Cyclin H Binding to the RAR Activation Function

Cyclin H binding to the RAR␣ activation function (AF)-2 domain directs phosphorylation of the AF-1 domain by cyclin-dependent kinase 7

Gae´ tan Bour*†, Emilie Gaillard*†, Nathalie Bruck*, Se´ bastien Laleve´ e*, Jean-Luc Plassat*, Didier Busso‡, Jean-Pierre Samama‡, and Ce´ cile Rochette-Egly*§

*De´partement de Biologie Cellulaire et de Transduction du Signal and ‡De´partement de Biologie et Geńomique Structurales, Institut de Geńe´ tique et de Biologie Molećulaire et Cellulaire, Centre National de la Recherche Scientifique͞Institut National de la Santeét de la Recherche Me´dicale͞Universite´Louis Pasteur, UMR 7104, BP 10142, 67404 Illkirch Cedex, France

Edited by Joan Massague, Memorial Sloan–Kettering Cancer Center, New York, NY, and approved October 3, 2005 (received for review July 1, 2005) The transcriptional activity of nuclear retinoic acid receptors altering the architecture of TFIIH results into a drop in the ability (RARs), which act as RAR͞retinoid X receptor (RXR) heterodimers, of RAR␣ to be phosphorylated by TFIIH and to activate tran- depends on two activation functions, AF-1 and AF-2, which are scription. targets for phosphorylations and synergize for the activation of The serine residues located in the N-terminal AF-1 domain of retinoic acid target genes. The N-terminal AF-1 domain of RAR␣ is RARs belong to a proline-rich motif (Fig. 1A) and could, in phosphorylated at S77 by the cyclin-dependent kinase (cdk)-acti- principle, be phosphorylated by all proline-dependent serine͞ vating kinase (CAK) subcomplex (cdk7͞cyclin H͞MAT1) of the threonine kinases, including cdks and mitogen-activated protein general transcription factor TFIIH. Here, we show that phosphor- kinases. The current thought is that specificity for the appropriate ylation of S77 governing the transcriptional activity of RAR␣ kinase arises from the presence of a docking site on the protein depends on cyclin H binding at a RAR␣ region that encompasses substrate. Our observation that RAR phosphorylation by cdk7 loop 8–9 and the N-terminal tip of helix 9 of the AF-2 domain. We results from an interaction between the receptor and TFIIH (8, 9) propose a model in which the structural constraints of this region raised the hypothesis that phosphorylation of the AF-1 domain of control the architecture of the RAR͞RXR͞TFIIH complex and there- RARs might require molecular recognition by another TFIIH fore the efficiency of RAR␣ phosphorylation by cdk7. To our subunit. Concomitantly, cyclins have been shown to contribute in knowledge, this study provides the first example of a cooperation both the catalytic activity of cdks and interactions with the substrate between the AF-2 and AF-1 domains of RARs through a kinase at docking sites distinct from the phosphorylation site, thereby complex. directing the kinase to phosphorylate specific residues (12, 13). Here, we provide evidence that cdk7 recruits RAR␣ through ͉ retinoic acid receptor transcription cyclin H binding to a specific region within the AF-2 domain corresponding to loop 8–9 and the N-terminal tip of helix 9 of the etinoic acid (RA) regulates the expression of specific networks LBD. Cyclin H binding controls the efficiency of AF-1 domain Rof genes through two families of nuclear receptors, the RA phosphorylation by cdk7, providing evidence for cooperation be- receptors (RARs) (␣, ␤, and ␥) and the retinoid X receptors tween the AF-2 and AF-1 domains through interaction with, and (RXRs) (␣, ␤, and ␥), which act as ligand-dependent heterodimeric phosphorylation by, a cdk͞cyclin complex. Our results suggest the RAR͞RXR transcription activators (1, 2). The transcriptional route for the formation of the RAR͞RXR͞TFIIH complex, lead- activity of RARs depends on activation function (AF) 1 and AF-2, ing to the phosphorylation of the AF-1 domain of RAR␣ and the which synergize for the activation of RA target genes. The C- transcriptional activity of the nuclear receptor. terminal AF-2 domain encompasses the ligand-binding domain (LBD), consisting of 11 ␣-helices (H1 and H3-H12) forming a Materials and Methods compact structure (3, 4). Ligand binding promotes complex allo- Plasmids and Reagents. The pSG5-based expression vectors for steric effects and conformational changes, the most striking one human (h) RAR␣1WT, hRAR␥1WT, and hRAR␥1S79A and being the swing of helix 12 (5, 6), which leads to dissociation of mouse (m) RXR␣ were previously described, as well as the corepressor complexes. It also generates an interaction surface for hRAR␤2-Luc and DR1-tk-CAT reporter genes (9, 14, 15). Vectors coactivators, which then recruit a battery of intermediary proteins, for hRAR␣S77A, S77E, P345G͞D346A, and L342T were con- including chromatin remodellers and modifiers. They act in a structed by double PCR amplification. The pXJ41-based expression ͞ coordinated and or combinatorial manner to decompact chroma- vectors for cyclin H and MAT1 were as described in ref. 8. tin and direct the RNA polymerase II and the general transcription Baculovirus expressing hRAR␣1WT as a flag fusion protein was factors to the promoter (7), leading to the activation of the RA constructed in the pSK278 vector (BD Biosciences). Baculoviruses responsive genes. expressing cdk7, MAT1, and His-tagged cyclin H were as described In the last several years, researchers have witnessed additional in refs. 16 and 17. The prokaryotic vectors encoding hRXR␣ and layers of regulation of transcription by RARs through their N- hRAR␣1 [WT, DEF (amino acids 153–462), DE (amino acids terminal AF-1 domain, which is targeted by phosphorylation pro- cesses. Indeed, the RAR␣1 and RAR␥1 isotypes are phosphorylated in their AF-1 domain at S77 and S79, respectively (Fig. 1A), Conflict of interest statement: No conflicts declared. by the cyclin-dependent kinase (cdk)-activating kinase (CAK) This paper was submitted directly (Track II) to the PNAS office. complex of the general transcription factor TFIIH (8, 9). TFIIH Abbreviations: cdk, cyclin-dependent kinase; CAK, cdk-activating kinase; AF, activation consists of 10 subunits (10) assembled into two subcomplexes: the function; RA, retinoic acid; RAR, RA receptor; RXR, retinoid X receptor; LBD, ligand-binding core complex and CAK (composed of the cdk7, cyclin H, and domain; siRNA, short interfering RNA; CAT, chloramphenicol acetyltransferase; SP, syn- MAT1 subunits). The key role of this phosphorylation process in thetic peptide. the RA response has been highlighted by studies performed with †G.B. and E.G. contributed equally to this work. patients carrying a mutation in one subunit [XPD (xeroderma §To whom correspondence should be addressed. E-mail: [email protected]. pigmentosum group D)] of the core of TFIIH (11). This mutation © 2005 by The National Academy of Sciences of the USA

16608–16613 ͉ PNAS ͉ November 15, 2005 ͉ vol. 102 ͉ no. 46 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0505556102 Downloaded by guest on September 23, 2021 Cell Lines, Transfections, Reporter Assays and in Vivo Phosphorylation. HeLa cells were grown as described in ref. 11. COS-1 cells were grown and transiently transfected in six-well plates by using DM- RIE-C reagent (Invitrogen) as in ref. 19. Whole-cell extracts were prepared, resolved by 10% SDS͞PAGE, electrotransferred to nitrocellulose membranes, and immunoprobed as described in ref. 19. Chloramphenicol acetyltransferase (CAT) assays were performed by using the ELISA method (19). Luciferase activities were determined according to standard procedures. The results were normalized to the CAT or luciferase activities in the absence of receptor and in the presence of ligand. For in vivo phosphorylation, transfected COS-1 cells were labeled with [32P]orthophosphate and processed as described in ref. 8.

Overexpression of Recombinant CAK Subunits and Purification of Binary and Ternary Complexes. Insect Sf9 cells were infected with baculoviruses expressing cdk7, His-cyclin H, MAT1, or FLAG- RAR␣, either alone or in combination, and extracts were prepared as described in refs. 16 and 17. Cyclin H was purified from the crude Sf9 extracts by metal chelate affinity chromatography (16, 17). cdk7 and MAT1 were purified by affinity chromatography with the corresponding antibodies cross-linked to protein A Sepharose (16). cdk7͞cyclin H binary and cdk7͞cyclin H͞MAT1 ternary complexes were purified essentially as described in ref. 20 by metal chelate affinity chromatography (Talon, BD Biosciences) followed by Mono S ion exchange chromatography. Then, purification was completed by a second ion exchange chromatography (Mono Q),

and the histidine tag fused to cyclin H was removed with thrombin BIOCHEMISTRY (Sigma). Finally, after addition of 5 mM Pefabloc (Roche Applied Science, Indianapolis), complexes were polished by gel filtration on an S200 column. Fractions containing the recombinant complexes were pooled and concentrated on Filtron 50 (Pall Filtron, North- Fig. 1. RARs interact with cyclin H. (A) Schematic representation of the borough, MA). The purity of the individual subunits and of the functional domains and the major phosphorylation sites of RAR␣1 and RAR␥1. binary and ternary complexes were analyzed by SDS͞PAGE, (B) GST, GST-RAR␣, and GST-RAR␥ proteins immobilized on glutathione- followed by Coomassie blue staining and immunoblotting. Sepharose beads were incubated with recombinant cdk7 (lanes 1–4) or cdk7͞ cyclin H binary complex (lanes 5–8) puriﬁed from Sf9 extracts (see Materials In Vitro Binding and Phosphorylation Assays. Equimolar amounts of and Methods). Bound cdk7 and cyclin H were analyzed by immunoblotting. GST and GST fusion proteins expressed in Escherichia coli were Lanes 1 and 5 correspond to 5% of the loaded material. (C) Immobilized purified onto gluthatione-Sepharose beads (Amersham Pharmacia GST-RXR␣, -RAR␣, and -RAR␥ proteins were incubated with recombinant Biosciences) and incubated with recombinant cyclin H, cdk7, cyclin H (free of cdk7, lanes 1–5) or cdk7 (free of cyclin H, lanes 6–10) puriﬁed ͞ from Sf9 extracts. Bound cdk7 or cyclin H were analyzed as in B.(D) Extracts MAT1, or the purified cdk7 cyclin H and CAK complexes in the Ϫ7 from Sf9 cells coinfected with baculoviruses encoding cyclin H and FLAG- presence or absence of RA (10 M) as described in ref. 19. Bound tagged RAR␣ were immunoprecipitated with FLAG antibodies and immuno- proteins were resolved by SDS͞PAGE and detected by immunoblotted with RAR␣ and cyclin H antibodies. Lane 1 corresponds to 10% of the blotting. In vitro phosphorylation of purified RAR␣ (8) or immo- loaded material. bilized GST-RAR␣ proteins by the highly purified cdk7͞cyclin H complex was performed as described in ref. 11. ⌬ ⌬ 153–421), LBD (amino acids 176–421), or DEF H12 ( aa 408– RNA Isolation and Real-Time RT-PCR. Total RNAs were isolated and 416)] fused to GST in the pGEX-2T plasmid (Amersham Phar- subjected to real-time quantitative RT-PCR as described in ref. 19. ␥ macia Biosciences) are described in ref. 18. The GST-RAR 1 The primers were as follows: 36B4, 5Ј-GAAGTCACTGTGC- vector was constructed by subcloning the cDNA into the BamHI CAGCCCA-3Ј and 5Ј-GAAGGTGTAATCCGTCTCCA-3Ј; ␣ ͞ site of pGEX-2T. The GST-RAR P345G D346A and L342T RAR␣2, 5Ј-CGAGGGGAAAGATGTACGAG-3Ј and 5Ј- vectors were constructed by subcloning the SacI-EcoRI fragment CCTTCTCTGG GAGCAAACAG-3Ј; cyclin H, 5Ј-GCATT- from the mutants in pSG5 into the same sites of pGEX-2T-RAR␣. GACGGATGCTTACCT-3Ј and 5Ј-TGACATCGCTCCAACT- The other GST-RAR␣ chimera were constructed by PCR ampli- TCTG-3Ј;RAR␣1, 5Ј-TGTCTGCCTCCCTTCTGACT-3Ј and 5Ј- fication using appropriate oligonucleotides with restriction sites and GGGGATGGTGTGCTATATCC-3Ј. cloned into the pGEX-2T vector. All constructs were generated by using standard cloning procedures and verified by restriction en- Short Interfering RNA (siRNA). The 19-nt RNA oligonucleotides zyme analysis and automated DNA sequencing. corresponding to cyclin H were purchased from Dharmacon All-trans and 9-cis RA were from Sigma-Aldrich. The synthetic (Lafayette, CO) and transfected into HeLa cells at a final concen- RAR␥ agonist (BMS961) was a gift from Bristol-Myers Squibb. tration of 50 nM by using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. At 48 h posttransfection (with Antibodies. Mouse monoclonal antibodies recognizing human cdk7, an intermediate retransfection at 24 h), the cells were treated with cyclin H, MAT1, RAR␥ [MAb4␥(F)], RAR␣ [MAb9␣(F)], and vehicle or RA and subjected to RNA and protein analysis. RAR␣ phosphorylated at S77 were as described in refs. 8, 9, 11, and 17. Mouse monoclonal anti-FLAG antibodies and goat polyclonal Results antibodies against ␤-actin were from Sigma and Santa Cruz Bio- Cyclin H Interacts in Vitro and in Vivo with RARs. RARs are phos- technology, respectively. phorylated by the cdk7 kinase within the ternary cdk7͞cyclin

Bour et al. PNAS ͉ November 15, 2005 ͉ vol. 102 ͉ no. 46 ͉ 16609 Downloaded by guest on September 23, 2021 H͞MAT1 complex named CAK (8, 9). We previously demonstrated that this phosphorylation process involves a direct interaction between cdk7 and the RAR␣ or RAR␥ isotypes (8, 9). Indeed, in GST pull-down experiments, purified recombinant cdk7 (free of cyclin H or MAT1) interacted with RAR␣ and RAR␥ (Fig. 1B, lanes 1–4). Because parts of CAK, cyclin H, and MAT1 contribute to the catalytic activity of cdk7 (21) and also to interactions with several cdk7 substrates (22–24), we investigated whether the interaction of RAR␣ and RAR␥ with cdk7 was more efficient when associated with cyclin H and MAT1. Our results show that both RAR isotypes had higher affinities to cdk7 present in the highly purified binary cdk7͞cyclin H (Fig. 1B, lanes 5–8) or ternary cdk7͞cyclin H͞MAT1 (data not shown) complexes than to cdk7 alone (Fig. 1B, lanes 1–4), suggesting that cyclin H strengthens the interaction of cdk7 with the receptors either by increasing the affinity of cdk7 for its substrate and͞or through direct binding to the substrate. Thus, the ability of purified recombinant cyclin H and MAT1 to interact with RARs was analyzed in GST pull-down experiments. MAT1 did not interact at all (data not shown), but cyclin H (free of any cdk7) interacted more efficiently than cdk7 with RARs (Fig. 1C, compare lanes 4 and 5 to lanes 9 and 10). In similar experiments, cyclin H, cdk7, and MAT1 did not form detectable complexes with RXR␣ (Fig. 1C, lanes 3 and 8, and data not shown). That cyclin H interacts with RARs has been corroborated in vivo in Sf9 insect cells coinfected with baculoviruses expressing cyclin H and FLAG- tagged RAR␣. Monoclonal anti-FLAG antibodies coimmunopre- ␣ cipitated RAR and cyclin H (Fig. 1D, lane 4). Fig. 2. Overexpressed cyclin H abrogates RAR␣ interaction with cdk7, RAR␣ phosphorylation, and RAR␣-mediated transcription. (A) COS-1 cells were co- Cyclin H Overexpression Inhibits RAR␣ Phosphorylation by cdk7 and transfected with the RAR␣, cyclin H, or MAT1 expression vectors and labeled RAR␣ Transcriptional Activity. To evaluate the importance of RAR␣ with [32P]. Whole-cell extracts were immunoprecipitated with MAb9␣(F) or binding to cyclin H for cdk7-dependent phosphorylation, compe- MAbcdk7 and analyzed by autoradiography and immunoblotting. Top cor- tition experiments were performed by cotransfecting COS-1 cells responds to aliquots of unprecipitated extracts immunoblotted with cyclin H with high concentrations of an expression vector for cyclin H, along or MAT1 antibodies. (B) COS-1 cells were cotransfected with the RAR␤2-Luc ␣ ␣ ␣ with RAR␣. Overexpression of cyclin H significantly decreased the reporter gene along with RAR WT, RAR S77A, RAR S77E, cyclin H, or MAT1 amount of RAR␣ associated with cdk7 as assessed by immuno- and RA-treated for 24 h. Extracts were analyzed for luciferase activity. The results represent the mean (ϮSD) of two independent experiments. (C) Same precipitation of the extracts with cdk7 antibodies (Fig. 2A Middle, as in B with RAR␥ and the RAR␥ agonist (BMS 961). (D) COS-1 cells were lane 2). In contrast, overexpression of MAT1 had no effect (Fig. 2A, cotransfected with the DR1-tk-CAT reporter and the RXR␣ and cyclin H vectors lane 3). and treated with 9-cis RA. Extracts were subjected to CAT ELISA. The consequences of cyclin H overexpression on RAR␣ phosphorylation were also analyzed by labeling the cells with [32P]orthophosphate and immunoprecipitation of the extracts with activity of RXR␣ homodimers on a DR1G-tk promoter was not RAR␣ antibodies. Overexpression of cyclin H, but not of MAT1, affected upon cyclin H overexpression (Fig. 2D), in line with the significantly decreased RAR␣ phosphorylation (Fig. 2A Bottom). absence of interaction of RXR␣ with cyclin H. According to these results, overexpressed cyclin H inhibits RAR␣ Collectively, these results suggest that cyclin H binding to RAR␣ interaction with, and phosphorylation by, cdk7 through competition mediates the recruitment of the substrate to CAK, thereby opti- with endogenous cyclin H within CAK. mizing RAR phosphorylation and transcriptional activity. The Because phosphorylation by the cdk7͞cyclin H complex governs results also indicate that the effects of complex formation between the transcriptional activity of RARs (8, 9, 11), we next asked cyclin H and RAR predominate over the possible increase in whether inhibition of RAR␣ phosphorylation subsequently to the binding affinity between RAR and the active form of cdk7 induced sequestration of RAR␣ by overexpressed cyclin H would also result by cyclin H. into a drop in its ability to induce the expression of a luciferase reporter gene under the control of a RA-inducible promoter, the Cyclin H Interacts with a Domain of the LBD Encompassing Loop 8–9 natural RAR␤2 promoter. In COS-1 cells cotransfected with and the N-Terminal Tip of Helix 9. We next aimed to identify the cyclin RAR␣ and the RAR␤2-Luc reporter gene, the luciferase activity H-binding region by testing the ability of a series of GST-deletion was increased in response to RA (Fig. 2B, lane 2). Substitution of mutants in RAR␣ (Fig. 3A) to pull down cyclin H. No interaction S77 with an alanine in RAR␣S77A decreased RAR␣-mediated was seen with the fusion proteins containing the N-terminal A͞B transcription, whereas substitution of S77 with a glutamic acid and C regions of RAR␣ (Fig. 3B, lane 3). In contrast, the remainder residue (RAR␣S77E) was without effect (Fig. 2B, lanes 7 and 8), of RAR␣ (RAR␣DEF) interacted with cyclin H (Fig. 3B, lane 4). in line with our previous reports (8, 11). Overexpression of cyclin Deletion of the F region in the GST-RAR␣DE chimera (amino H significantly reduced RAR␣-mediated transcription (Fig. 2B, acids 153–421) and of part of the D region in GST-RAR␣LBD lanes 5 and 6), but MAT1 overexpression had no effect (Fig. 2B, (amino acids 176–421) did not affect this interaction (Fig. 3B, lanes lanes 3 and 4). Similar results were obtained for RAR␥-mediated 5 and 6), indicating that cyclin H interacts with the LBD containing transcription (Fig. 2C). Interestingly, the observed transcription helices 1–12. This interaction of RAR␣ with cyclin H did not defect observed was similar upon overexpression of cyclin H and depend on RA (Fig. 3C, lanes 3 and 4) and thus on structural upon mutation of RAR␣ or RAR␥ at their cdk7 phosphorylation rearrangements induced by ligand binding. Accordingly, deletion in site (RAR␣S77A and RAR␥S79A) (Fig. 2 B, compare lanes 6 and RAR␣DEF of helix 12 (amino acids 408–416), which has been 7, and C, compare lanes 4 and 5). In contrast, the transcriptional shown to abrogate the ligand-dependent recruitment of the coac-

16610 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0505556102 Bour et al. Downloaded by guest on September 23, 2021 Fig. 3. Cyclin H interacts with the LBD of RAR␣.(A) Schematic representation of the truncated and deleted GST-RAR␣ chimeric proteins. (B) The immobilized GST-RAR␣ proteins described in A were incubated with recombinant Fig. 4. Cyclin H interacts with a surface overlapping loop 8–9 and the

cyclin H purified from Sf9 extracts. Bound cyclin H was detected by immuno- BIOCHEMISTRY ␣ blotting. Lane 1 corresponds to 5% of the amount of loaded material. (C) Same N-terminal tip of helix 9. (A) Schematic representation of the RAR LBD. Boxes ␣ as in B with the GST-RAR␣DEF WT and ⌬H12 proteins in the absence or represent the -helices. The residues in loop 8–9, helix 9, loop 9–10, and the presence of RA. N-terminal tip of H10 are aligned. Arrowheads mark the residues that have been mutated, and asterisks mark the exposed residues. The SPs used in competition experiments are indicated. (B) Immobilized GST and GST-RAR␣ tivator proteins (18), had no effect on cyclin H binding (Fig. 3C, proteins were incubated with recombinant cyclin H purified from Sf9 extracts in the absence or presence of increasing amounts of SP1, SP2, or SP3. Bound lanes 5 and 6). This finding indicates that the recruitment of cyclin cyclin H was analyzed by immunoblotting. Lanes 1, 8, and 15 correspond to 5% H involves a region that is distinct from the coactivator-binding site of the amount of loaded extract. (C) Fragments encompassing loop 8–9 and and that does not undergo a major structural reorganization upon helix 9 and fused to GST were immobilized, incubated with purified recom- ligand binding. binant cyclin H, and processed as in B.(D) Same as in C with GST-RAR␣ WT, According to the tridimensional structure of the LBD (6, 25), P345G͞D346A, or L342T. loop 8–9, helix 9, and loop 9–10 depict exposed surfaces that are not involved in heterodimerization with RXR␣ and are not drastically reorganized upon ligand binding. Together with helix 8, these were exposed to solvent (Fig. 5A). We addressed the importance of secondary structure elements carry charged residues involved in the conformational dynamics of this region in governing complex salt-bridge interactions, which constitute a signature motif of the formation between RARs and cyclin H. We first intended to decrease the flexibility of loop 8–9 through mutation of L342, the class II nuclear receptors to which RARs belong (26). To test only residue whose side chain is oriented toward the protein core whether this part of the LBD encompasses the cyclin H-binding site, (Fig. 5B). According to modeling considerations, substitution of a series of synthetic peptides (SPs) were generated (Fig. 4A) and this Leu with a Thr residue is accommodated without modification assayed for their ability to prevent the interaction between RAR␣ in the main-chain architecture of the native structure, and the and cyclin H in GST pull-down assays. SP1 overlapping loop 7–8 side-chain methyl group preserves the hydrophobic contact require- and helix 8 (amino acids 319–333) was inert (Fig. 4B, lanes 3–7), as ments (Fig. 5B). However, the side-chain hydroxyl group comes at was SP3 overlapping the C-terminal part of helix 9, loop 9–10, and a hydrogen-bond distance to the main-chain oxygen atom of R339, the N-terminal part of helix 10 (amino acids 356–376) (Fig. 4B, which should rigidify the conformation of the loop (Fig. 5B Right). lanes 17–21). However, SP2 corresponding to loop 8–9 and the Our results clearly show that the L342T mutation in RAR␣L342T N-terminal part of helix 9 (amino acids 335–351) efficiently dis- resulted in the abrogation of cyclin H binding (Fig. 4D, lane 5). ␣ rupted the interaction between RAR and cyclin H (Fig. 4B, lanes Second, we intended to induce the opposite structural effect by 10–14). constructing a double mutant with P345 and D346 substituted with ␣ To further delineate the cyclin H-binding site in RAR , frag- Gly and Ala residues, respectively (RAR␣P345G͞D346A). Indeed, ments of the RAR␣ LBD were fused to GST and tested for their the substitution of Pro to Gly both releases the constraint and favors ability to interact in vitro with cyclin H. The fragment corresponding the local flexibility of the polypeptide chain. Accordingly, the cyclin to loop 8–9 and helix 9 (amino acids 335–366) retained the cyclin H-binding activity of this mutant was higher than that of the WT H-binding property (Fig. 4C, lane 3). However, deletion of loop 8–9 counterpart (Fig. 4D, lane 4). (in fragment 345–366) decreased cyclin H-binding efficiency (Fig. The major consequences of the L͞T and PD͞GA mutations 4C, lane 4), and further deletion of the first two amino acids of helix suggest an induced-fit interaction (entropically driven) between 9 (in fragment 347–366) abrogated cyclin H binding (Fig. 4C, lane RAR␣ and cyclin H. 5), emphasizing the importance of loop 8–9 and the first amino acids of helix 9 for cyclin H binding. In Vitro and in Vivo Phosphorylation of RARs by cdk7 Depends on The first two amino acid residues of helix 9 (P345 and D346) and Cyclin H͞RAR Complex Formation. Using a monoclonal antibody amino acids Q340, D341, E343, and Q344 of loop 8–9 (Fig. 4A) specific for RAR␣ phosphorylated at S77, the cdk7 phosphor-

Bour et al. PNAS ͉ November 15, 2005 ͉ vol. 102 ͉ no. 46 ͉ 16611 Downloaded by guest on September 23, 2021 Fig. 5. The cyclin H-binding surface. (A) Close-up view of the RAR (yellow)͞ Fig. 6. Cyclin H binding controls RAR␣ phosphorylation. (A) Bacterially RXR (blue) heterodimer and the loop (red) connecting helix 8 and helix 9. (B) expressed RAR␣ was phosphorylated by the cdk7͞cyclin H binary complex Detailed conformation of loop 8–9 (Left). Upon mutation of L342 to threonine puriﬁed from Sf9 extracts, with or without preincubation with increasing (Right), a hydrogen bond is established between the hydroxyl group and the amounts of SP2. RAR␣ phosphorylation was analyzed by immunoblotting with main-chain oxygen atom of R339 (dotted line). In the L342T mutant, this bond, antibodies recognizing RAR␣ or its phosphorylated form at S77 (P-RAR␣). (B) together with proline 345, restrains drastically the ﬂexibility of the loop. Immobilized GST-RAR␣ WT, L342T, and P345G͞D346A proteins were phosphorylated by cdk7͞cyclin H as in A.(C) Equal amounts of COS-1 cell extracts overexpressing RAR␣ WT, P345G͞D346A, or L342T were immunoblotted with ylation site, we found that RAR␣ phosphorylation by the antibodies recognizing RAR␣ or its phosphorylated form. (D) COS-1 cells were ␤ ␣ purified cdk7͞cyclin H complex was markedly inhibited in the cotransfected with the RAR 2-Luc reporter gene, along with RAR WT, S77A, or L342T, and treated with RA for 24 h. Extracts were analyzed for luciferase presence of a 50-fold molar excess of SP2, shown above to disrupt activity. The results represent the mean (ϮSD) of two independent the interaction between cyclin H and the receptor (Fig. 6A, lanes experiments. 3–6). We also investigated the phosphorylation of the RAR␣ L342T and P345G͞D346A mutant proteins by cdk7͞cyclin H. The GST-RAR␣L342T mutant showed a severe drop in S77 targeting of cyclin H did not affect the basal transcription of phosphorylation (Fig. 6B, lane 4), in agreement with its inability RAR␣2 (Fig. 7C) or the transcription of other genes that are RA to bind cyclin H. In contrast, the P345G͞D346A mutant was unresponsive (data not shown), indicating that the observed more efficiently phosphorylated at S77 than RAR␣ WT (Fig. 6B, effect does not reflect an inhibition of general transcription. lane 8), in line with its increased cyclin H-binding capacity. The in vivo phosphorylation patterns are in accordance with Discussion the biochemical results. RAR␣ WT overexpressed in COS-1 Our previous observation that RARs interact with TFIIH raised cells was phosphorylated at S77 (Fig. 6C, lane 1), whereas the the hypothesis that the efficient phosphorylation of these nuclear ␣ ͞ ␣ RAR P345G D346A and RAR L342T mutants were more receptors by cdk7 might result from the recruitment of RARs and less efficiently phosphorylated, respectively (Fig. 6C, lanes through another TFIIH subunit. In the present study, we iden- 2 and 3). Collectively, these results indicate that the efficiency of ␣ ␣ tified cyclin H as the TFIIH subunit interacting with RAR and RAR phosphorylation by cdk7 is modulated by the molecular highlighted the importance of this interaction for RAR phos- recognition of the substrate by cyclin H. Underphosphorylation phorylation and RAR-mediated transcription. We found that of the RAR␣L342T mutant correlates with a deficiency in the cyclin H-binding region in RARs encompasses loop 8–9 and transcription (Fig. 6D, lane 4), in agreement with our previous the N-terminal tip of helix 9 of the LBD domain, a region distal observations that phosphorylation of S77 is crucial for RAR␣ transcriptional activity (8, 11). from the AF-1 domain, which contains the phosphorylation site. Finally, we analyzed the consequences of siRNA-mediated This solvent-exposed polypeptide stretch is part of a helical knock-down of cyclin H in HeLa cells with respect to RAR␣ hairpin motif involved in two salt bridges that define a signature phosphorylation and transactivation. Transfection of siRNA of the class II heterodimeric nuclear receptors (26). We took targeting cyclin H significantly reduced endogenous cyclin H at advantage of modeling considerations to select mutations that the mRNA (Fig. 7A) and protein (Fig. 7B) levels, as shown by should preserve the architecture of the native structure but RT-PCR and immunoblotting, respectively. In these sicyclin H either favor the flexibility or constrain the conformation of this knock-down cells, RAR␣ phosphorylation at S77 was decreased, region. Favoring the flexibility improved complex formation whereas RAR␣ and cdk7 levels were not affected (Fig. 7B). between RAR␣ and cyclin H, likely by facilitating the comple- Consequently, RAR␣-mediated transcription of endogenous mentarity of the protein–protein interface. Constraining the target genes was also decreased, as evidenced by the low conformation was detrimental to complex formation, suggesting RA-induced expression of RAR␣2 transcripts (Fig. 7C). siRNA that the conformational features of loop 8–9 and the N-terminal

16612 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0505556102 Bour et al. Downloaded by guest on September 23, 2021 Similar mechanisms for eliciting substrate specificity have been described for several activators of transcription and other cdk͞ cyclin complexes (12, 13) or mitogen-activated protein kinases (28–30), in agreement with the concept that docking sites allow the recruitment of the right kinase by the right substrate. In that context, it must be noted that the sequence of loop 8–9 and the N-terminal tip of helix 9, the region in RARs involved in cyclin H binding, differs from that of other cyclin docking sites (31). More- over, although it is conserved between the three RAR isotypes, this sequence is not conserved in other cdk7 targets, which do not interact with cyclin H but do interact with other TFIIH subunits. As an example, the estrogen receptor and E2F-1 instead interact with the p62 subunit of the core of TFIIH (32, 33), and the POU domain of octamer factors interacts with the MAT1 subunit of CAK (24). Thus, the diversity in TFIIH subunits appears to provide a clue to the substrate specificity of cdk7. In conclusion, the present study sheds light on mechanisms of regulation of RAR activity through a cooperation between the AF-2 and AF-1 domains, involving interaction with, and phosphorylation by, the cyclin H͞cdk7 complex. This finding raises the question of whether this cooperativity might be altered in certain pathologies. In that context, it would be interesting to investigate whether the deficient phosphorylation of RAR␣ observed in cells Fig. 7. Knock-down of cyclin H with siRNA decreases RAR␣ phosphorylation and RAR␣-mediated transcription. (A and B) Knock-down of cyclin H by siRNA from XPD (xeroderma pigmentosum group D) patients might in HeLa cells as demonstrated by quantitative RT-PCR (A) and by immunoblot- result from an altered docking of cyclin H subsequently to the ting (B). RAR␣ RNA, RAR␣ protein levels, and phosphorylation at S77 and cdk7 mutations in the XPD subunit and an altered architecture of TFIIH levels were analyzed as a control. For protein loading controls, ␤-actin levels (11). Reciprocally, it is not excluded that cyclin H binding might also

were determined after reprobing the membrane. (C) HeLa cells transfected influence the activity of RARs through propagation of allosteric BIOCHEMISTRY with the cyclin H siRNA or untransfected were RA-treated for 3 h, and modifications that may in turn affect the recruitment of coregula- transcripts for RAR␣2 were analyzed by quantitative RT-PCR. The presented tors. Further biochemical and structural studies are required to results are the mean of two individual experiments. determine the possible occurrence and consequences of these structural transitions. tip of helix 9 of the LBD are critical for the recruitment of We thank D. Moras for helpful discussions; J. M. Egly [Institut de cyclin H. Geńe´tique et de Biologie Molećulaireet Cellulaire (IGBMC)] for the We propose that recruitment of the general transcription factor gifts of the cdk7, cyclin H, and MAT1 expression vectors and antibodies; TFIIH at the promoter (27) promotes cyclin H binding to the AF-2 A. Poterszman (IGBMC) for the baculoviruses expressing cyclin H; domain of RARs. This interaction brings the proline-rich motif of N. Rochel (IGBMC) for purified recombinant GST-RAR␣ and GST- the AF-1 domain and the cdk7 active site in position for phosphor- RXR␣ proteins; P. Eberling for preparation of SPs; and I. Kolb-Cheynel ylation of S77 by the kinase, allowing full transcriptional activity of for production of recombinant baculoviruses. We also thank members of the cell culture and oligonucleotide facilities at IGBMC for help and the nuclear receptor. According to this proposal, the cyclin H- ␥ binding region located in the AF-2 domain appears to play a key Bristol-Myers Squibb for the gift of the RAR agonist. This work was ␣ supported by the Centre National de la Recherche Scientifique, the roleinRAR -mediated transcription by directing the phosphory- Institut National de la Santeét de la Recherche Me´dicale (G.B.), the lation of the AF-1 domain by cdk7. To our knowledge, this is the Association pour la Recherche sur le Cancer, the Ministe`redela first example of cooperation between the AF-2 and AF-1 domains Recherche et de l’Enseignement Supe´rieur(E.G.), the Ligue Nationale through kinase complexes. Contre le Cancer (E.G. and N.B.), and the Region Alsace (G.B.).

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Bour et al. PNAS ͉ November 15, 2005 ͉ vol. 102 ͉ no. 46 ͉ 16613 Downloaded by guest on September 23, 2021