Nuclear retinoic acid receptors (RARs) are ligand-dependent transcription factors that regulate the expression of retinoic acid target genes. Although the importance of RAR phosphorylation in their N-terminal domain is clearly established, the underlying mechanism for the phosphorylation-dependent transcriptional activity of the receptors had not been elucidated yet. Here, using a yeast two-hybrid system, we report the isolation of vinexin ␤ as a new cofactor that interacts with the N-terminal A/B domain of the RAR␥ isotype. Vinexin ␤ is a multiple SH3 motif-containing protein associated with the cytoskeleton and also present in the nucleus. We demonstrate that vinexin ␤ colocalizes with RAR␥ in the nucleus and interacts with the non-phosphorylated form of the AF-1 domain of RAR␥. We also show that this interaction is prevented upon phosphorylation of the AF-1 domain. Using F9 cells stably overexpressing vinexin ␤ or vinexin knockdown by RNA interference, we demonstrate that vinexin ␤ is an inhibitor of RAR␥-mediated transcription. We propose a model in which phosphorylation of the AF-1 domain controls RAR␥-mediated transcription through triggering the dissociation of vinexin ␤.Retinoic acid (RA), 1 the most potent biologically active metabolite of vitamin A, influences the proliferation, differentiation, and apoptosis of a variety of cell types through modifications of expression of subsets of RA target genes (1-3). The effects of RA are mediated by two classes of nuclear receptors, the retinoic acid receptors (RAR␣, RAR␤, and RAR␥) and the retinoid X receptors (RXR␣, RXR␤, and RXR␥), which function as ligand-dependent heterodimeric RAR/RXR transcription activators (4 -6). RARs and RXRs exhibit a conserved modular structure (see Fig. 1A) with a central DNA-binding domain and two activation domains (AF-1 and AF-2) that synergize for the activation of RA target genes.Ligand-induced conformational changes in the AF-2 domain of RARs bound at cognate response elements (RA response elements) located in the promoter of target genes cause the dynamic, coordinated, and combinatorial recruitment of coactivators and large complexes with chromatin-modifying and chromatin-remodeling activity, which will decompact repressive chromatin to allow positioning of the transcription machinery at the promoter (2, 7). Other proteins are also recruited and serve as connections with the transcription machinery. In line with this, RARs interact with the general transcription factor TFIIH (8, 9). This results in the phosphorylation of one residue located in their N-terminal AF-1 domain (Ser 77 in RAR␣1, Ser 79 in RAR␥1, and Ser 68 in RAR␥2) (see Fig. 1A) by the Cdk7 subunit of TFIIH, which has cyclin H-dependent kinase activity. This phosphorylation process, which has been extensively studied especially in the case of RAR␣ (10), plays a critical role in the response to RA.However, in the particular case of the RAR␥ isotype, phosphorylation by TFIIH, although necessary, is not sufficient. Indeed, to be transcriptionally active, RAR␥ needs...

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Nuclear retinoic acid receptors (RARs) work as ligand-dependent heterodimeric RAR͞retinoid X receptor transcription activators, which are targets for phosphorylations. The N-terminal activation function (AF)-1 domain of RAR␣ is phosphorylated by the cyclindependent kinase (cdk) 7͞cyclin H complex of the general transcription factor TFIIH and the C-terminal AF-2 domain by the cAMP-dependent protein kinase A (PKA). Here, we report the identification of a molecular pathway by which phosphorylation by PKA propagates cAMP signaling from the AF-2 domain to the AF-1 domain. The first step is the phosphorylation of S369, located in loop 9 -10 of the AF-2 domain. This signal is transferred to the cyclin H binding domain (at the N terminus of helix 9 and loop 8 -9), resulting in enhanced cyclin H interaction and, thereby, greater amounts of RAR␣ phosphorylated at S77 located in the AF-1 domain by the cdk7͞cyclin H complex. This molecular mechanism relies on the integrity of the ligand-binding domain and the cyclin H binding surface. Finally, it results in higher DNA-binding efficiency, providing an explanation for how cAMP synergizes with retinoic acid for transcription.cAMP͞nuclear retinoid receptors R etinoic acid (RA) has essential roles in cell growth and differentiation (1) and 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 RAR͞ RXR transcription activators (2-4). During the last decade, the molecular rationale for RAR and RXR action has been established by the determination of the crystal structure of the ligand-binding domain (LBD) (5) and by evidence that RAR and RXR are phosphoproteins (6).The LBD is composed of 11 ␣-helices (H1 and H3-H12) that form a compact structure. It is functionally complex, containing the ligand-binding pocket, the main dimerization domain, and activation function (AF)-2 (Fig. 1A). Ligand binding promotes allosteric effects, such as the propagation of signals across the heterodimerization surface (7,8). It also induces conformation changes, the most striking one being the swing of helix 12 (9-11), which leads to corepressor complex dissociation (12). It also generates a new interaction surface for coactivators, which then recruit a battery of chromatin remodelers and modifiers acting in a coordinated and͞or combinatorial manner to decompact chromatin and direct RNA polymerase II and the general transcription factors to the promoter (13-15), leading to transcription initiation.In the last several years, it has been demonstrated that RARs are also targeted for phosphorylation processes (Fig. 1 A), which regulate their transcriptional activity (6). 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 th...

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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...

The transcriptional activity of nuclear retinoic acid receptors (RARs) relies on the association/dissociation of coregulators at the ligand-binding domain. However, we determined that the N-terminal domain (NTD) also plays a role through its phosphorylation, and we isolated vinexinβ, a cytoskeleton protein with three SH3 domains, as a new partner of the RARγ NTD. Here we deciphered the mechanism of the interaction and its role in RARγ-mediated transcription. By combining molecular and biophysical (surface plasmon resonance, NMR, and fluorescence resonance energy transfer) approaches, we demonstrated that the third SH3 domain of vinexinβ interacts with a proline-rich domain (PRD) located in RARγ NTD and that phosphorylation at a serine located in the PRD abrogates the interaction. The affinity of the interaction was also evaluated. In vivo, vinexinβ represses RARγ-mediated transcription and we dissected the underlying mechanism in chromatin immunoprecipitation experiments performed with F9 cells expressing RARγ wild type or mutated at the phosphorylation site. In the absence of retinoic acid (RA), vinexinβ does not occupy RARγ target gene promoters and sequesters nonphosphorylated RARγ out of promoters. In response to RA, RARγ becomes phosphorylated and dissociates from vinexinβ. This separation allows RARγ to occupy promoters. This is the first report of an RAR corepressor association/dissociation out of promoters and regulated by phosphorylation.

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