The nuclear retinoic acid (RA) receptor alpha (RARa) is a transcriptional transregulator that controls the expression of specific gene subsets through binding at response elements and dynamic interactions with coregulators, which are coordinated by the ligand. Here, we highlighted a novel paradigm in which the transcription of RARa target genes is controlled by phosphorylation cascades initiated by the rapid RA activation of the p38MAPK/MSK1 pathway. We demonstrate that MSK1 phosphorylates RARa at S369 located in the ligand-binding domain, allowing the binding of TFIIH and thereby phosphorylation of the N-terminal domain at S77 by cdk7/cyclin H. MSK1 also phosphorylates histone H3 at S10. Finally, the phosphorylation cascade initiated by MSK1 controls the recruitment of RARa/TFIIH complexes to response elements and subsequently RARa target gene activation. Cancer cells characterized by a deregulated p38MAPK/MSK1 pathway, do not respond to RA, outlining the essential contribution of the RA-triggered phosphorylation cascade in RA signalling.

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...

All-trans retinoic acid (ATRA) is the only clinically useful differentiating agent, being used in the treatment of acute promyelocytic leukemia (APL). The use of ATRA in other types of acute myelogenous leukemia (AML) calls for the identification of novel strategies aimed at increasing its therapeutic activity. Here, we provide evidence that pharmacological inhibition of the mitogen-activated protein kinase, p38a, or silencing of the corresponding gene sensitizes APL and AML cell lines, as well as primary cultures of AML blasts to the anti-proliferative and cyto-differentiating activity of ATRA and synthetic retinoids. P38a inhibits ligand-dependent transactivation of the nuclear retinoic acid receptor, RARa, and the derived chimeric protein expressed in the majority of APL cases, PML-RARa. Inhibition is the consequence of ligand-independent binding of p38a, which results in stabilization of RARa and PML-RARa via blockade of their constitutive degradation by the proteasome. The inhibitory effect requires a catalytically active p38a and direct physical interaction with RARa and PML-RARa. Ser-369 in the E-region of RARa is essential for the binding of p38a and the ensuing functional effects on the activity of the receptor.

Arsenite trioxide (As 2 O 3 ) induces apoptosis in several cell lines by disturbing key signal transduction pathways through its oxidative properties. Here, we report that As 2 O 3 also induces the phosphorylation of the retinoid receptor RXRa, subsequent to oxidative damages and the activation of the stress-activated protein kinases cascade (JNKs). We also report that RA amplifies both As 2 O 3 -induced phosphorylation of RXRa and apoptosis. Taking advantage of 'rescue' F9 cell lines expressing RXRa mutated at its phosphorylation sites, in an RXRa null background, we provide evidence that RXRa is a key element involved in that potentiating effect. Finally, we demonstrate that As 2 O 3 also abrogates the transactivation of RA-target genes.

The induction of the granulocytic differentiation of leukemic cells by all-trans retinoic acid (RA) has been a major breakthrough in terms of survival for acute promyelocytic leukemia (APL) patients. Here we highlight the synergism and the underlying novel mechanism between RA and the granulocyte colony-stimulating factor (G-CSF) to restore differentiation of RA-refractory APL blasts. First, we show that in RA-refractory APL cells (UF-1 cell line), PML-RA receptor alpha (RAR␣) is not released from target promoters in response to RA, resulting in the maintenance of chromatin repression. Consequently, RAR␣ cannot be recruited, and the RA target genes are not activated. We then deciphered how the combination of G-CSF and RA successfully restored the activation of RA target genes to levels achieved in RA-sensitive APL cells. We demonstrate that G-CSF restores RAR␣ recruitment to target gene promoters through the activation of the extracellular signalregulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway and the subsequent derepression of chromatin. Thus, combinatorial activation of cytokines and RARs potentiates transcriptional activity through epigenetic modifications induced by specific signaling pathways.

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.