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 Ge´nomique Structurales, Institut de Ge´ne´ tique et de Biologie Mole´culaire et Cellulaire, Centre National de la Recherche Scientifique͞Institut National de la Sante´et 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 phosphory- lated 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 per- formed 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.