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The AF-1 and AF-2 Activating Domains of Retinoic Acid Receptor- (RAR) and Their Phosphorylation Are Differentially Involved in Parietal Endodermal Differentiation of F9 Cells and Retinoid-Induced Expression of Target Genes

Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP/Collège de France BP 163, 67404 ILLKIRCH Cedex CU de Strasbourg, France

Retinoic acid (RA) induces the differentiation of F9 cells cultured as monolayers into primitive endodermal-like cells, whereas a combination of RA and cAMP leads to parietal endodermal differentiation. In RA receptor -null F9 cells (RAR^-/- cells), RA still efficiently triggers RAR-mediated primitive endodermal differentiation, but parietal endodermal differentiation is markedly delayed. To investigate the role of RAR1 activation functions AF-1 and AF-2 and of their phosphorylation sites during RA- and cAMP-induced parietal differentiation, cell lines reexpressing WT or mutated RAR1 were established in RAR^-/- cells. We have found that the protein kinase A (PKA) phosphorylation site and the AF-2AD core (helix 12) of RAR1 are required for efficient parietal endodermal differentiation, whereas the AF-1 proline-directed kinase phosphorylation site is dispensible. Interestingly, deletion of the AF-1 activating domain (the A/B region), but not of the AF-2AD core, generates a dominant negative mutant that abrogates primitive endodermal differentiation when expressed in RAR^-/- cells. We also show that the RAR AF-1 and AF-2 activation functions, but not their phosphorylation sites, are involved in the induction of RA-responsive genes in a differential promoter context-dependent manner. is the most potent biologically active form of vitamin A. RA exhibits a wide range of activities and influences the proliferation and differentiation of a variety of cell types. In that context, F9 embryonal carcinoma (EC) cells represent a well established cell-autonomous model system for investigating retinoid signaling in vivo, as upon RA treatment and depending on culture conditions, they differentiate into three distinct cell types resembling primitive, parietal, and visceral endodermal extraembryonic cells (1–4). RA- induced differentiation of F9 EC cells is accompanied by an apoptotic response and a decrease in the rate of proliferation, as well as by the induction of expression of a number of genes (3, 5–7).

RA exerts its pleiotropic effects through two classes of nuclear ligand-dependent transregulators: the retinoic acid receptors (RAR, RARß, and RAR isotypes and their isoforms) activated by either all-trans-RA or its 9-cis-isomer and the retinoid X receptors (RXR, RXRß and RXR) activated by 9-cis-RA only (8–11). F9 cells express all RARs and RXRs (12–14), and two strategies have been used to investigate their roles in the response of F9 EC cells to RA treatment. Firstly, using homologous recombination, we engineered F9 cells in which either the RAR, the RAR, or the RXR gene, both the RAR and RXR genes or both the RAR and RXR genes, are knocked out (6, 7, 15, 16). Secondly, wild-type (WT) and mutant F9 cells were treated with pan-RXR- and RAR isotype (, ß, or )-selective retinoids (7, 17–20). It was established that RAR is indispensible for RA-induced differentiation of F9 EC cells into primitive endoderm-like cells, whereas RAR is additionally required for efficient parietal endodermal differentiation that occurs in the presence of RA and cAMP (20), and for which primitive endodermal differentiation is a prerequisite. These studies also demonstrated that RAR/RXR heterodimers are the functional units transducing the retinoid signal in vivo (7, 19).

RARs and RXRs possess two transcriptional activation functions (AFs): AF-1, located in the N-terminal A/B region, and AF-2, associated with the ligand binding domain (LBD) (region E) and activated by agonistic ligands (8, 21–24). The integrity of a conserved amphipatic -helix, referred to as the AF-2AD core, is required for AF-2 activity (23, 25–28, and references therein). The AF-2AD core is located in the C-terminal helix of the LBD (helix 12) that is indispensible for the formation of the coactivator-binding surface generated during the LBD transconformation triggered by ligand binding (8, 29–31).

In addition, RARs and RXRs are phosphorylated in their AF-1 domain that contains sites for proline- directed kinases (20, 32, 33). In RAR1, the phospho-rylated residue in the AF-1domain has been identified as serine 77 (32). Furthermore, RAR1 can be phosphorylated by PKA at serine 369 that is located in the LBD/AF-2 domain (34). Interestingly, RAR1 is phosphorylated at these two residues in F9 cells, as well as in transfected COS cells (20, 34). Similar phospho-rylation sites are present in other RARs and have been shown to be phosphorylated in RAR2, which is the major RAR isoform in F9 cells (34, 35). The role of RAR phosphorylation in retinoid-induced events has been studied in RAR^-/- F9 cells, by establishing rescue cell lines reexpressing either RARWT or RAR mutated at its phosphorylation sites (20). The RAR AF-1 domain and the proline-directed phosphorylation sites located in this domain were found to be required for rescuing the differentiation of F9 cells into primitive endoderm-like cells, whereas the PKA phosphorylation site was dispensible (20). As we had previously established that overexpression of RAR in RAR^-/- cells could also restore their primitive and parietal endodermal differentiation (14), the role of RAR phosphorylation was similarly studied using overexpressed RAR mutants. We concluded that both the RAR AF-1 and AF-2 phosphorylation sites were not required for allowing overexpressed RAR to functionally replace RAR for primitive endodermal differentiation, but they were apparently mandatory for parietal differentiation (20).

Thus, RAR and its phosphorylation sites could be selectively required for parietal endodermal differentiation. However, an unequivocal demonstration of this RAR-selective function requires to establish rescue cell lines reexpressing RAR (either WT or mutated) in RAR^-/- F9 EC cells that can still differentiate into primitive endodermal cells (16) but whose parietal endodermal differentiation is greatly delayed (20), thus providing a model for directly analyzing the contribution of RAR AF-1 and AF-2 and of their phospho-rylation sites in parietal endodermal differentiation. Furthermore, such lines also offer the possibility to study which RAR mutations may generate dominant negatives, preventing endogenous RAR from mediating primitive endodermal differentiation. Rescue lines were therefore established with RAR mutated at either one of its phosphorylation sites, as well as with AF-1 or AF-2AD core deletion mutants. We demonstrate here that the PKA phosphorylation site and the AF-2AD core (helix 12 of the LBD) of RAR are required for parietal endodermal differentiation, whereas the AF-1 proline-directed kinase phosphorylation site is dispensible. None of the corresponding mutants behaved as dominant negatives, preventing primitive endodermal differentiation, whereas deletion of the AF-1 activating domain (the A/B region) generated such a dominant negative mutant. The present study also shows that RAR AF-1 and AF-2 are differentially implicated in the induction of RA-responsive genes, whereas their phosphorylation sites are not involved.

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