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J. Biol. Chem., Vol. 278, Issue 36, 34458-34466, September 5, 2003
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The AF-1 and AF-2 Domains of RAR
2 and RXR
Cooperate for Triggering the Transactivation and the Degradation of RAR2/RXR Heterodimers*
From the Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC) CNRS INSERM ULP, UMR 7104, BP 10142, 67404 Illkirch Cedex, France and ¶Laboratorio di Biologia Molecolare, Istituto di Ricerche Farmacologiche Mario Negri, Via Eritrea 62, 20157 Milano, Italy
Received for publication, May 12, 2003 , and in revised form, June 23, 2003.
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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In eukaryotic cells, liganded RAR
2/RXR
heterodimers activate
the transcription of retinoic acid (RA) target genes and then are degraded
through the ubiquitin-proteasome pathway. In this study, we dissected the role
of the RAR2 and RXR partners as well as of their respective AF-1
and AF-2 domains in the processes of transactivation and degradation.
RAR2 is the "engine" initiating transcription and its own
degradation subsequent to ligand binding. Integrity of its AF-2 domain and
phosphorylation of its AF-1 domain are required for both the degradation and
the transactivation of the receptor. Deletion of the whole AF-1 domain does
not impair these processes but shifts the receptor toward other proteolytic
pathways through RXR. In contrast, RXR plays only a modulatory
role, cooperating with RAR2 through its AF-2 domain and its
phosphorylated AF-1 domain in both the transcription activity and the
degradation of the RAR2/RXR heterodimers. Our results underline
that the AF-1 and AF-2 domains of each heterodimer partner cooperate with one
other and that this cooperation is relevant for both the transcription and
degradation processes. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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In response to retinoic acid (RA),1 target genes are regulated by two families of nuclear receptors, the RARs (
, 
,
and ) and the RXRs (, , and ) that bind as RAR/RXR
heterodimers to response elements located in their promoters
(1–3).
RARs and RXRs are modular proteins (Fig.
1) with a highly conserved central DNA-binding domain and a less
conserved ligand-binding domain that is composed of 11 -helices (H1 and
H3-H12), loops, and two short -strands (Ref.
4 and references therein) with
a dimerization interface formed mainly by helices H9 and H10
(5). The recent comparison of
the crystal structures of the ligand-binding domain of unliganded and liganded
RARs and RXRs (1,
6,
7) shed light on the molecular
mechanism underlying the structural reorganization that accompanies ligand
binding. The ligand-induced conformational changes in the ligand-binding
domain result in the release of corepressors and in conformational
rearrangements that affect mostly the N-terminal part of H3, H11, and the
highly conserved amphipathic helix 12, which carries the autonomous activation
function AF-2 (8). The new
conformation generates an interaction surface for coactivators
(9), which then recruit
multiprotein complexes and lead to the activation of responsive genes
(10,
11). Through this surface,
RARs also interact with SUG-1
(12), which belongs to the 19
S regulatory complex of the 26 S proteasome
(13).
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In RAR/RXR heterodimers, RXR is subordinated to the nonliganded RAR and therefore cannot autonomously induce transcription upon binding of a cognate agonist (14). However, in the presence of both RAR and RXR ligands, RXR synergizes with RAR for the recruitment of coactivators and thus for the transcription of RA target genes.
The N-terminal region of RARs and RXRs contains another transcription
activation domain called AF-1, which acts autonomously and
ligand-independently (15). The
interesting feature of this AF-1 domain is that it contains consensus
phosphorylation sites for proline-dependent kinases (for a review, see Ref.
16). The AF-1 domain of
unliganded RAR2 (Fig. 1)
is phosphorylated at serine 68
(17) by cdk7/cyclin
H-associated to transcription factor IIH, a general transcription factor also
involved in DNA repair (18).
Phosphorylation of this serine is required for RA-induced transcription
initiation (17). In response
to RA, however, RAR2 can also be phosphorylated at the nearby serine 66
by p38MAPK (12,
19). Phosphorylation of
RXR by MAPKs (Fig. 1) at
three residues located in the AF-1 domain (Ser-61, Ser-75, and Thr-87) has
been also reported (20).
We have recently shown that liganded RAR2 is degraded by the
ubiquitin-proteasome pathway when heterodimerized with RXR and engaged
in transcription of RA target genes
(12) according to the
following model. The fraction of RAR2 that is bound to cognate response
elements as heterodimers with RXR is phosphorylated by the cdk7 subunit
of transcription factor IIH and activates transcription, which increases up to
24–48 h and then reaches a plateau. This restriction in transcription is
concomitant to the RA-induced activity of p38MAPK, which leads to further
phosphorylation of the AF-1 domain of RAR2. This marked increase in
phosphorylation acts as a permissive signal, paving the way to RAR2
degradation through an increase in its ubiquitylation and subsequent
recognition by the proteasomal SUG-1 subunit bound at helix 12. Liganded
RXR is also degraded by the proteasome pathway
(21–23),
but whether this process is controlled by its AF-1 domain and/or its
phosphorylation remains unknown. Most interestingly, both phosphorylation and
the ubiquitinproteasome machineries also play a role in RAR-mediated
transcription. Therefore, we postulated that phosphorylation of the AF-1
domain would play a dual role, programming on the one hand the transcriptional
pattern and on the other hand the receptor degradation, to prevent a single
RAR/RXR heterodimer from performing endless rounds of
transcription of the cognate RA target genes and therefore controlling the
magnitude of transcription.
In the present study, we have investigated how, within
RAR2/RXR heterodimers, the AF-1 and AF-2 domains of each
receptor regulate, in addition to their transcriptional properties, their own
degradation and that of the partner. We show that the AF-2 domains of each
receptor are crucial for their autonomous degradation. The phosphorylated AF-1
domain of RAR2 is a fundamental determinant of the degradation and the
transactivation of both partners. Unexpectedly, deletion of the whole AF-1
domain does not impair these processes but targets RAR2 and its
dimerization partner RXR toward different mechanisms of regulation. We
also show that the AF-1 of RXR can be phosphorylated in response to RA.
This phosphorylation has only a modulatory effect on the degradation of
RXR and its cooperation with RAR2 for transcription.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Plasmids, Antibodies, and Chemicals—The pSG5-based expression vectors for mouse (m) RAR
2WT, mRAR
AF-1,
mRAR2AF-2, mRXRWT, mRXRAF-1,
mRXRAF-2, mRXRHet expression vectors and the
DR5-tk-CAT reporter gene have been described elsewhere
(21,
24,
25). The expression vectors
for RAR2S66/68A and for RXRS61/S75/T87A were as described
(17,
20). Rabbit polyclonal
antibodies raised against the F region of RAR, RP(F), have been
described previously (26). The
polyclonal and monoclonal antibodies raised against the A region of
RXR, RPRX(A), and the E region of RXR, Ab4RX3A2,
respectively, were also described previously
(27). MG-132 and z-VAD-fmk were from Calbiochem. The synthetic retinoids BMS961 and BMS649 were a gift from Bristol-Myers Squibb.
Cell Lines, Transfections, and CAT Assays—F9 cells were
grown in Dulbecco's modified Eagle's medium under 7% CO2 with a
change of medium every 48 h, as described
(28). F9 cells ablated for
RXR (RXR–/– cells)
and F9 cells reexpressing RARAF-1 in a RAR-null
background (RARAF-1 rescue line) were described previously
(29,
30). Rescue lines expressing
RARAF-1 in a RAR/RXR-null background were
established as described
(30).
COS-1 cells were grown in Dulbecco's modified Eagle's medium supplemented
with 5% fetal calf serum, plated in 6-well plates, and transiently transfected
using the DMRIE-C reagent, according to the manufacturer's protocol
(Invitrogen). All transient transfections were carried out in OPTIMEM
(Invitrogen) and contained the DR5-tk-CAT reporter gene (1 µg/well), the
pSG5-based expression vectors for mRAR2 and mRXR (0.05 µg of
each/well), Bluescript as a carrier, and the -galactosidase expression
vector pCH110 (0.5 µg/well) to correct for variations in transfection
efficiency. After a 16-h incubation with the DNA, the cells were washed and
incubated for a further 48 h in medium in the absence or presence of RA
(10–6 M), the RAR agonist
(BMS961, 10–7 M), the pan-RXR agonist
(BMS649, 10–6 M), or the combination of
both agonists. CAT assays were performed using the enzyme-linked immunosorbent
assay method (CAT enzyme-linked immunosorbent assay, Roche Applied Science).
All assays were normalized to equal -galactosidase activity, and the
results were expressed as pg of CAT/unit of -galactosidase.
Extracts and Immunoblotting—Whole cell extracts (WCEs) were prepared from F9 cells or transfected COS-1 cells as described previously (31). Proteins were resolved by SDS-10% PAGE, transferred onto nitrocellulose membranes by semidry blotting, and immunoprobed. The protein-antibody complexes were detected by chemiluminescence according to the manufacturer's protocol (Amersham Biosciences).
RNA Isolation and Real-time Reverse Transcriptase-PCR—Total
RNAs were isolated using the guanidinium thiocyanate method, and aliquots (500
ng) were subjected to real-time quantitative reverse transcriptase-PCR by
using the SYBR Green Light cycler detection system (Roche Applied Science and
Idaho Technologies). Transcript levels were normalized according to 36B4
transcripts, which are unresponsive to retinoids. The oligonucleotides for
36B4, HNF3, and HNF1 and were as described
(12,
30).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The AF-1 and AF-2 Domains of RAR
2 Control the
Degradation of RAR2/RXR
Heterodimers—To investigate how the activation functions of
RAR2 influence the degradation of both partners within
RAR2/RXR heterodimers, COS-1 cells were cotransfected with a
RAR2 expression vector and a CAT reporter gene controlled by a DR5
RA-responsive element (DR5-tk-CAT) in the presence or absence of an RXR
expression vector and treated with RA. When concomitantly overexpressed,
RAR2 and RXR bound as RAR2/RXR heterodimers to the
DNA-response element, and RA treatment resulted in the degradation of
RAR2 that was maximal at 48 h
(12)
(Fig. 2A, lane
2). However, in the absence of overexpressed RXR, RAR2WT
was degraded less efficiently (Fig.
2A, lane 4), confirming that RXR
modulates RAR2 degradation and indicating that endogenous RXR is
present in limited amounts in COS-1 cells. The degradation of RAR2WT
was reversed by the proteasome inhibitor MG132
(Fig. 2A, lane
8) but less by the caspase inhibitor z-VAD-fmk
(Fig. 2A, lane
10).
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Consistent with our previous results
(12,
21), RAR2 deleted for
helix 12 (RAR2H12) and RAR2 mutated at the
phosphorylation sites located in the AF-1 domain (RAR2S66/68A) were
refractory to RA-induced degradation, regardless of the presence of
overexpressed RXR (Fig.
2B). Unexpectedly, we found that RAR2 deleted for
its N-terminal AF-1 domain (RARAF-1) was significantly degraded
when cotransfected with RXR (Fig.
2C, lane 2). However, RARAF-1 was
resistant to RA-induced degradation when overexpressed in the absence of
RXR (Fig. 2C,
lane 6) or when cotransfected with RXR mutated at its
dimerization surface (mRXRD364A/E384A/E399A/Y402A/E406A/R426A/E439A)
(RXRHet) (21)
(Fig. 2C, lane
4). This is different from what was observed with the other RAR isotype,
RAR, which does not require the phosphorylation sites located in the
AF-1 domain for degradation
(21). Indeed,
RARAF-1 and RAR mutated at the phosphorylation sites
(RARS77A) were degraded as efficiently as RARWT, regardless of
RXR overexpression (data not shown). The degradation of
RARAF-1 was neither reversed by MG132
(Fig. 2D, compare
lanes 2 and 4) nor reversed by z-VAD-fmk
(Fig. 2D, lane
6), suggesting that deletion of the AF-1 domain targets RAR2
toward a proteolytic pathway that is distinct from those controlled by the
proteasome or by caspases.
In transfected COS cells, RXR was also degraded at 48 h of RA
treatment, whether its heterodimerization partner, RAR2, was WT
(Fig. 2A, lanes
1–4), AF-2 (Fig.
2B, lanes 1–4), or AF-1
(Fig. 2D, lanes
1 and 2). The only exception was when RXR heterodimerized
with RAR2S66/68A (Fig.
2B, lanes 5–8). The degradation of
RXR was reversed by MG132 in the context of heterodimers with
RAR2WT (Fig.
2A, compare lanes 6 and 8) but not with
RARAF-1 (Fig.
2D, compare lanes 2 and 4). However, in
both cases, z-VAD-fmk promoted the accumulation of RXR, indicating that
the turnover of RXR is sensitive to caspases
(Fig. 2, A (lanes
9 and 10) and D (lanes 5 and 6)).
Similar results were obtained in F9 embryocarcinoma cells with the
endogenous RAR2 and RXR receptors. Indeed, in these cells, the
degradation of RAR2 that occurs at 48 h of RA treatment
(12,
21)
(Fig. 3A, lanes
1 and 2) required the presence of RXR as
RXR–/– F9 cells did not
show any evidence of RAR2 degradation
(Fig. 3A, lanes
9 and 10). In agreement with the results obtained with
transfected COS-1 cells, RARAF-1 expressed in RAR-null
cells (i.e. in the presence of RXR) was degraded in response
to RA (Fig. 3A,
lanes 5 and 6). This degradation was neither reversed by
MG132 (Fig. 3A,
compare lanes 6 and 8) nor reversed by z-VAD-fmk
(Fig. 3B, lanes
6 and 8). However, degradation did not occur when
RARAF-1 was expressed in the double knock-out
RAR–/–/RXR–/–
cells (Fig. 3A,
lanes 11 and 12).
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The Degradation of RARAF-1 Involves the AF-2
Domain of RXR—To gain insight into the mechanisms
through which RXR participates in the degradation of RAR2,
either WT or deleted of its AF-1 domain, synthetic agonistic ligands specific
for RAR (BMS961) or RXRs (BMS649) were tested. The consequences of
deleting the AF-1 and AF-2 domains of RXR (in the RXRAF-1
and RXRH12 mutants respectively) were also tested.
When dimerized with RXRWT, RAR2WT was degraded in response to
its cognate ligand (Fig.
4A, lane 3) but not in response to the pan-RXR
agonist (Fig. 4A,
lane 4). The combination of the two ligands was as efficient as the
RAR agonist alone in inducing RAR2 degradation
(Fig. 4A, lane
5). Deletion of the AF-1 or AF-2 domains of RXR, in RXRAF-1
and RXRAF-2, respectively, did not affect this process
(Fig. 4A, lanes
6–15).
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Similarly, RARAF-1 underwent degradation upon binding of its
cognate ligand (Fig.
4B, lane 3) but not in response to the pan-RXR
agonist (Fig. 4B,
lane 4). Deletion of the AF-1 domain of RXR had no influence
(Fig. 4B, lanes
6–10). However, deleting the AF-2 domain of RXR made the
RAR ligand unable to signal degradation
(Fig. 4B, lane
13), indicating that the helix 12 of RXR may be of functional
importance for the proteolysis of liganded RARAF-1. However, the
absence of this domain could be compensated either partially or totally upon
addition of the two agonists (Fig.
4B, lane 15) or of RA
(Fig. 4B, lane
12), respectively. Thus, one can hypothesize that the two agonists, as
well as RA and its 9-cis metabolite, cooperate for inducing allosteric
transconformation changes (7)
that would overcome the absence of the AF-2 domain of RXR.
The Autonomous Degradation of RXR Requires, in Addition
to Its AF-2 Domain, the AF-1 Domains of Both Partners—The
degradation of RXR heterodimerized with RAR2, either WT or
AF-1, was also considered. When paired with RAR2WT, RXRWT
was also degraded in response to its specific ligand, a pan-RXR agonist
(Fig. 4A, lane
4), and not in response to the RAR agonist
(Fig. 4A, lane
3). The combination of the two agonists did not influence the degrading
activity of the RXR agonist (Fig.
4A, lane 5). Thus, RXRWT can be
autonomously degraded. Accordingly, RXR degradation still occurred upon
deletion of helix 12 in the RAR2 partner
(Fig. 2B, lanes
1 and 2) or in the absence of RAR2 overexpression
(21).
The pan-RXR agonist-induced degradation of RXR was abrogated upon
deletion of the AF-2 domain (Fig.
4A, lane 14), confirming that helix 12 is as
crucial for the autonomous degradation of RXR as for that of
RAR. However, in contrast to the same RAR mutant,
RXRH2 was not totally resistant since its degradation could be
triggered by the liganded RAR partner
(Fig. 4A, lanes
13 and 15).
Deletion of the AF-1 domain also made RXR refractory to its
agonist-induced degradation (Fig.
4A, lane 9). Importantly, deletion of the AF-1
domain of the RAR2 partner led to the same effect
(Fig. 4B, lane
4), indicating that the autonomous degradation of RXR is regulated
through mechanisms involving the AF-1 domains of both partners. However, in
both cases, the resistance was overcome upon addition of the two agonists
(Fig. 4, A (lane
10) and B (lane 5)) or of RA
(Fig.4A(lane 7) and
B (lane 2)).
Phosphorylation of the AF-1 Domain Also Accounts for the Autonomous
Degradation of RXR—Because the AF-1 domain plays a
crucial role in the degradation of RXR and phosphorylation serving as a
positive signal for the degradation of several proteins
(32–35),
including RAR2 (12,
21), we investigated whether
it was the same for RXR. The AF-1 domain of RXR can be
phosphorylated by MAPKs at three residues (Ser-61, Thr-75, and Ser-87) located
in the AF-1 domain (20).
Interestingly, we found that either in transfected COS-1 cells
(Fig. 5A, lanes
1–4) or in F9 WT cells (Fig.
5B), RA induces an upward shift in the electrophoretic
mobility of the receptor. This shift, which appears within 2 h of RA treatment
and disappears at 24 h, reflects the phosphorylation of the three residues
located in the AF-1 domain
(20) as it is abrogated upon
their mutation into alanine in the RXRS61A/T75A/S87A mutant
(RXRm) (Fig.
5A, lanes 5–8).
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RXR mutated at the phosphorylation sites was degraded after 48 h of
RA treatment, as efficiently as RXR WT
(Fig. 5C, compare
lanes 2 and 4 and lanes 6 and 8),
indicating that phosphorylation is not required for signaling the degradation
of RXR within RAR2/RXR heterodimers. Mutation of the
RXR phosphorylation sites did not affect the degradation of the
RAR2 partner, either WT or AF-1
(Fig. 5C, lanes 3,
4, 7, and 8).
However, mutation of the phosphorylation sites made RXR less
efficiently degraded in response to its agonist
(Fig. 5D, compare
lanes 3 and 7), as did the deletion of the AF-1 domain. This
could be overcome upon addition of the two agonists
(Fig. 5D, lane
8).
RARAF-1 Heterodimerized with RXR
Is Transcriptionally Active—Because RAR2 degradation and
transactivation are intimately linked
(12,
19), we compared the
transcriptional activity of RAR2WT and RARAF-1.
RAR2/RXR heterodimers bind to response elements located in the
promoters of RA target genes, and upon ligand binding, they induce
transcription. Accordingly, in COS-1 cells coexpressing RAR2WT,
RXR, and a CAT reporter gene under the control of a DR5-RA-response
element (DR5-tk-CAT), RA induces a 10-fold increase in CAT activity
(Fig. 6A, lane
2). This induction was less efficient in the absence of overexpressed
RXR, in agreement with the known heterodimer requirement for activation
of transcription (Fig.
6A, lane 1). In contrast, RAR2S66/68A and
RAR2H12 were inactive regardless of the presence of
overexpressed RXR (Fig.
6A, lanes 3–6), confirming that the
transcriptional properties of RAR2/RXR heterodimers are strictly
dependent on the integrity of the AF-1 and AF-2 domains of RAR2.
|
Importantly, RARAF-1 cotransfected with RXR was
transcriptionally active (Fig.
6A, lane 8). However, RARAF-1 was
unable to activate transcription when cotransfected with RXRHet
(Fig. 6A, lane
9) or when overexpressed in the absence of RXR
(Fig. 6A, lane
7). All together, these results highlight the importance of the
heterodimerization partner RXR, not only for the degradation but also
for the transactivation of RARAF-1.
Transactivation by RAR2, either WT or AF-1, was abrogated by
the proteasome inhibitor MG132 (Fig.
6B, lanes 2 and 5), supporting the
concept that the proteasome can also carry out non-proteolytic tasks and
modulate transcription (36).
In contrast, z-VAD-fmk increased transcription mediated by RAR2, either
WT or AF-1 (Fig.
6B, lanes 3 and 6), in agreement with
the accumulation of RXR (Fig. 2,
A and C).
The above observations were recapitulated in F9 embryocarcinoma cells. In
these cells, the transactivation of RA target genes such as HNF1 and
HNF3 was maximal and reached a plateau at 48 h
(Fig. 7, A and
B, lane 1). In agreement with the results
obtained with transfected COS-1 cells, the RA-induced expression of these
genes was completely impaired in
RXR–/– cells
(Fig. 7, A and
B, lane 3) and in F9 cells expressing
RARAF1 in a double RXR/RAR-null background
(Fig. 7, A and
B, lane 4). However, it was only decreased in F9
cells expressing RARAF-1 in a RAR-null background
(i.e. in the presence of endogenous RXR)
(Fig. 7, A and
B, lane 2). As in transfected COS cells, MG132
abrogated the RA-induced expression of these RA target genes in F9 cells
expressing either RAR2WT or RARAF1
(Fig. 7, A and
B, lanes 1 and 2), whereas z-VAD-fmk
increased transcription in both cell lines
(Fig. 7, A and
B, lanes 1 and 2).
|
RAR2 and RXR Synergize for
Transcription through the Phosphorylation of the AF-1 Domain of
RXR—The mechanisms through which the activation
domains of each partner participate in the transactivation of
RAR2/RXR heterodimers were further investigated by using the
same strategy as that described for degradation. Within
RAR2WT/RXRWT heterodimers, liganded RAR2 is able to
induce transcription, whereas RXR is subordinated to RAR
(14). Accordingly, RXR
cannot autonomously induce transcription in response to its cognate ligand
(Fig. 8A, lane
1) but synergizes with liganded RAR
(Fig. 8A, lane
1). Therefore, deleting helix 12 impaired the ability of RXR to
synergize with RAR2 (Fig.
8A, lane 3). Deletion of the AF-1 domain of
RXR also abrogated the synergy between RAR and RXR ligands
(Fig. 8A, lane
2). Interestingly, mutation of the three phosphorylation sites located in
the AF-1 domain of RXR had the same effect
(Fig. 8B).
|
In the context of RARAF-1/RXR heterodimers,
transcription was still efficiently activated by the RAR agonist.
However, synergy with liganded RXR was abrogated, indicating that the
AF-1 domain of RAR2 also accounts for this process
(Fig. 8A, lane
4). It is noteworthy that the autonomous activity of
RARAF-1 was not affected upon deletion of the AF-1 domain of
RXR (Fig. 8A,
lane 5) but was impaired subsequently to the deletion of the AF-2
domain of RXR (Fig.
8A, lane 6), indicating that the helix 12 of the
partner accounts for the transactivation of liganded RARAF-1.
However, some transcriptional activity of the
RARAF-1/RXRH12 heterodimers could be detected in
the presence of the two agonists or of RA
(Fig. 8A, lane
6).
|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Within RAR/RXR heterodimers (Fig. 9A), the AF-2 domain of each partner cooperates for the recruitment of multiprotein complexes that act in coordinated and/or combinatorial manner to decompact chromatin and direct RNA polymerase II and the general transcription factors to the promoter (10, 11). According to recent studies, the AF-1 domains would also recruit intermediary proteins (37). Here we demonstrate that the AF-1 and AF-2 domains of each partner are key elements that cooperate with one other for controlling not only their own transcriptional activity and degradation but also that of the partner. We also demonstrate that deletion of one AF within one partner renders each one dependent on the other, highlighting the complex cooperation mechanisms between the AF-1 and AF-2 domains of each receptor.
|
The AF-2 Domain of RAR2 but Not That of RXR
Is Strictly Required for the Degradation and Transactivation of
RAR2/RXR
Heterodimers—Within transcriptionally active
RAR2/RXR heterodimers, both RAR2 and RXR are
autonomously degraded in response to their cognate ligand, through their AF-2
domains. However, the role played by the AF-2 domain of each partner is
different. Considering RAR2 (Fig.
9B), the integrity of its AF-2 domain is strictly
required for the degradation of the receptor, in accordance with our previous
observation that the 26 S proteasome is recruited through this domain
(12). In addition, RAR2
degradation is not influenced by the AF-2 domain of RXR, excluding a
cooperative role for the liganded partner in the recruitment of the
degradation machinery. Thus, one can suggest that RXR might rather
influence RAR2 degradation through promoting its binding to the cognate
response elements (38). In
contrast, RXR deleted for this same domain was not refractory to
degradation as it could be degraded in response to the ligand of its
partner.
The contribution of the AF-2 domains of RAR2 and RXR in the
transcriptional properties of the heterodimers is also somehow different.
Indeed, upon deletion of the AF-2 domain of RAR2, transcription cannot
be induced by RA or any agonist, either alone or in combination
(Fig. 9B). In
contrast, deletion of the AF-2 domain of the RXR partner did not affect
the autonomous activity of RAR2 but impaired the synergy between the
two receptors. These results confirm the model according to which, within
RAR/RXR heterodimers, liganded RXR is subordinated to the liganded RAR partner
for the dissociation of co-repressors and the recruitment of coactivators
(14).
In conclusion, the AF-2 domain of RAR2 plays an
"indispensable" role as it promotes both transactivation and
degradation, and its absence cannot be compensated by the AF-2 of the
RXR partner. In contrast, the AF-2 domain of RXR instead appears
to play a "permissive" role as it only cooperates with RAR2
during transcription and can be substituted by the AF-2 of RAR2 in the
degradation process.
Phosphorylation of the AF-1 Domain of RAR2 Is Strictly
Required for the Degradation and Transactivation of
RAR2/RXR Heterodimers, whereas That
of RXR Plays Only a Cooperative Role—The AF-1
domain of RAR2 is also crucial to the degradation process through its
phosphorylation and ubiquitylation
(12,
21). Accordingly,
RAR2S66/68A is completely refractory to RA-induced degradation
(Fig. 9C).
Interestingly, when heterodimerized to this mutant, RXR was also
refractory to degradation, suggesting that the phosphorylated AF-1 of
RAR2 signals the degradation of both partners. The phosphorylated AF-1
domain also plays a crucial role in transcription
(15,
17), probably through helping
the recruitment of coactivators and/or intermediary proteins, in cooperation
with the AF-2 domain, as already demonstrated for RAR
(37) and other nuclear
receptors
(39–41)
and/or with the RXR partner (compare
Fig. 9, A and
C).
Our results also illuminate a role for the AF-1 domain of RXR,
through its phosphorylation, in the degradation and transactivation of the
heterodimers. We observed that RXR responds to RA through an increase
in the phosphorylation of its AF-1 domain. However, the role of this
phosphorylated AF-1 domain (Fig.
9D) differs from that of RAR2 as it is not
critical but rather influences its own degradation as well as its cooperation
with its partner for transcription.
First, mutation of the phosphorylation sites (similarly to the deletion of
the AF-1 domain) did not influence the degradation of the RAR2 partner.
Further, it did not make RXR refractory to degradation. However,
RXR degradation was no more autonomous and required the influence of
its liganded partner. Thus, one can postulate that the phosphorylation sites
located in the AF-1 domain of RXR cooperate with RAR for the
recruitment and/or the action of the degradation machinery and that this
action can be substituted by liganded RAR2.
Second, mutation of the RXR phosphorylation sites did not affect the
ability of RAR2/RXR heterodimers to transactivate RA target
genes. However, it made RXR unable to synergize with liganded
RAR2 for transcription, indicating that not only the AF-2 domain but
also the phosphorylation sites located in the AF-1 domain of RXR
cooperate with RAR for the recruitment of cofactors and maximal
transcriptional activity.
Deletion of the AF-1 Domain Does Not Make RAR2
Refractory to RA-induced Degradation Transactivation but Renders It Dependent
on RXR—The novelty of this study is that
unexpectedly, upon deletion of the whole AF-1 domain of RAR2, the
degradation and the transcriptional properties of the heterodimers were not
abrogated but were regulated through different molecular mechanisms
(Fig. 9E). First,
RARAF-1 required the AF-2 domain of RXR to activate
transcription and to be degraded in response to its cognate ligand. However,
in the absence of this domain, both processes could occur in response to the
combination of both the RAR and RXR agonists, probably due to
allosteric transconformations changes
(7). In that context, the
degradation of RXR WT, AF-1, or AF-2 also required
allosteric transconformations induced by the two liganded partners. Thus,
deletion of the AF-1 domain renders RAR2 dependent on its partner.
Second, RARAF-1 degradation did not involve the proteasome but
involved other proteases that remain to be characterized. Thus, in contrast to
the mutation of the RAR2 phosphorylation sites that makes the
heterodimers completely refractory to proteasomal degradation, deletion of the
whole AF-1 domain shifts the receptors toward other proteolytic pathways
through RXR. Finally, RARAF-1 was unable to synergize
with liganded RXR for transcription, indicating that not only the AF-2
domain but also the AF-1 domain of RAR2 cooperates with RXR for
the recruitment of cofactors and maximal transcriptional activity.
In conclusion, our results highlight the crucial role played by the
phosphorylated AF-1 domain of RAR2 in the autonomous degradation of
each partner and their cooperation for transcription. As many cancers
characterized by aberrant kinase activities
(42) are resistant to
retinoids (43), it is tempting
to speculate that the phosphorylation of RARs and/or RXRs is affected in these
cells, leading to an aberrant degradation of the receptors and a deficient
transcription of the RA target genes. To gain insight in the mechanisms
regulating these processes, we are currently investigating which proteins are
interacting with the AF-1 domains of RAR2 and RXR.
|
FOOTNOTES |
|---|
* This work was supported by funds from CNRS, INSERM, the Collège de France, the Hôpital Universitaire de Strasbourg, the Association pour la Recherche sur le Cancer, and Bristol-Myers Squibb. This work was also supported by the "Associazione Italiana per la Ricerca contro il Cancro," the "Progetto Finalizzato Oncologia," Consiglio Nazionale delle Ricerche-Ministero dell'Università e della Ricerca Scientifica e Technologica, and the "Fondo d'Investimento per la Ricerca Biotecnologica." 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 indicate this fact.
Supported by short term fellowships from Human Frontier Science Program,
the Association pour la Recherche sur le Cancer, and the Fondazione Italiana
per la Ricerca sul Cancro. Present adress: Laboratorio di Biologia Molecolare,
Istituto di Ricerche Farmacologiche Mario Negri, Via Eritrea 62, 20157 Milano,
Italy.
Supported by the "Ligue Nationale contre le Cancer." Present
address: Neurogénétique Moléculaire, E.O223,
Génopole, 2 rue Gaston Crémieux, 91057 Evry Cedex, France.
|| To whom correspondence should be addressed: IGBMC, BP 10142, 67 404 Illkirch Cedex, CU de Strasbourg, France. Tel.: 33-3-88-65-34-59; Fax: 33-3-88-65-32-01; E-mail: cegly{at}igbmc.u-strasbg.fr.
1 The abbreviations used are: RA, retinoic acid; RAR, retinoic acid receptor;
RXR, retinoic X receptor; AF, activation function domain; CAT, chloramphenicol
acetyltransferase; MAPK, mitogen-activated protein kinase; WCE, whole cell
extract; WT, wild type; Z, benzyloxycarbonyl; fmk, fluoromethyl ketone; m,
mouse.
|
ACKNOWLEDGMENTS |
|---|
We acknowledge J. Bastien and S. K. Biswas for critically reading the manuscript. We are grateful to Annie Bauer for technical help. We thank E. Kopf, J. Clifford, H. Chiba, D. Metzger, and P. Chambon for providing mutant F9 cell lines. We also thank members of the cell culture and oligonucleotides facilities for help.
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