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Down-regulation of the PI3K/Akt pathway is involved in RA-

induced phosphorylation, degradation and transcriptional activity

of RAR 2

M. Giannì*, E. Kopf**, J. Bastien, M. Oulad-Abdelghani, E. Garattini***, P.

Chambon and C. Rochette-Egly#.

Institut de Génétique et de Biologie Moléculaire et Cellulaire. CNRS/INSERM

/ULP/Collège de France, BP 163, 67404 ILLKIRCH Cedex, FRANCE and ***

Laboratorio di Biologia Molecolare, Istituto di Ricerche Farmacologiche Mario

Negri, Via Eritrea 62, 20157 Milano, Italia

Present adresses:

*Laboratorio di Biologia Molecolare, Istituto di Ricerche Farmacologiche Mario

Negri, Via Eritrea 62, 20157 Milano, Italia.

**Sigma Israël, Plaut 3, Park-Rabin, Rehovot, Israël 76100.

# Corresponding author:

Dr. C. Rochette-Egly

IGBMC, BP 163, 67 404 Illkirch Cedex, CU de Strasbourg, FRANCE

Phone: (33) 3 88 65 34 59

Fax: (33) 3 88 65 32 01

E-Mail: cegly@igbmc.u-strasbg.fr

Running title : RA-induced RARγ phosphorylation through PI3K/Akt inhibition.

Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on May 24, 2002 as Manuscript C200230200 by guest on A

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ABSTRACT

Nuclear retinoic acid receptors (RARs) are phosphorylated at conserved

serine residues located in their N-terminal domain. Phosphorylation of RARγ2 at

these residues is increased in response to RA subsequently to the activation of

p38MAPK. We show here that this RA-induced phosphorylation of RARγ2 results

from the down-regulation of the PI3K/Akt pathway. By overexpressing Akt and by

using agents activating or inhibiting the PI3K/Akt pathway, we also demonstrate that

the RA-induced down-regulation of the PI3K/Akt pathway targets not only the

phosphorylation of RARγ2, but also the turn-over and transcriptional activity of the

receptor. Altogether, these data indicate that the PI3K/Akt pathway plays an

important role in retinoic acid signaling.

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INTRODUCTION

The effects of retinoic acid (RA) are mediated by two families of nuclear

receptors, the Retinoic Acid Receptors (RARα, β and γ) and the Retinoid X

Receptors (RXRα, β and γ) which are ligand-dependent transcriptional regulators

functioning as RAR/RXR heterodimers both in vivo and in vitro (1-3). A ligand-

independent activation domain called AF-1 which is present in the N-terminal A/B

region of RARs, contains serine residues (see Fig. 2A) which are constitutively (i.e. in

the absence of ligand) phosphorylated by the cdk7 subunit of the general

transcription factor TFIIH (4,5). We recently demonstrated that phosphorylation of

RARγ2 at these residues is markedly increased in response to RA through activation

of p38MAPK (Giannì et al., manuscript submitted). This RA-induced phosphorylation

is important for both RARγ2-mediated transcription of RA-target genes and

degradation of the receptor by the ubiquitin-proteasome pathway. The aim of the

present study was to investigate how p38MAPK is activated in response to RA.

Activation of p38MAPK has been traditionally associated with stress

responses, through a cascade of phosphorylation reactions involving upstream

kinases (MAPKKK, MAPKK and MAPK) (6-9 and references therein). However, it has

been recently reported that p38MAPK activity could be regulated through cross-talks

with the PI3K/Akt pathway (10-13). We show here that the RA-induced activation of

p38MAPK and therefore the subsequent increase in RARγ2 phosphorylation result

from the inhibition of the PI3K/Akt pathway. This down-regulation of the PI3K/Akt

pathway, is crucial for RA-induced degradation and transactivation activity of RARγ2

indicating that it is a key step in retinoid signaling.

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EXPERIMENTAL PROCEDURES

Reagents, Plasmids and Chemicals: The pSG5-based expression vectors for

mouse (m) RARγ2, mRARγ2S66/68A, and the DR5-tk-CAT reporter gene were

previously described (5,14). All-trans Retinoic Acid, LY294002 and Wortmanin were

from Sigma-Aldrich. STI571 was a gift from Dr. Barbara Willi (Novartis Pharma AG,

CH). The vectors for dominant active and negative Akt containing a Myc-tag were

purchased from Upstate Biotechnology Inc. The cDNA for p38MAPK was provided

by P. Cohen (Dundee, UK) and cloned into the pSG5 expression vector.

Rabbit polyclonal antibodies against RARγ, RPγ(F), have been described (15).

P-Tyr 4G10 monoclonal antibodies were from Jackson Immuno Research Lab, Inc.

Rabbit polyclonal antibodies against p38MAPK and Akt and their active

phosphorylated forms, P-p38MAPK (Thr180/Tyr182) and P-Akt (Ser473) were from

Cell Signaling Technology, Inc. (USA). Rabbit polyclonal antibodies against c-Abl (K-

12), c-Abl antibodies conjugated to agarose beads and goat polyclonal antibodies

against Actin (C-11) were from Santa Cruz Biotechnology Inc. Anti-Myc-tag

antibodies were from Upstate Biotechnology Inc.

Polyclonal antibodies specific to RARγ2 phosphorylated at ser66 or ser68

were prepared by immunizing rabbits with synthetic phosphopeptides followed by

column chromatography with sulfolink gel columns (Pierce, USA) coupled to the

corresponding immunizing phosphorylated peptide. After elution, the antibodies

reacting with unphosphorylated RARγ were depleted by chromatography on a column

coupled to the unphosphorylated peptide.

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Cell lines, transfections and CAT assays: F9 cells were cultured and treated

with RA 10-7M as described (16). COS-1 cells were grown and transiently transfected

in six-wells plates using the DMRIE-C reagent, according to the manufacturer’s

protocol (GIBCO-BRL-Life Technologies). All transfections contained 2.5µg DNA

including the DR5-tk-CAT reporter gene (1 µg), the RARγ2pSG5-based expression

vector (0.05 µg), the β-galactosidase expression vector pCH110 (0.5 µg) to correct

for variations in transfection efficiency and bluescript as a carrier. After a 16 h

incubation with DNA, cells were washed and incubated for a further 48 h in medium

with or without RA (10-6 M). CAT assays were performed using the enzyme-linked

immunosorbent assay method (CAT ELISA, Roche Molecular Biochemicals). All

assays were normalized to equal β-galactosidase activity and the results were

expressed as pg CAT per unit of β-galactosidase.

Extracts, immunoprecipitations and immunoblotting: Whole cell extracts

(WCEs) were prepared as previously described (4) and, when required,

immunoprecipitated with protein A-Sepharose beads cross-linked with the indicated

antibodies. For the detection of the phosphorylated forms of RARγ2, p38MAPK, Akt,

or c-Abl, WCEs were prepared in phosphorylation lysis buffer (PBL) (17). Proteins,

with or without prior immunoprecipitation, were resolved by SDS-10% PAGE,

electrotransferred to nitrocellulose (NC) membranes and immunoprobed. All

antibodies were diluted in PBS-0.05% Tween, containing 5% non-fat milk, except

antibodies against phosphorylated proteins which were diluted in TBS-0.05% Tween

containing 2% BSA. The protein-antibody complexes were detected by

chemiluminescence according to the Amersham-Pharmacia Biotech protocol.

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RESULTS

RA-induced activation of p38MAPK results from the down-regulation of

the PI3K/Akt pathway.

In RARγ2-transfected COS-1 cells, p38MAPK phosphorylation is induced after

24h of RA treatment as assessed by WB analysis with specific antibodies recognizing

the active phosphorylated form of the kinase (P-p38) (Fig.1A, compare lanes 1 and

2). To investigate whether this increase in p38MAPK activity involves Akt, we

activated or inhibited the PI3K/Akt pathway. First, a constitutively active form (da) of

Akt was coexpressed with RARγ2 in COS-1 cells. This markedly decreased the RA-

induced activation of p38MAPK (Fig. 1A, lanes 3-6). On the other hand,

overexpression of a dominant negative (dn) form of Akt enhanced p38MAPK

phosphorylation (Fig. 1A, lanes 7-10). These results were corroborated by using

STI571 (18), an inhibitor of the non-receptor tyrosine kinase c-Abl that down-

regulates the PI3K/Akt pathway (19,20). As expected, STI571 (10µM) decreased the

level of cAbl tyrosine-phosphorylation (Fig. 1B) while it increased the amount of

active phosphorylated Akt (P-Akt, Fig. 1B) as assessed by WB with antibodies

specific for the phosphorylated form of Akt. STI571 also suppressed the RA-induced

increase in phosphorylated p38MAPK (Fig. 1C, compare lanes 2 and 4). The effects

of PI3K inhibitors (LY292002 and Wortmanin) on p38MAPK phosphorylation were

also evaluated. LY292002 (10µM) and Wortmanin (100nM) decreased the amount of

constitutively phosphorylated and activated Akt (P-Akt, Fig. 1D) and increased the

activation of p38MAPK induced by RA (Fig.1E, compare lanes 2 and 3).

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RA also activated p38MAPK in mouse embryocarcinoma cells (F9 cells)

(Giannì et al, manuscript submitted, and Fig. 1C, lane 2). Moreover, as in transfected

COS-1 cells, STI571 and LY294002 abrogated (Fig.1C, compare lanes 2 and 4) and

increased (Fig. 1E, compare lanes 2 and 3), respectively, the activation of p38MAPK

induced by RA in these cells. Altogether, these results indicate that the RA-induced

activation of p38MAPK involves the inhibition of the PI3K/Akt pathway.

Inhibition of the PI3K/Akt pathway increases RA-induced RAR 2

phosphorylation.

In transfected COS-1 cells, RARγ2 is constitutively (i.e. in the absence of RA)

phosphorylated at serine residues 66 and 68 located in the N-terminal A/B region

(Fig. 2A) (5). Moreover, [32P]-labeling experiments demonstrated that the amount of

RARγ2 phosphorylated at these residues is increased in response to RA (Giannì et

al., manuscript submitted). These results were confirmed by WB analysis using

antibodies recognizing specifically RARγ2 phosphorylated at serine 66 (P-RARγS1)

or at serine 68 (P-RARγS2). Both antibodies detected an increase in the amount of

phosphorylated RARγ2 upon RA treatment (Fig. 2B, lane 2), indicating that RA

increases the phosphorylation of both residues. No increase was observed in COS-1

cells expressing a RARγ2 mutant in which the two serine residues are mutated into

alanine (RARγS66/68A) (Fig. 2B, lane 4).

The RA-induced increase in phosphorylated RARγ2 detected with the phospho

RARγ antibodies was impaired upon incubation of the transfected cells with

SB203580 (10µM), a highly specific inhibitor of p38MAPK (21) (Fig. 2C, lane 3), while

the MEK1 inhibitor PD98059 (5µM) had no effect (Fig. 2C, lane 4). Additionally, the

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increase in RARγ2 phosphorylation was enhanced upon overexpresion of p38MAPK

(Fig. 2D, compare lanes 2 and 4) and appeared earlier (at 10h instead of 24h).

Altogether, these results further support the conclusion that the RA-induced increase

in RARγ2 phosphorylation results from the activation of p38MAPK (Giannì et al.,

manuscript submitted).

We then investigated whether, as expected from the above results, modulating

the activity of the PI3K/Akt pathway would affect RA-induced RARγ2 phosphorylation.

Overexpression of the constitutively active (da) form of Akt inhibited the increase in

RARγ2 phosphorylation (Fig.3A, lanes 3 and 4), whereas a dominant negative (dn)

form of Akt enhanced RARγ2 phosphorylation (Fig. 3A, lanes 5 and 6). Addition of

STI571 also blocked the RA-induced increase in RARγ2 phosphorylation (Fig. 3B,

compare lanes 2 and 4), while in contrast, it was enhanced by the PI3K inhibitors,

LY294002 and Wortmanin (Fig. 3C, compare lanes 2 and 4). Similar results were

obtained with F9 cells (data not shown).

Collectively, these results indicate that the RA-induced RARγ2 phosphorylation

results from the activation of p38MAPK through inhibition of the PI3K/Akt pathway.

RA-induced down-regulation of the PI3K/Akt pathway is involved in

RAR 2 degradation and transactivation.

Agonistic ligands convert RARγ2 into a strong transcriptional activator.

Concomitantly, RARγ2 is degraded by the ubiquitin-proteasome pathway (22). We

recently demonstrated that the RA-induced increase in RARγ2 phosphorylation that is

mediated through activation of p38MAPK, is required for both degradation and

transactivation ot the receptor (Gianni et al. manuscript submitted). Therefore, we

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investigated whether modulating the PI3K/Akt pathway would also affect RA-induced

RARγ2 degradation and transcriptional potential.

Expression of active (da) Akt (Fig. 4A, lanes 5 and 6) blocked RA-induced

RARγ2 degradation that occurs at 48 h in transfected COS-1 cells. Addition of

STI571 had the same effect in COS-1 cells and F9 cells (Fig. 4C, compare lanes 2

and 4). In contrast, overexpression of a dominant negative form (dn) of Akt (Fig. 4A,

lanes 3 and 4), as well as the PI3K inhibitors, LY294002 and Wortmanin (Fig. 4E,

compare lanes 4 and 6 to lane 2 and data not shown) increased RARγ2 degradation.

The transcriptional activity of RARγ2 was similarly affected. Indeed, in COS-1

cells expressing RARγ2 and a CAT reporter gene under the control of a DR5-RA

response element (DR5-tk-CAT), the expression of active (da) Akt (Fig. 4B)

decreased the RA-induced increase in CAT activity. Addition of STI571 had similar

effects (Fig. 4D). In contrast, the PI3K inhibitors LY294002 and Wortmanin,

enhanced CAT activity (Fig. 4F).

Collectively, these results indicate that the down-regulation of the PI3K/Akt

pathway is involved in both RA-induced degradation and transcriptional activity of

RARγ2.

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DISCUSSION

We previously found that the RA-induced increase in RARγ2 phosphorylation

is mediated through activation of p38MAPK (Giannì et al., manuscript submitted).

Here we report that this activation implicates the down-regulation of the PI3K/Akt

pathway. Indeed, blocking PI3K with Wortmanin or LY294002 amplifies the observed

RA-induced increase in p38MAPK activity and RARγ2 phosphorylation. Reciprocally,

stimulation of the PI3K/Akt pathway upon STI571 treatment or overexpression of

active (da) Akt, down-regulates these processes.

Our present results are in agreement with recent reports demonstrating that

Akt negatively regulates p38MAPK (10-12), and that disruption of the PI3K/Akt

pathway prevents these effects, resulting in the activation of the p38MAPK (13). How

does RA inhibit the PI3K/Akt pathway was recently elucidated in mouse

embryocarcinoma cells (F9 cells) by Bastien et al. (manuscript submitted) who have

shown that RA acts at two levels, phosphorylation of the phosphatase PTEN and

inhibition of PI3K through its p85α subunit, both of them leading to Akt inhibition.

Interestingly, our present study demonstrates that the RA-induced down-

regulation of the PI3K/Akt pathway targets not only the phosphorylation of RARγ2

through the activation of the p38MAPK, but also its transcriptional activity and its

degradation by the proteasome. Thus, RARγ2 phosphorylation, RARγ2 turnover and

RARγ2-mediated transcription of RA-target genes are inter-related events resulting

from the RA-induced down-regulation of the PI3-K/Akt pathway which therefore plays

an important role in RA signaling.

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It is interesting to note that Akt is a mediatior of cell growth and survival, while

RA has pronounced antiproliferative potential which is usually linked to its capacity to

induce differentiation. In keeping with this activity, RA is used in the treatment of

several cancers (23,24). As a number of tumoral processes have been correlated

with constitutively high Akt activity (11,25,26) and therefore to aberrant downstream

kinase activities, one can speculate that inhibition of this pathway would improve the

efficiency of RA therapy. Note in that respect that STI571 which does not only inhibit

c-Abl tyrosine kinase, but also other receptor tyrosine kinases which are often

amplified in carcinoma (27) and lead to increased activation of the PI3K/Akt pathway,

is currently used in cancer therapy (28-31). Moreover, it synergizes with retinoids in

terms of cytodifferentiation and growth inhibition (32) and is capable of partially

reversing the RA-resistance of some APL cells (32). Altogether these results strongly

suggest that the combination of retinoids with agents that affect the PI3K/Akt

pathway could be exploited at the clinical level to improve retinoids therapy and/or

reverse RA resistance. In conclusion, this study will provide new insights not only into

retinoid signaling but also into retinoid therapy.

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ACKNOWLEDGMENTS

We aknowledge Dr. Barbara Willi for a generous gift of STI571 and Dr. P.

Cohen for providing the p38MAPK expression vector. We thank Jean-Luc Plassat,

and Annie Bauer for help. We also thank P. Eberling for preparation of the synthetic

phosphopeptides, G. Duval for the rabbit injections and A. Tarrade for critical reading

of the manuscript. This work was supported by funds from the Centre National de la

Recherche scientifique (CNRS), the Institut National de la Santé et de la Recherche

Médicale (INSERM), the Collège de France, the Hôpital Universitaire de Strasbourg,

the Association pour la Recherche sur le Cancer, and Bristol-Myers Squibb. MG was

supported by short term fellowships from Human Frontier Science Program, the

Association pour la Recherche sur le Cancer and by FIRC (Fondazione Italiana per la

Ricerca sul Cancro). JB was supported by the Ministère de la Recherche et de

l’Enseignement Supérieur. E.K was supported by Fondation Chateaubriand and by

an INSERM fellowship.

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LEGENDS TO FIGURES

Fig. 1. Down-regulation of PI3K/Akt is involved in RA-induced activation

of p38MAPK. A, COS-1 cells were cotransfected with the RARγ2 expression vector,

in the absence or presence of either the dominant negative (dn) or the dominant

active (da) Akt expression vector, and treated with vehicle or RA. WCEs were

immunoprecipitated with immobilized p38MAPK antibodies and immunoblotted with

antibodies against p38MAPK or its phosphorylated form (P-p38 MAPK). B, COS-1

cells were cultured in the absence or presence of 10µM STI571. WCEs were

immunoprecipitated with immobilized c-Abl antibodies and immunoblotted with

antibodies against either c-Abl or phosphotyrosine. WCEs were also

immunoprecipitated with immobilized Akt antibodies and immunoblotted with

antibodies against Akt or its phosphorylated form (P-Akt). C, RARγ2-transfected

COS-1 cells and F9 cells were treated with RA or STI571, either alone or in

combination as indicated. WCEs were immunoprecipitated with immobilized

p38MAPK antibodies and immunoblotted with antibodies against p38MAPK or its

phosphorylated form. D, transfected COS-1 cells and F9 cells were grown without or

with addition of Wortmanin (100 nM) or LY294002 (10 µM) for 16 h or 2 h

respectively, before harvesting. WCEs were immunoblotted with antibodies against

Akt or its phosphorylated form. E, transfected COS-1 cells and F9 cells were treated

with vehicle (lane 1) or RA (lanes 2 and 3). In lane 3, LY294002 (10 µM) was added

2 h before harvesting. WCEs were immunoprecipitated with immobilized p38MAPK

antibodies and immunoblotted with antibodies against p38MAPK or its

phosphorylated form.

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Fig. 2. RA increases RAR 2 phosphorylation through activation of

p38MAPK. A, schematic representation of the RARγ2 protein with the A to F regions

(not to scale). The target sequence for phosphorylation by proline-directed kinases in

the B region is shown and the corresponding serine residues which have been

mutated to alanine (Ser66 and Ser68) are indicated. B, COS-1 cells transfected with

the RARγ2 expression vector, either WT or S66/68A were treated for 24 h with

vehicle or with RA as indicated. WCEs containing equal amounts of RARγ2, as

checked by immunoblotting with RPγ(F), were immunoblotted with antibodies

recognizing RARγ2 phosphorylated at serine 66 (P-RARγS1) or at serine 68 (P-

RARγ-S2). C, RARγ2-transfected COS-1 cells were treated with RA in combination or

not with SB203580 (10µM) or PD98059 (5 µM) as indicated. WCEs were

immunoblotted with P-RARγS1 and RPγ(F). D, COS-1 cells cotransfected with the

RARγ2 and p38MAPK expression vectors were treated with RA for 10 h. WCEs were

immunoblotted with P-RARγS1, RPγ(F) and p38MAPK antibodies.

Fig.3. Inhibition of the PI3K/Akt pathway is involved in RA-induced

RAR 2 phosphorylation. A, COS-1 cells were cotransfected with the mRARγ2

expression vector, in the absence or presence of either the dominant negative (dn) or

the dominant active (da) Akt expression vector, and treated with vehicle or RA.

WCEs were immunoblotted with P-RARγS1, RPγ(F) or actin antibodies. Akt

overexpression was checked by immunoblotting with antibodies recognizing the Myc-

tag. B, RARγ2-transfected COS-1 cells were treated with RA or STI571 either alone

or in combination as indicated. WCEs containing equal amounts of RARγ2 were

immunoblotted with P-RARγS1 and RPγ(F) antibodies. C, RARγ2-transfected COS-1

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cells were treated with vehicle or RA and LY294002 (10 µM) was added 2 h before

harvesting, as indicated. WCEs were immunoblotted with P-RARγS1, RPγ(F) or actin

antibodies.

Fig. 4. RA-induced down-regulation of the PI3K/Akt pathway is involved

in degradation and transciptional activity of RAR 2. COS-1 cells were

cotransfected with the mRARγ2 expression vector and the DR5-tk-CAT reporter

construct and treated for 48 h with vehicle or RA, as indicated. In A, C and E, WCEs

were resolved by SDS-10% PAGE and immunoblotted with RPγ(F) or actin

antibodies. In B, D and F, extracts were analyzed for CAT activity. The results which

are the mean ±SEM of two independent experiments correspond to the fold activation

relative to control cells. A and B, transfection was performed in the absence or

presence of either the dominant negative (dn) or the dominant active (da) Akt

expression vector. C and D, transfected cells were treated with vehicle, RA or

STI571 either alone or in combination, as indicated. E and F, transfected cells were

treated with vehicle or RA. Wortmanin (100 nM) and LY294002 (10 µM) were added

for 16 h and 2 h, respectively, before harvesting.

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Garattini, Pierre Chambon and Cecile Rochette-EglyMaurizio Giannì, Eliezer Kopf, Julie Bastien, Mustapha Oulad-Abdelghani, Enrico

phosphorylation, degradation and transcriptional activity of RARg2Down-regulation of the PI3K/Akt pathway is involved in RA-induced

published online May 24, 2002J. Biol. Chem. 

  10.1074/jbc.C200230200Access the most updated version of this article at doi:

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