The AF-1 and AF-2 activating domains of retinoic acid receptor-alpha (RARalpha) and their phosphorylation are differentially involved in parietal endodermal differentiation of F9 cells and retinoid-induced expression of target genes.
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 alpha-null F9 cells (RARalpha-/- cells), RA still efficiently triggers RARgamma-mediated primitive endodermal differentiation, but parietal endodermal differentiation is markedly delayed. To investigate the role of RARalpha1 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 RARalpha1 were established in RARalpha-/- cells. We have found that the protein kinase A (PKA) phosphorylation site and the AF-2AD core (helix 12) of RARalpha1 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 RARalpha-/- cells. We also show that the RARalpha 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.
Rochette-Egly
C
C
Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP/College de France, Illkirch, CU de Strasbourg. cegly@igbmc.u-strasbg.fr
Plassat
J L
JL
Taneja
R
R
Chambon
P
P
D000818:Animals; D001665:Binding Sites; D002454:Cell Differentiation; D002460:Cell Line; D000242:Cyclic AMP / Q000502:physiology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004707:Endoderm / Q000166:cytology / Q000502:physiology; D051379:Mice; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011965:Receptors, Glucocorticoid / Q000378:metabolism; D017471:Receptors, Interferon / Q000235:genetics / Q000378:metabolism*; D018168:Receptors, Retinoic Acid / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D000072482:Retinoic Acid Receptor alpha; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014212:Tretinoin / Q000494:pharmacology
C490116:Ifngr2 protein, mouse; D009687:Nuclear Proteins; C000607072:Rara protein, mouse; D011965:Receptors, Glucocorticoid; D017471:Receptors, Interferon; D018168:Receptors, Retinoic Acid; D011994:Recombinant Proteins; D000072482:Retinoic Acid Receptor alpha; D014212:Tretinoin; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases