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

Abstract

The transcriptional activity of nuclear retinoic acid receptors (RARs), which act as RAR͞retinoid X receptor (RXR) heterodimers, depends on two activation functions, AF-1 and AF-2, which are targets for phosphorylations and synergize for the activation of retinoic acid target genes. The N-terminal AF-1 domain of RAR␣ is phosphorylated at S77 by the cyclin-dependent kinase (cdk)-activating kinase (CAK) subcomplex (cdk7͞cyclin H͞MAT1) of the general transcription factor TFIIH. Here, we show that phosphorylation of S77 governing the transcriptional activity of RAR␣ depends on cyclin H binding at a RAR␣ region that encompasses loop 8 -9 and the N-terminal tip of helix 9 of the AF-2 domain. We propose a model in which the structural constraints of this region control the architecture of the RAR͞RXR͞TFIIH complex and therefore the efficiency of RAR␣ phosphorylation by cdk7. To our knowledge, this study provides the first example of a cooperation between the AF-2 and AF-1 domains of RARs through a kinase complex.retinoic acid receptor ͉ transcription R etinoic acid (RA) regulates the expression of specific networks of genes through two families of nuclear receptors, the RA receptors (RARs) (␣, ␤, and ␥) and the retinoid X receptors (RXRs) (␣, ␤, and ␥), which act as ligand-dependent heterodimeric RAR͞RXR transcription activators (1, 2). The transcriptional activity of RARs depends on activation function (AF) 1 and AF-2, which synergize for the activation of RA target genes. The Cterminal AF-2 domain encompasses the ligand-binding domain (LBD), consisting of 11 ␣-helices (H1 and H3-H12) forming a compact structure (3, 4). Ligand binding promotes complex allosteric effects and conformational changes, the most striking one being the swing of helix 12 (5, 6), which leads to dissociation of corepressor complexes. It also generates an interaction surface for coactivators, which then recruit a battery of intermediary proteins, including chromatin remodellers and modifiers. They act in a coordinated and͞or combinatorial manner to decompact chromatin and direct the RNA polymerase II and the general transcription factors to the promoter (7), leading to the activation of the RA responsive genes.In the last several years, researchers have witnessed additional layers of regulation of transcription by RARs through their Nterminal AF-1 domain, which is targeted by phosphorylation processes. Indeed, the RAR␣1 and RAR␥1 isotypes are phosphorylated in their AF-1 domain at S77 and S79, respectively (Fig. 1A), by the cyclin-dependent kinase (cdk)-activating kinase (CAK) complex of the general transcription factor TFIIH (8, 9). TFIIH consists of 10 subunits (10) assembled into two subcomplexes: the core complex and CAK (composed of the cdk7, cyclin H, and MAT1 subunits). The key role of this phosphorylation process in the RA response has been highlighted by studies performed with patients carrying a mutation in one subunit [XPD (xeroderma pigmentosum group D)] of the core of TFIIH (11). This mutation altering the architecture of TFIIH resu...

Phosphorylation by PKA potentiates retinoic acid receptor α activity by means of increasing interaction with and phosphorylation by cyclin H/cdk7

“…We previously demonstrated that cyclin H binding controls the phosphorylation of the AF-1 domain at S77 by cdk7 (18). Therefore, the above observations predicted that S369 phosphorylation would enhance the ability of RAR␣ to be phosphorylated at S77.…”

“…The prokaryotic vectors encoding RAR␣ and RXR␣ in the pGEX-2T plasmid, as well as the baculoviruses expressing cyclin H or hRAR␣1WT as a FLAG fusion protein, are described in ref. 18. Baculoviruses expressing hRAR␣S369A and S369E were constructed by subcloning the SacI͞BamHI fragment from the same mutants in pSG5 into the same sites of pSK278-hRAR␣.…”

“…The pSG5-based expression vectors for human (h) RAR␣1 WT, S77A, or L342T, as well as the DR5-tk-CAT reporter gene, are described in ref. 18. hRAR␣S369A and S369E in pSG5 were constructed by double PCR amplification reactions to generate a SacI͞BamHI fragment containing the appropriate mutation.…”

“…The LBD of the RAR␣ isotype can be phosphorylated at S369, located in loop 9 -10 (16), by the cAMP-dependent protein kinase A (PKA), whereas the N-terminal AF-1 domain is phosphorylated at S77 (17) by the cyclin-dependent kinase (cdk)-activating kinase complex of the general transcription factor TFIIH, which is composed of the kinase cdk7, cyclin H, and MAT1 subunits. Recently, we demonstrated that phosphorylation by the cdk7 kinase is controlled by the binding of cyclin H to a specific region of the RAR␣ LBD corresponding to loop 8 -9 and the N terminus of helix 9 (18) (Figs. 1B and 2 A).…”

“…Insect Sf9 cells were infected with baculoviruses expressing cyclin H or FLAG-RAR␣ either alone or in combination, and extracts were prepared as described in ref. 18. For coimmunoprecipitations, aliquots were incubated with Sepharose beads cross-linked with anti-FLAG mAbs in lysis buffer (20 mM Tris⅐HCl, pH 7.8͞100-500 mM KCl͞10% glycerol͞0.1 mM EDTA͞1 mM DTT͞0.1% Nonidet P-40).…”