dishes 48 h after transfection were scored under
fluorescence microscopy for the appearance of cells with distinct
apoptotic morphology. Mean values were derived from three experiments.
B, suppression of the endogenous expressed
-arrestin 2 using RNA interference technology in Saos2 cells. C,
inhibition of the expression of
-arrestin 2 attenuated the
p53-induced apoptosis. Saos2 cells were transfected with GFP with
-arrestin 2 RNAi plasmids or control vector. The apoptosis was
performed as in A. D, induction of cell death by
overexpression of
-arrestin 2. After infection with indicated
adenovirus, the cell death of L02 cells was examined with trypan blue
staining at indicated time. Mean values were derived from three
experiments. E, enhancement of apoptosis in L02 but not in
Saos2 cells by overexpression of
-arrestin 2. L02 and Saos2 cells
were infected with adenovirus expressing
-arrestin 2 or
-galactosidase. 48 h after infection, cells were exposed to UV
or left unexposed. Apoptosis induction was monitored 24 h after
irradiation. F, restoration of p21 protein level by
-arrestin 2. 48 h after transfection with indicated plasmids,
Saos2 cells were lysed and subjected to Western blotting analysis with
anti-p21 antibody and anti-actin antibody.
-arrestin 2 on p53-induced apoptosis. We transfected Saos2
cells with RNAi plasmid of
-arrestin 2 or the control vector, and
the expression of endogenous
-arrestin 2 was remarkably reduced
compared with the control level as detected by Western blotting (Fig.
7B), while the expression of actin was not affected under
the same conditions. Further experiments showed that when
-arrestin
2 expression was reduced by RNAi, the p53-mediated apoptosis was also
attenuated (Fig. 7C).
-arrestin 2 overexpression was examined using another
independent assay that measures cell death. In L02 cells that express
wild type p53, the infection with
-arrestin 2 adenovirus caused a
significantly higher ratio of cell death than that with the comparable
level of
-galactosidase adenovirus (Fig. 7D). It has been
known that Mdm2/p53 loop is regulated by UV irradiation, and our
results from DNA fragmentation assays showed that overexpression of
-arrestin 2 significantly enhanced the apoptosis in both
UV-irradiated or control L02 cells. In contrast, overexpression of
-arrestin 2 exerted no effect on apoptosis in p53-null Saos2 cells
(Fig. 7E). These data clearly show that
-arrestin 2 affects cell apoptosis in a p53-dependent manner.
-arrestin 2 expression on p53-mediated
apoptosis, the expression level of p21, which is strongly transactivated by p53, was analyzed under the same conditions. In Saos2
cells, transfection of p53 strongly induced the expression of
endogenous p21, and this could be inhibited by cotransfected Mdm2.
Further coexpression of
-arrestin 2 was able to restore the protein
levels of p21 transactivated by p53 while V54D
-arrestin 2 mutant
failed to do so, as shown in Fig. 7F.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Arrestin 2 has been established as an important modulator of
GPCR signaling and Mdm2 as the master regulator of p53 function, but
little is known regarding cross-talk between GPCR pathways and p53
apoptotic apparatus. Under normal circumstances, the cellular concentration of Mdm2 is maintained at low levels and most are distributed in the nucleus to control the function of p53 (33, 34).
Mdm2 in the cytoplasm is present at much lower levels compared with the
nucleus, and little Mdm2 is detected at the plasma membrane to regulate
GPCRs (35). In this study, we demonstrated that under physiological
conditions
-arrestin 2 not only physically interacted with
oncoprotein Mdm2 but also functionally modulated the subcellular
distribution of Mdm2, thus providing a solid basis for the functional
consequence of
-arrestin 2-Mdm2 interaction. Our study further
revealed that the association of
-arrestin and Mdm2 was greatly
promoted by activation of GPCRs and thus led to reduction of Mdm2
function in the feedback loop of Mdm2/p53. According to the results of
this study and Shenoy et al. (16), which demonstrates that
Mdm2 functionally regulates GPCR trafficking by interacting with and
ubiquitinating
-arrestin 2, a bi-directional regulatory model of
-arrestin 2/Mdm2 interaction can be proposed as following.
Activation of GPCRs by various extracellular signals significantly
increases the binding of
-arrestin 2 and Mdm2 and thus effectively
shifts the equilibrium of Mdm2 subcellular distribution from nucleus to
plasma membrane. The functional consequences of the enhanced
-arrestin 2/Mdm2 interaction are in one direction to promote
ubiquitination of
-arrestin 2 and assist internalization of GPCRs
and in the other direction to modulate Mdm2/p53 feedback loop and
reduce the ubiquitination and degradation of p53. In summary, the
current study strongly suggests a potential role for GPCR signaling in
modulation of p53-mediated apoptosis and clearly indicates that
-arrestin 2 may serve as a signaling linker between the GPCR and p53 pathways.
-arrestin 2 binding site on Mdm2 (residues
383-410) was also located in this important region, and the
interaction of
-arrestin with this small segment was critical to
functionally inhibit Mdm2-mediated p53 degradation as in the case of Rb
(Mdm2-(272-320)) or ARF (Mdm2-(210-244)). The
-arrestin 2 binding
site on Mdm2 identified in this study is a little different from the
-arrestin 2 binding site on Mdm2 reported previously (16) (which may
be due to the different source of Mdm2), but both these sites are in
this important region. Furthermore, it is worth pointing out that the
-arrestin 2 binding site is adjacent to the RING finger domain of
Mdm2 (435-489), which is necessary for its ubiquitin ligase activity
for itself and p53 (40). One of the Mdm2 regulators, Mdmx, is
demonstrated to bind to the RING finger domain of Mdm2 and to stabilize
both p53 and Mdm2 (41). Thus one of the molecular mechanisms for
-arrestin 2 to inhibit Mdm2 function could be hypothesized as that
the binding of
-arrestin 2 to Mdm2 somehow interferes with its
ubiquitin ligase activity and thus consequently attenuates the
self-ubiquitination of Mdm2 and ubiquitination of p53, as observed in
this study. Moreover, our previous report shows that the
-(S/T)X4-5(S/T)- motif is critical for
-arrestin 2 to bind to DOR (42). Interestingly, the
-arrestin 2 binding region in Mdm2 identified in this study also contains several such motifs, and this may provide the structural basis for
-arrestin 2/Mdm2 interaction.
-arrestin 2, if any, should be
much weaker than that between Mdm2 and
-arrestin 2. Moreover, our
data also revealed that the presence of MdmX did not interfere with the
interaction between the
-arrestin 2 and Mdm2. Taken together, the
current study indicates that MdmX is unlikely involved in the
-arrestin 2 regulation of Mdm2 function.
-arrestin 2 and Mdm2 was significantly enhanced by
specific stimulation of GPCRs. The enhancement of
-arrestin 2/Mdm2
interaction by GPCR activation has greatly shifted the distribution
equilibrium of Mdm2 from nucleus to cytoplasm and thus effectively
reduces the Mdm2-mediated p53 degradation. There exist at least two
possible explanations on how
-arrestin 2/Mdm2 interaction is
enhanced by GPCR activation. It has been well established that
stimulation of GPCRs leads to the phosphorylation of receptors and the
subsequent enhancement of
-arrestin binding to the phosphorylated
GPCRs. Moreover, the association of
-arrestin with its partner, such
as dishevelled protein (Dvl), is also significantly augmented by
phosphorylation of Dvl (47). Since Mdm2 can be phosphorylated by
several protein kinases such as ATM or Akt, and importantly Akt can be
activated efficiently by GPCR stimulation (48). So one possibility may
be that the potential phosphorylation of Mdm2 stimulated by GPCR
signaling enhances the
-arrestin 2/Mdm2 interaction. In addition,
the current study demonstrated that Mdm2, mediated by
-arrestin 2, could be recruited to the activated GPCRs at cytoplasm membrane where
they were colocalized and that the activated receptor,
-arrestin 2, and Mdm2 could be precipitated together in an
agonist-dependent manner. Therefore, the other possibility
may be that after stimulation of GPCRs the activated receptor,
-arrestin 2, and Mdm2 form a ternary complex that greatly enhances
the association between
-arrestin 2 and Mdm2. This may also explain
why stimulation of either Gi-, Gs-, or
Gq-coupled receptors can enhance the
-arrestin 2-Mdm2
interaction. However, the actual scenario, in which both mechanisms
described above may work simultaneously or other unknown ones may be
involved, remains to be further investigated.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. D. Bohmann for the HA-ubiquitin construct and Dr. Yang Shi for BS/U6 vector. We thank Jiyong Wang, Yue Sun, Jiali Li, and Shunmei Xin for their technical assistance, and Xin Ge, Yaya Wang, Yujie Li, Xiaohui Zhao, and Peihua Wu for kind help. We also thank Wenbo Zhang and Nanjie Xu for helpful discussions.
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FOOTNOTES |
---|
* This work was supported by Grants from the Ministry of Science and Technology (G1999053907 and G1999054003), Chinese Academy of Sciences (KSCX2-2 and KSCX2-SW), and the National Natural Science Foundation of China (30021003).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work.
To whom correspondence should be addressed: Institute of
Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, People's Republic of China. Tel.: 86-21-64716049; Fax: 86-21-64718563; E-mail: gpei@sibs.ac.cn.
Published, JBC Papers in Press, December 17, 2002, DOI 10.1074/jbc.M210350200
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ABBREVIATIONS |
---|
The abbreviations used are:
GPCR, G-protein-coupled receptor;
HEK, human embryonic kidney;
HA, hemagglutinin;
CMV, cytomegalovirus;
GFP, green fluorescent protein;
DOR, opioid receptor;
nt, nucleotide;
RNAi, RNA-mediated
interference.
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