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J. Biol. Chem., Vol. 275, Issue 29, 21896-21904, July 21, 2000

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TFIIH Interacts with the Retinoic Acid Receptor  and Phosphorylates Its AF-1-activating Domain through cdk7^*

Julie Bastien, Sylvie Adam-Stitah, Thilo Riedl^§, Jean-Marc Egly, Pierre Chambon, and Cécile Rochette-Egly^¶

From the Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/Université Louis Pasteur/Collège de France, BP 163, 67404 Illkirch Cedex, France

Received for publication, March 8, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Retinoic acid receptor  (RAR) is phosphorylated in COS-1 cells at two conserved serine residues located in the N-terminal region (serines 77 and 79 in RAR1 and serines 66 and 68 in RAR2) that contains the activation function AF-1. These serines are phosphorylated in vitro by cdk7, a cyclin-dependent kinase associated to cyclin H and MAT1 in the CAK complex (cdk7·cyclin H·MAT1), that is found either free or as a component of the transcription/DNA repair factor TFIIH. RAR is more efficiently phosphorylated by TFIIH than by CAK and interacts not only with cdk7 but also with several additional subunits of TFIIH. RAR phosphorylation and interaction with TFIIH occur in a ligand-independent manner. Our data demonstrate also that phosphorylation of the AF-1 function modulates RAR transcriptional activity in a response gene-dependent manner.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The pleiotropic effects of retinoids are transduced by two nuclear receptor families, the retinoic acid receptors (RARs)¹ and the retinoid X receptors (RXRs), that are ligand-dependent transregulators belonging to the nuclear receptor superfamily (1-4). RARs are activated by all-trans and 9-cis retinoic acid, whereas RXRs are activated by 9-cis retinoic acid only. There are three RAR (, , and ) and three RXR (, , and ) isotypes, and for each isotype there are at least two main isoforms that differ in their N-terminal region (1, 5, 6).

As do other members of the nuclear receptor superfamily, RARs and RXRs exhibit a conserved modular structure with six variably conserved regions (A to F) (Fig. 1) (1, 5). The N-terminal A/B region of RARs contains a ligand-independent transcriptional activation function, AF-1 (7, 8). Although the B regions of the three RAR isotypes are moderately conserved, their A regions are unrelated and differ for each isoform of a given RAR isotype (5). The highly conserved C region encompasses the central DNA binding domain. The function of region F, if any, is unknown. Region E is more complex, as it contains the ligand binding domain, a dimerization interface, and the ligand-dependent transcriptional activation/repression domain AF-2 (1, 9). The activity of AF-2 is entirely dependent on the integrity of a conserved sequence referred to as the AF-2 AD core, located in -helix 12 at the C-terminal end of the ligand binding domain. Ligand binding induces a major conformational change that includes helix 12 and creates a new surface for coactivator binding while corepressors are released, thus resulting in a transcriptional-competent nuclear receptor relayed to the transcriptional machinery and the chromatin template (1, 10-12). The AF-2 and AF-1 activities synergize with each other in a response element- and promoter context-dependent manner (1, 8, 13).

RARs and RXRs are phosphoproteins (14-16), and their phosphorylation involves several kinases. RAR can be phosphorylated in its AF-1-containing B region by the cyclin-dependent kinase cdk7 (14), which together with MAT1 and cyclin H forms the CAK complex that is found either free or as a component of the general transcription/DNA repair factor TFIIH (17-20). This phosphorylation, which results from an interaction with cdk7, is crucial for RAR transcriptional activity and modulates its ligand-induced degradation by the ubiquitin-proteasome pathway.² RAR can also be phosphorylated by protein kinase A at a residue located in the ligand binding domain (15), and this phosphorylation is required for differentiation of mouse embryonal carcinoma F9 cells into parietal endoderm-like cells upon RA and cAMP treatment (13). Similarly, RXR was found to be phosphorylated in its N-terminal A/B region and shown to be hyperphosphorylated in the same region by c-Jun N-terminal kinases upon UV activation (16).

Mutations of putative phosphorylation sites located in the AF-1 domain of mRAR2 were found to prevent the RA-induced differentiation of F9 cells (13), thus indicating that RAR2 phosphorylation in this domain could be required for this differentiation. Moreover, phosphorylation in the same domain was recently shown to be crucial for the ligand-induced degradation of RAR by the ubiquitin-proteasome pathway.² Because of our previous demonstration that RAR can be phosphorylated by cdk7 present within TFIIH, we assumed that RAR could also be phosphorylated by TFIIH. In the present study, we demonstrate that the B region of the two major human or mouse RAR isoforms, RAR1 and RAR2, are phosphorylated in a ligand-independent manner, by the cyclin H- and MAT1-dependent protein kinase cdk7. We also show that phosphorylation of RAR is more efficient when cdk7 is present within TFIIH. In addition, we reveal the existence of multiple RAR-TFIIH interactions that involve not only cdk7 but also additional subunits of TFIIH. Finally, phosphorylation of the A/B region was found to modulate RAR-induced transcription in a response gene-dependent manner.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Plasmids and Construction of Receptor Mutants-- The pSG5-based expression vectors for human (h) RAR1, mouse (m) RAR1, mRAR2, and mRAR AB were previously described (7, 21-23). hRAR1S76A, S77A, S79A, and S77A/S79A in pSG5 were constructed by double PCR amplification reactions according to Ho et al. (24) to generate a MscI/SacI fragment containing the appropriate mutation. The external oligonucleotides were 5'-CAG CTG CCA TGG CCA CCA AT-3' and 5'-GGT GAT GAG CTC TTC TAA CTG-3', encompassing the MscI and SacI sites, respectively. Internal oligonucleotides used in the PCR reaction encoded alanine (Ala) instead of serine (Ser) at positions 76, 77, and/or 79. The MscI/SacI fragments containing the mutation were inserted into MscI/SacI-restricted pSG5 hRAR1. hRARAB was prepared by PCR amplification of the fragment encoding amino acids 90-454 of hRAR1 using oligonucleotides primers 5'-CGGAATTCATGTGCTTCGTGTGCAATGACA-3' and 5'-CGGGATCCTCAGGCTGGGGACTTCAGG-3' and subcloning the amplified product into EcoRI-BamHI-digested hRAR1.

mRAR2S66A/S68A in pSG5 was constructed by subcloning the KpnI/MscI fragment from RAR₂S66A/S68A in pD402A (13) into the same sites of pSG5. mRAR2F in pSG5 was constructed by exchanging the MscI/SacI fragment of the mRAR2 expression vector with a MscI/SacI-digested PCR-amplified fragment encoding amino acids 223-410. mRAR2S440A was constructed by double PCR amplification reaction to generate a MscI-SacI fragment containing the appropriate mutation. The external nucleotides were 5'- CAGCGAGCTGGCCACCAAATGCATC-3' and 5'-CTTTGGCAAAATAACGAGCTC-3'.

Vectors encoding the chimeric proteins hRAR1(A/B)-ER(C) and Gal4-mRAR(DEF) in pSG5 were as described (8). hRAR1(A/B)-ER(C) mutated at serines 77 and/or 79 were created by cloning the MscI/AvaI fragment from pSG5 hRAR1 into the same sites of the chimeric construct.

The procaryotic vectors pET3d hRAR1WT, S77A, S79A, and S77A/S79A were constructed by subcloning the NcoI-BamHI fragments from the corresponding pSG5 vectors into the same sites of pET3d. Escherichia coli expressed wild type, and mutated proteins were purified as described (25). Purified hRAR1WT and RARAB were gifts from H. Gronemeyer (IGBMC, Illkirch, France).

The expression vectors for cdk7 and cdk7m were as described (14). Recombinant cdk7 and CAK were produced and purified from baculovirus-infected Sf9 cells as described (19, 26). Highly purified TFIIH hydroxylapatite fractions were prepared from HeLa cells (27). Baculoviruses allowing the expression of single subunits of the TFIIH core (XPB, XPD, p62, p52, p44, and p34) and of CAK (cdk7, cyclin H, and MAT1) were as described (17). Baculovirus encoding for hRARAB as an His-tagged fusion protein was constructed in the pVL 1392 vector (Pharmingen). Baculovirus expressing hRAR1 as a flag fusion protein was constructed in the pSK278 vector (28). The reporter genes mRAR2-chloramphenicol acetyltransferase (CAT), (TRE3)3tk-CAT, and mCRBPII (17 m-ERE) have been described (7, 8, 29).

Antibodies-- Mouse monoclonal antibodies against the F region of mRAR [mAb2(F)], hRAR [mAb4(F)], the A1 region of m- and hRAR1 [mAb1(A1)], and the D2 region of m- and hRAR [mAb5(D2)] were as described (30). Mouse monoclonal antibodies against the A2 region of mRAR2 [mAb10(A2)] were raised against synthetic peptide SP82 (amino acids 22-34). Rabbit polyclonal antibodies against the F regions of RAR [RP(F)] and RAR [RP(F)] as well as mouse monoclonal antibodies against the F region of RAR [mAb9(F)] were previously described (31, 32). Mouse monoclonal antibodies recognizing human cdk7, cyclin H, MAT1, and the XPB, XPD, p62, p52, p44, and p34 subunits of TFIIH were as described (33). Mouse monoclonal anti-FLAG antibodies were from Sigma. Mouse monoclonal antibodies recognizing mouse cdk7 were raised against synthetic peptide PC 135 (amino acids 1-22), and mAb raised against glutathione S-transferase were as described (14).

Cells, Transfections, and CAT Assays-- Mouse embryocarcinoma F9 cells (WT and RAR^/) were cultured as monolayers (34, 35). COS-1 cells were grown and transiently transfected by using the Ca²⁺ phosphate precipitation technique (36). In addition to expression vectors and reporters described in figures, all transfections included Bluescript DNA as a carrier and the -galactosidase expression vector pCH110 (1 µg) to correct for variations in transfection efficiency. After a 20-h incubation with calcium phosphate-precipitated DNA, cells were washed and incubated for an additional 20 h in the appropriate medium in the absence or presence of RA (10⁷ M) or the RAR agonist (BMS961) (10⁷ M). Cells were harvested 48 h after transfection, and CAT assays were performed using the enzyme-linked immunosorbent assay method (Roche Molecular Biochemicals). All assays were normalized to equal -galactosidase activity, and the results were expressed as pg of CAT/unit of -galactosidase.

Immunoprecipitations-- Sf9 cells were infected with combinations of baculoviruses, and whole cell extracts were prepared (17). Nuclear extracts were prepared from F9 cells as described (30). Extracts were incubated with protein A-Sepharose beads cross-linked with the indicated monoclonal antibodies in lysis buffer (20 mM Tris-HCl, pH 7.8, 200-250 mM NaCl, 10% glycerol, 0.1 mM EDTA, 1 mM dithiothreitol, and 0.1% Nonidet P-40) (14, 19). The beads were washed several times with the same buffer, resuspended in Laemmli buffer, and boiled, and the immunoprecipitated proteins were resolved by SDS-PAGE and revealed by immunoblotting and chemiluminescence.

In Vitro and in Vivo Phosphorylation-- In vitro phosphorylation reactions were performed with purified bacterially expressed WT or mutated hRAR1 proteins (14, 15). Reaction were initiated by the addition of either purified recombinant CAK complex (cdk7·cyclin H·MAT1) (19, 26), highly purified TFIIH (hydroxylapatite fraction) (27), or p44^MAPK (20 ng, Upstate Biotechnology Inc., Lake Placid, NY). Phosphorylated proteins were resolved by SDS-PAGE, electrotransferred to nitrocellulose filters, and visualized by autoradiography or by chemiluminescence after reaction with specific antibodies (14, 15).

For phosphorylation in transfected cells, COS-1 cells were transfected with wild type or mutated RAR expression vectors using the standard calcium phosphate procedure and labeled with [³²P]orthophosphate as described (15). Whole cell extracts were immunoprecipitated and resolved by SDS-PAGE, and after electrotransfer, the proteins were revealed by autoradiography and immunoreaction. Two-dimensional phosphoamino acid and tryptic phosphopeptide separations were carried out on thin layer cellulose plates using the Hunter thin-layer electrophoresis (HTLE) system as described (15).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Both Human and Mouse RAR Overexpressed in COS-1 Cells Are Phosphorylated in Their N-terminal A/B Region, whereas Mouse RAR Is Additionally Phosphorylated in Its F Region-- To determine whether wild type human RAR1 (hRAR1WT) is a phosphoprotein, COS-1 cells were transfected with the corresponding expression vector and labeled with [³²P]orthophosphate in the absence or presence of RA (10⁷ M). Whole cell extracts were immunoprecipitated with a RAR-specific monoclonal antibody and resolved by SDS-PAGE, and the phosphorylated proteins were analyzed either by autoradiography or by immunoblotting. hRAR1 was phosphorylated irrespective of the addition of RA to the culture medium (Fig. 2A, lanes 1 and 2). Phosphoamino acid analysis indicated that this phosphorylation was restricted to serine residues (Fig. 2B).

Tryptic phosphopeptide mapping yielded 3 phosphopeptides a, b, and c (Fig. 2C, panel 1). To characterize these phosphopeptides, COS-1 cells were transfected with hRAR₁ deleted for the A/B region (hRARAB) and labeled with [³²P]orthophosphate. hRARAB was not phosphorylated (Fig. 2A, lane 3), suggesting that phosphopeptides a, b, and c are located in the A/B region. Similarly, transfection in COS-1 cells of a chimeric construct expressing the A/B region of hRAR1 fused to the DNA binding domain (C) of the human estrogen receptor (ER) showed that the corresponding chimeric protein, hRAR1(AB)-ER(C), was phosphorylated (Fig. 2A, lane 8) and yielded the three phosphopeptides a, b, and c (Fig. 2C, panel 2).

Because the above data suggested that the A/B region of hRAR1 is a target for phosphorylation, the three serines belonging to potential phosphorylation sites for proline-directed kinases were individually mutated to alanine. The corresponding mutants, hRAR₁S76A, S77A, S79A, and S77A/S79A (Fig. 1) were transfected in COS-1 cells and analyzed by phosphopeptide mapping. hRAR1S77A, S79A, and S77A/S79A exhibited differences in their phosphorylation level (Fig. 2A, compare lanes 4-7), and phosphopeptide mapping analysis revealed that hRAR1S77A lacked phosphopeptide a (Fig. 2C, panel 3), whereas hRARS79A and hRARS77A/S79A lacked all three phosphopeptides (Fig. 2C, panel 4, and data not shown). Similar results were obtained with the chimeric construct hRAR1(A/B)-ER(C) in which serines 77 and 79 were mutated (data not shown). However, phosphorylation of the hRAR1S76A mutant was similar to that of hRAR1WT (data not shown), indicating that serine 76 is not phosphorylated.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Schematic representation of the major phosphorylation sites of RAR1 (human and mouse) and mouse RAR 2. The putative phosphorylation sites are underlined, and the actually phosphorylated serines indicated by an asterisk. The sequences of the different mutants are indicated. The phosphorylation site in the B region of mRAR1 is also shown. LBD, ligand binding domain; DBD, DNA binding domain.

View larger version (26K): [in this window] [in a new window]   Fig. 2.   hRAR 1 overexpressed in COS-1 cells is phosphorylated in its A/B region. A, COS-1 cells transfected with either hRAR1WT (lanes 1, 2, and 4), hRARAB (lane 3), hRAR1S77A (lane 5), S79A (lane 6), S77A/S79A (lane 7), or the chimeric construct hRAR1(AB)-ER(C) (lane 8) were labeled with 32P, and whole cell extracts were immunoprecipitated with mAb4(F) (lanes 1-7) or mAb1(A1) (lane 8). Immunoprecipitates were resolved by SDS-PAGE, electrotransferred to nitrocellulose membranes, and analyzed by autoradiography ([32P]) and chemiluminescence after immunoblotting (WB) with RP(F) (lanes 1-7) or mAb1(A1) (lane 8). In lane 2, cells were treated with RA (107 M) for 4 h. B, two-dimensional phosphoamino acid analysis of 32P-immunoprecipitated hRAR1. P-, phosphorylated. C, two-dimensional tryptic phosphopeptide maps of 32P-labeled immunoprecipitated hRAR1WT, S77A, S79A, and hRAR1(AB)-ER(C) as indicated.

Altogether, our results indicate that hRAR1WT is phosphorylated at serines 77 and 79. They also indicate that phosphopeptide a contains serine 77, whereas phosphopeptides b and c contain serine 79. These two b and c peptides may be partial digestion products due to the presence of low efficiency sites for trypsin cleavage in human RAR1 (37). In addition, the fact that mutation of serine 79 eliminates all phosphopeptides suggests that phosphorylation of serine 77 depends on that of serine 79 (37). Finally, as both serines 77 and 79 are located in a same tryptic peptide, it is not excluded that peptides a, b, and c are phosphoisomers, spot "a" representing the diphosphorylated peptide (at serines 77 and 79) and spots "b" and "c," the monophosphorylated one (at serine 79) (37).

The phosphorylation sites that are located in the B region of RAR are conserved between human and mouse and identical in the RAR1 and RAR2 isoforms (21, 23) (Fig. 1). In contrast, human and mouse regions F are different (Fig. 1) (21). Therefore, the two mouse RAR isoforms (mRAR1 and mRAR2) were overexpressed in COS-1 cells and labeled with [³²P]orthophosphate. Both receptors were phosphorylated in a RA-independent manner (Fig. 3A, lanes 1-4). Tryptic phosphopeptide mapping of mRAR1 yielded, in addition to phosphopeptides a, b and c described above for hRAR1, two other phosphopeptides, d and e (compare Fig. 3B, panel 1 to Fig. 2C, panel 1). The same d and e peptides were present in the tryptic digest of mRAR2, whereas phosphopeptides a' and b' differed from phosphopeptides a and b of RAR1, and phosphopeptide c was not seen (Fig. 3B, compare panels 1 and 2; see also panel 6), most probably because regions A of these two RAR isoforms are unrelated in their sequence (23). As expected, mRARAB was less phosphorylated than mRAR2WT (Fig. 3A, compare lanes 5 and 6) and lacked phosphopeptides a' and b' (Fig. 3B, panel 4). Moreover, the tryptic digest of mRAR2S66A/S68A lacked phosphopeptides a' and b' (Fig. 3B, panel 5), indicating that mRAR2 is phosphorylated in its A/B region at serines 66/68.

View larger version (44K): [in this window] [in a new window]   Fig. 3.   mRAR1 and mRAR2 overexpressed in COS-1 cells are phosphorylated in both their A/B and F regions. A, COS-1 cells were transfected with either mRAR1 WT (lanes 1 and 2), mRAR2 WT (lanes 3-5, 7, and 9), mRARAB (lane 6), mRARF (lane 8), mRAR2S66A/S68A (lane 10), or the chimeric construct Gal4-mRAR(DEF) (lane 11) and labeled with 32P. Whole cell extract were prepared, immunoprecipitated with mAb2(mF) (lanes 1-6 and 9-11) or mAb10(A2) (lanes 7 and 8) and processed as in Fig. 2A for autoradiography and immunoblotting with RP(F) (lanes 1-6 and 9-11) or mAb10(A2) (lanes 7 and 8). B, tryptic phosphopeptide maps of 32P-labeled WT and mutated mRAR proteins, as indicated. C, F9 WT cells grown as monolayers were labeled with 32P, followed by immunoprecipitation (IP) with either a control antibody (lanes 1 and 3) or with mAb10(A2) (lanes 2 and 4) and processed as in Fig. 2A. The phosphorylated proteins were visualized by autoradiography ([32P]) (lanes 1 and 2) and immunoblotting (WB) with RP(F) (lanes 3 and 4). D, two-dimensional tryptic phosphopeptide mapping of 32P-labeled immunoprecipitated mRAR2 from F9 cells.

Deletion of the F region in mRAR2F decreased the overall phosphorylation level (Fig. 3A, lane 8), and phosphopeptides d and e disappeared (Fig. 3B, panel 6). Moreover, a chimeric receptor construct expressing the DEF regions of mRAR fused to the DNA binding domain of the yeast transactivator Gal4 [Gal4-mRAR(DEF)] was also phosphorylated when overexpressed in COS-1 cells (Fig. 3A, lane 11) and yielded phosphopeptides d and e (Fig. 3B, panel 3). When the region F serine 440, which belongs to a potential phosphorylation site for proline-dependent kinases (Fig. 1), was mutated to alanine, the corresponding mutant construct, mRAR2S440A, did not yield the two phosphopeptides d and e (data not shown), which, as mentioned above, may correspond to partial trypsin digestion products (37). Thus, mouse RAR contains a phosphorylation site in its F region, that is absent in human RAR.

Mouse RAR2 is the main RAR isoform present in F9 embryonal carcinoma cells (21, 23). This endogenous RAR isoform was also phosphorylated (Fig. 3C) in its B and F regions, as it yielded an array of tryptic phosphopeptides similar to that observed with recombinant mRAR2 overexpressed in COS-1 cells (compare Fig. 3D with Fig. 3B, panel 2).

RAR Is Phosphorylated in Vitro by cdk7 Present in the CAK Complex and TFIIH-- We then investigated whether like RAR (14), RAR could be phosphorylated by cdk7 contained in either CAK (cdk7·cyclin H·MAT1 complex) or the general transcription/DNA repair factor TFIIH (17). Purified bacterially expressed hRAR1 was phosphorylated in vitro by CAK either free or as a component of TFIIH (Fig. 4A, lanes 1-3). Mutation of the cdk7 ATP binding site (cdk7 m in CAKm) suppressed hRAR1 phosphorylation, demonstrating that this phosphorylation was mediated by cdk7 (Fig. 4A, lanes 4-6). Interestingly, hRAR1 was more efficiently phosphorylated by TFIIH than by CAK (Fig. 4A, compare lanes 2 and 3, autoradiography and Western blot), irrespective of the presence of RA (data not shown).

View larger version (41K): [in this window] [in a new window]   Fig. 4.   In vitro phosphorylation of hRAR1 by cdk7 within CAK and TFIIH and by p44MAPK. A, purified bacterially expressed hRAR1WT was phosphorylated with purified recombinant CAK containing wild type cdk7 (rCAK, lanes 2 and 6) or mutated cdk7 (rCAKm, lane 5) or with purified TFIIH (lane 3). Phosphorylated proteins were resolved by SDS-PAGE, electrotransferred onto nitrocellulose membranes, and visualized by autoradiography [([32P]) and immunoblotting (WB) with RP(F) or mAb-cdk7. Lanes 1 and 4 are controls without any kinase. B, bacterially expressed hRAR1WT (lanes 1 and 4), S77A (lanes 2 and 5), S79A (lanes 3 and 6), or S77A/S79A (lane 7) were phosphorylated with either purified recombinant CAK (lanes 1-3) or p44MAPK (lanes 4-7). Phosphorylated proteins were processed as in A and visualized by autoradiography and immunoblotting with RP(F). C, two-dimensional tryptic phosphopeptide maps of hRAR1WT and S77A by TFIIH and p44MAPK, as indicated.

hRAR1 phosphorylated in vitro by either CAK (Fig. 4C, panel 1) or TFIIH (data not shown) yielded the same phosphopeptide pattern as hRAR1 overexpressed in COS-1 cells. hRAR1S77A was less phosphorylated (Fig. 4B, lane 2) and lacked phosphopeptide a (Fig. 4C, panel 2). Both hRAR1S79A and hRAR1S77A/S79A were not phosphorylated (Fig. 4B, lane 3 and data not shown) and migrated as single species corresponding to the faster migrating non-phosphorylated form of hRAR1. Altogether, these data indicate that hRAR1 is a substrate for cdk7 within CAK and TFIIH.

Because serines 77 and 79 belong to consensus motifs for proline-directed kinases (38-40), we also investigated whether RAR could be a substrate for mitogen-activated protein kinases. Although hRAR1 was phosphorylated by p44^MAPK (Fig. 4B, lane 4), tryptic phosphopeptide mapping yielded only phosphopeptide a (Fig. 4C, panel 3). Only the S77A mutation and not the S79A one abrogated this RAR phosphorylation (Fig. 4B, compare lanes 4 to 7). Thus, although RAR can be a substrate for MAPK in vitro, phosphorylation by this kinase is different from that achieved with cdk7.

RAR and RAR Bind Both to cdk7 and to Several Subunits of the Core of TFIIH-- As previously reported in the case of RAR (14), RAR directly interacted with cdk7. Purified bacterially expressed hRAR1 was indeed bound by recombinant cdk7 immunoabsorbed onto protein A-Sepharose beads cross-linked with cdk7 antibodies, (Fig. 5, lane 2). Similarly to RARAB (14), RARAB was also bound by cdk7 (Fig. 5, lane 3), indicating that cdk7 does not bind to the A/B region.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Interaction of purified bacterially expressed RAR with purified recombinant cdk7 in vitro. Purified RARWT (lanes 1 and 2) or RARAB (lanes 3 and 4) were incubated with cdk7 immunoadsorbed onto protein A beads cross-linked with mAb-cdk7. Bound proteins were probed with RP(F) and mAb-cdk7. Controls were performed in the absence of cdk7 (lanes 1 and 4). Aliquots of unprecipitated RAR and cdk7 are shown in lanes 5 and 6. mAbIP, mAb-immunoprecipitation.

The observation that RAR was more efficiently phosphorylated by cdk7 within TFIIH than within CAK prompted us to investigate whether this resulted from a tighter binding of RAR to TFIIH than to free CAK. TFIIH is a multisubunit complex composed of CAK and of six additional subunits (p34, p44, p52, p62, XPB, and XPD) referred to as the core of TFIIH (17). Sf9 cells were therefore coinfected with combinations of baculoviruses expressing RARAB and either the whole TFIIH (nine subunits, IIH9) or some TFIIH subcomplexes, such as the "core TFIIH/XPD" (six subunits, IIH6), the core TFIIH (IIH5), or CAK (17). Infected cell extracts were then immunoprecipitated with monoclonal antibodies directed against either the p44 subunit of TFIIH or RAR. The immunoprecipitated fractions (mentioned as "Bound (B)" in Fig. 6) were resolved by SDS/PAGE and analyzed by immunoblotting using antibodies against either XPB and p62 subunits of TFIIH or cdk7, cyclin H, and MAT1 subunits of CAK. mAb-p44 retained the corresponding IIH complexes (Fig. 6A, lanes 1-8) and also RAR (Fig. 6A, lanes 4, 6, and 8). Conversely, mAb-RAR retained not only RAR but also the TFIIH subcomplexes, whether the core of TFIIH was produced or not in association with CAK (Fig. 6B, lanes 2 and 6), indicating that RAR can bind not only CAK but also the core of TFIIH. RAR also coimmunoprecipitated with cdk7, cyclin H, and MAT1 when coinfected with CAK only (Fig. 6B, lane 10).

View larger version (50K): [in this window] [in a new window]   Fig. 6.   hRAR1 interacts with cdk7 associated to CAK and with the core of TFIIH. A, Sf9 cells were coinfected with baculoviruses encoding for hRARAB and for each TFIIH subunit that form either the IIH9, IIH6, or IIH5 complexes. Equal amounts of extracts were immunoprecipitated (IP) with mAbp44 and immunoblotted with RP(F) (lower panel) or with antibodies raised against the representative TFIIH subunits (upper panels, p62, XPB, cdk7, cyclin H, and MAT1). Controls were performed in the absence of RAR (lanes 1 and 2). Indicated are the loaded (L) and bound (B) materials. B, extracts from Sf9 cells coinfected with baculoviruses encoding for hRARAB and for each TFIIH subunit that form either the IIH9, IIH6, or CAK complexes were immunoprecipitated with mAb4(F) and immunoblotted as in A. Controls were performed in the absence of RAR (lanes 3, 4, 7, 8, 11, and 12). L and B are as in A. C, Sf9 cells were coinfected with baculoviruses encoding for FLAG-tagged full-length RAR1 and for each TFIIH subunit that form either the IIH9, IIH6, or IIH5 complexes. Extracts were immunoprecipitated with mAbp44 and immunoblotted with RP(F) (lower panel) or with antibodies against the TFIIH subunits (upper panel) as in A. Controls were performed in the absence of RAR (lanes 1, 2, 11, and 12). L and B are as in A. D, extracts from Sf9 cells coinfected with baculoviruses encoding for FLAG-tagged full-length RAR1 and for each TFIIH subunit that form either the IIH9, IIH5, or CAK complexes were immunoprecipitated with monoclonal anti-FLAG antibodies and immunoblotted with RP(F) (lower panel) or with antibodies against the TFIIH subunits (upper panel) as in A. Controls were performed in the absence of RAR (lanes 3, 4, 11, and 12) or in the absence of TFIIH (lanes 1 and 2). L and B are as in A. E, extracts from Sf9 cells coinfected with baculoviruses encoding for FLAG-tagged RAR1 and for either XPB, XPD, p62, or p52 were immunoprecipitated with monoclonal anti-FLAG antibodies and immunoblotted with RP(F) (lower panel) or with antibodies against the TFIIH subunit (upper panel). Controls were performed in the absence of RAR (lanes 3, 4, 7, 8, 11, 12, 15, and 16). L and B are as in A.

Similarly, when Sf9 cells were coinfected with FLAG-tagged RAR and either IIH9, IIH6, or IIH5, mAb-p44 coimmunoprecipitated RAR whether it was coexpressed with the entire TFIIH or only with the TFIIH core (Fig. 6C, lanes 4, 6, and 8) (14). Reciprocally, monoclonal anti-FLAG antibodies coimmunoprecipitated RAR and subunits from the TFIIH core, whether the latter was produced or not in association with CAK (Fig. 6D, lanes 6 and 8). RAR also coimmunoprecipitated with the three subunits of CAK when coinfected with CAK only (Fig. 6D, lane 10).

Collectively, these results show that RAR and RAR interact with TFIIH through both CAK and the TFIIH core. Moreover, a direct interaction between RAR (Fig. 6E) and RAR (data not shown) and either one of the core TFIIH subunits, XPB, XPD, p52 (Fig. 6E, lanes 2, 6, and 14), p44, or p34 (data not shown), could be revealed, indicating that these subunits may interact with RARs within TFIIH. Note that no interaction was observed between p62 and RARs, whether the coimmunoprecipitations were carried out with mAb-p62 or mAb-RAR (Fig. 6E, lane 10, and data not shown).

To investigate whether RAR was also intracellularly associated with TFIIH, we used the murine F9 cell line in which mRAR2 is phosphorylated (see above). Nuclear extracts from F9 cells were immunoprecipitated with monoclonal antibodies against p44 and analyzed by SDS-PAGE/immunoblotting using antibodies against either p62, cdk7, or RAR. mAb-p44 coimmunoprecipitated TFIIH and RAR (Fig. 7A, lane 3). Reciprocally, mAb-RAR coimmunoiprecipitated RAR and cdk7 (Fig. 7B, lane 5). These interactions were specific, as they were not revealed in control immunoprecipitations performed with mAb against the unrelated glutathione S-transferase protein (Fig. 7B, lanes 3 and 4) or with nuclear extracts from RAR null F9 cells (RAR^/ F9 cells) (Fig. 7B, lanes 2 and 6 and Fig. 7A, lane 4). Similar results were observed with RAR in the same cells (data not shown). Altogether these data indicate that a fraction of RAR2 and RAR is associated with TFIIH in F9 cells.

View larger version (22K): [in this window] [in a new window]   Fig. 7.   Coimmunoprecipitation of endogenous RAR2 with cdk7 and TFIIH in F9 cells extracts. A, nuclear extracts from WT and RAR/ F9 cells (1 mg) were immunoprecipitated (IP) with mAb-p44 (lanes 3 and 4) and immunoprobed with either mAb-p62, mAb-cdk7, or RP(F). Lanes 1 and 2 correspond to aliquots of the unprecipitated extracts. B, nuclear extracts from WT and RAR/ F9 cells were immunoprecipitated with either mAb5(D2) (lanes 5 and 6) or mAb-glutathione S-transferase (lanes 3 and 4) and probed with RP(F) and mAb-cdk7. Aliquots of unprecipitated extracts are shown in lanes 1 and 2.

Phosphorylation by cdk7 Modulates the Transcriptional Activity of RAR-- To investigate whether phosphorylation of the A/B region of RAR could play a role in the ligand-independent AF-1 transactivation function, we first tested the activity of hRAR1(AB)-ER(C) (a fusion of the A/B region of hRAR1 with the ER DNA binding domain, see above) in transient transfection assays using the CAT reporter construct, mCRBPII(17m-ERE)-CAT (8). hRAR1(AB)-ER(C) activated 3-fold the expression of the reporter, whereas mutation of serines 77 and 79 into alanine abrogated this stimulation, indicating the importance of these serines for AF-1 activity (Fig. 8).

View larger version (21K): [in this window] [in a new window]   Fig. 8.   Phosphorylation is crucial for the AF-1 function of RAR. COS-1 cells were cotransfected with the mCRBPII (17 m-ERE)-CAT reporter gene (5 µg) without (lane 1) or with the vector (1 µg) encoding for the chimeric protein hRAR1(AB)-ER(C) in which the DNA binding domain of ER is fused to the A/B region (either WT or S77A/S79A mutated) of hRAR1. The results are the mean of two independent experiments.

The role of phosphorylation of the AF-1 domain on the transactivation properties of RAR was further investigated using the full-length receptor and a reporter construct containing the CAT gene under the control of a RA-inducible promoter, the natural mRAR2 promoter (7). Transcription from this promoter was stimulated by hRAR1WT in the presence of a selective ligand (the RAR agonist, BMS961) at 10⁷ M (Fig. 9A). Deletion of the A/B region abrogated transcriptional activation, in agreement with previous reports (7). Mutation into alanine of Ser-77 and Ser-79 located in the A/B region reduced the transcriptional activity of hRAR1 (Fig. 9A), confirming that these phosphorylation sites are required for optimal transcription. That phosphorylation of serines 77 and 79 by cdk7 could be responsible for efficient transcription was further supported by the observation that overexpressed cdk7 significantly enhanced transcription by hRAR1WT but not by hRAR1S77A/S79A (Fig. 9B). This increase did not occur with cdk7 devoid of kinase activity through mutation within its ATP binding site (cdk7m) (Fig. 9C). Importantly, overexpression of other proline-dependent kinases such as cdk1 or p44^MAPK did not enhance the transcriptional activity of hRAR1 (data not shown).

View larger version (27K): [in this window] [in a new window]   Fig. 9.   Transactivation of the mRAR2 promoter by RAR is reduced by mutation of the phosphorylation sites located in the B region and is increased by overexpressed cdk7. A, COS-1 cells were cotransfected with the mRAR2-CAT (5 µg) reporter gene without or with increasing amounts (0.1, 0.2, and 0.5 µg) of hRAR1 WT, S77A/S79A, or AB expression vectors and treated with a RAR-specific ligand (BMS 961) at 107 M for 20 h. The results are the mean of three independent experiments. B, increasing concentrations of hRAR1WT or S77A/S79A expression vectors were cotransfected with the mRAR2-CAT reporter as in A in the presence or absence of the cdk7 vector (0.5 µg). The results correspond to a representative experiment among three. C, COS-1 cells were cotransfected as in A without (lane 1) or with hRAR1WT expression vector (0.2 µg) in the absence (lane 2) or presence of the cdk7 wt (lane 3) or mutated (cdkm, lane 4) expression vectors. The results are the mean of two independent experiments. D, the mRAR2-CAT reporter gene was cotransfected as in A without or with increasing amounts of mRAR2WT, AB, S66A/S68A, F, and S440A expression vectors. The results correspond to a representative experiment among three. E, cotransfections were performed as in B with increasing concentrations of mRAR2WT and S66A/S68A expression vectors and in the presence or absence of the cdk7 vector. The results correspond to a representative experiment among three.

Because the phosphorylation sites located in the B region are the same in RAR1 and RAR2 isoforms and are conserved between human and mouse (see above and Fig. 1), similar results were expected with mRAR2. In fact, transactivation by mRAR2 was also reduced by mutation of serines 66 and 68 into alanine (Fig. 9D) and overexpressed cdk7-enhanced transcription by mRAR2WT but not by mRAR2S66A/S68A (Fig. 9E). Interestingly, mRAR2F and mRAR2S440A were as active as mRAR2WT (Fig. 9D), indicating that, at least under these conditions, the phosphorylation site located in region F is dispensable for the transcriptional activity of mouse RAR.

Phosphorylation Modulates the Transcriptional Activity of RAR in a Responsive Gene-dependent Manner-- Because the transactivation activity of the A/B region of RARs has been shown to be responsive gene-dependent (7, 8), the role of the phosphorylation of this region was also studied with another RA-inducible promoter, the synthetic (TRE3)3tk promoter (29). Both hRAR1 and mRAR2 stimulated transcription in the presence of the ligand (Fig. 10, A and B). However, with this promoter, hRAR1S77A/S79A and mRAR2S66A/S68A exhibited higher activities (Fig. 10, A and B). Similar results were obtained with hRARAB and mRARAB (data not shown). Moreover, overexpressed cdk7 was without effect (Fig. 10A and data not shown). Note, however, that as described above with the mRAR2 promoter, mRAR2F and mRAR2S440A were as active as mRAR2WT (Fig. 10B). Thus, phosphorylation of the A/B region of RAR modulates transcription in a responsive gene-dependent manner, as it clearly affects differentially transcription from the (TRE3)3tk and mRAR2 promoters.

View larger version (23K): [in this window] [in a new window]   Fig. 10.   Transactivation of the (TRE3)3tk promoter by RAR is enhanced by mutation of the phosphorylation sites located in the B region. A, COS-1 cells were cotransfected with the (TRE3)3tk CAT (1 µg) reporter gene without or with increasing amounts (0.1, 0.2, and 0.5 µg) of hRAR1 WT or S77A/S79A expression vectors in the presence or absence of the cdk7 WT expression vector (0.5 µg) and treated with all-trans (107 M) for 20 h. B, COS-1 cells were cotransfected as in A without or with increasing amounts of mRAR2WT, S66A/S68A, F, and S440A expression vectors and treated with all-trans (107 M) for 20 h. The results in A and B are the mean of four independent experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this study, we have demonstrated that RAR is phosphorylated in the N-terminal region that contains the activation function AF-1 (8) and plays an essential role in RA-induced primitive endodermal differentiation of F9 cells (13). This phosphorylation, which involves two serine residues, is ligand-independent and appears to be most efficiently performed by the cyclin H-dependent kinase cdk7, a component of the general transcription/DNA repair factor TFIIH (17, 41, 42). Interestingly, this phosphorylation modulates the activity of AF-1 in a responsive gene-dependent manner.

RAR Is Phosphorylated by cdk7 in Its B and F Regions-- RAR is phosphorylated at two phosphorylation sites located in the B region. These sites are present in both the 1 and 2 isoforms and are conserved between human and mouse (21, 23). They have been identified to serines 66 and 68 in RAR2 and to serines 77 and 79 in RAR1 (see Fig. 1). In this latter case, our data show that phosphorylation of serine 77 depends on that of serine 79. As previously reported for RAR (14), we demonstrate that these sites are phosphorylated by cdk7, a cyclin H- and MAT1-dependent kinase. Indeed, overexpression of wild type cdk7, but not of cdk7 mutated at its ATP binding site or of any other cdk, results in a higher level of phosphorylation of the various RARs so far tested (Ref. 14).³ Moreover, similarly to RAR, the pattern of phosphorylation of RAR appears to be independent of the phases of the cell cycle (Ref. 14).³

As previously reported for RAR, RAR is also phosphorylated in its F region. However, this phosphorylation concerns only mouse RAR (either the 1 or the 2 isoforms) and not its human counterpart, due to the lack of conservation of this region between human and mouse (see Fig. 1). In contrast to phosphorylation of region B, no role has yet been found for this F region phosphorylation, either in RAR or in RAR.

RAR Is More Efficiently Phosphorylated by TFIIH than by Free CAK-- Cdk7 is associated with cyclin H and MAT1 in the CAK complex, and in the cell CAK is found either free or complexed with TFIIH, a general transcription factor also involved in DNA repair (17-20). Interestingly, we have shown that RAR is more efficiently phosphorylated in vitro by cdk7 when included in TFIIH rather than in CAK, as previously reported for RAR (14) and for the CTD of RNA polymerase II (19, 41, 43, 44). That RARs are more efficiently phosphorylated by TFIIH than by CAK may result from RAR interactions not only with cdk7, as previously reported (Ref. 14 and Fig. 5), but also with core subunits of TFIIH. Indeed, in coimmunoprecipitations experiments performed with insect cells coinfected with baculoviruses expressing different subunits of TFIIH and either RAR or RAR, we found that both RARs interact not only with cdk7 in CAK and TFIIH but also with several subunits of the TFIIH core. Thus, these multiple interactions may account for more efficient phosphorylation by cdk7 within TFIIH than within free CAK. Note that other transcription factors such as p53 and E2F-1, which have been shown to be phosphorylated by cdk7, also interact with the core subunits of TFIIH (45-47).

How cdk7 and the different TFIIH subunits interact with RARs remains to be investigated, but it is already clear from this and previous (14) studies that the N-terminal A/B region is not mandatory for these interactions. Moreover, the interaction of TFIIH with RARs is not sensitive to deletion of the AF-2AD core/helix 12,³ which is involved in the coactivator binding surface of the ligand binding domain (1). This is in accordance with our observation that the interaction of RARs with cdk7 and TFIIH is ligand-independent. Moreover, it suggests that the interaction between RARs and TFIIH involves another surface and is therefore mechanistically distinct from that described between RARs and coactivators (see the Introduction).

Regulation of Transcription by RAR through Phosphorylation of the AF-1 Domain by TFIIH-associated cdk7-- To investigate whether phosphorylation of the B region of RARs could modulate the ligand-induced activation of transcription, two reporter genes under the control of different responsive elements and promoters were tested: the natural mRAR2 promoter, which contains a RARE with directly repeated motifs separated by 5 nucleotides (DR5), and the synthetic (TRE3)3tk promoter, which contains inverted (palindromic) repeated motifs. In both cases, RAR activated transcription upon ligand binding. However, mutation of the phosphorylation sites located in the A/B region reduced transcription from the mRAR2 promoter-based reporter gene, whereas it enhanced that from the (TRE3)3tk promoter-based reporter gene. The three-dimensional conformation of bound RXR/RAR heterodimers is most likely different on the two types of response elements. This may result in distinct steric conformations of the AF-1-activating domain and, therefore, in different interactions with putative AF-1 coactivators, which could be differentially modulated by phosphorylation. In this respect we note that interactions between coactivators and the AF-1-activating domain of either the estrogen receptor ER or the nuclear receptor SF-1 have been recently shown to be modulated by AF-1 phosphorylation (48, 49). In any event, such a possibility is in accordance with our previous report showing that phosphorylation of RAR AF-1 is differentially required for RA-induced expression of target genes in F9 cells (13). Additionally, RAR phosphorylation may modulate the activity of TFIIH-associated cdk7 and/or regulate the enzymatic activity of some TFIIH subunits such as XPB and XPD that possess ATPase and helicase activity (18, 50-54) and are involved in distinct transcriptional steps (17).

In conclusion, we have demonstrated that, as other transcriptional regulators such as p53 and E2-F (45, 46, 55), RARs are targets for phosphorylation by cdk7 and interact with TFIIH. Thus, phosphorylation by TFIIH may be a general way of modulating the activity of transcriptional regulators. However, our present data also show that another proline-dependent kinase, p44^MAPK, can also phosphorylate RAR in vitro. Even though phosphorylation by this kinase is different from that achieved with cdk7 and overexpression of MAPK in COS cells does not affect the phosphorylation level and the transactivation properties of RAR, our present data do not rule out the possibility that p44^MAPK could modulate RAR activity in other cell types upon activation of the growth factor/MAPK cascade, as previously reported for ER (56).

	ACKNOWLEDGEMENTS

We are grateful to L. Penna and J-L. Plassat for excellent technical assistance. We are also grateful to C. Leborgne. We thank J. M. Chipoulet for purified TFIIH, I. Kolb-Cheynel for production of recombinant baculoviruses, and S. Vicaire for DNA sequencing. We thank E. A. Nigg (Swiss Institute for Experimental Cancer Research) for cdk7 expression vector, Dr. P. Reczek (Bristol-Myers Squibb Pharmaceutical Research Institute) for a gift of the synthetic retinoids and S. Nagpal (Allergan Inc.) for numerous plasmids.

	FOOTNOTES

^* This work was supported by CNRS, INSERM, Collège de France, Hôpital Universitaire de Strasbourg, Association pour la Recherche sur le Cancer (ARC), and Bristol-Myers Squibb Co.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.

Equal first authors, supported by the Ministère de la Recherche et de l'Enseignement Supérieur.

^§ Supported by the Ernst Schering Research foundation.

^¶ To whom correspondence should be addressed. Tel: 33 3 88 65 34 59; Fax: 33 3 88 65 32 01; E-mail: cegly@igbmc.u-strasbg.fr.

Published, JBC Papers in Press, March 29, 2000, DOI 10.1074/jbc.M001985200

² E. Kopf, J. L. Plassat, V. Vivat, H. de Thé, P. Chambon, and C. Rochette-Egly, submitted for publication.

³ J. Bastien, S. Adam-Stitah, and C. Rochette-Egly, unpublished results.

	ABBREVIATIONS

The abbreviations used are: RAR, retinoic acid receptor; RXR, retinoid X receptor; CAK, cdk-activating kinase; h-, human; m-, mouse; PCR, polymerase chain reaction; mAb, monoclonal antibody; CAT, chloramphenicol acetyltransferase; WT, wild type; PAGE, polyacrylamide gel electrophoresis; ER, estrogen receptor; MAPK, mitogen-activated protein kinase.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES