Rochette-Egly C, Plassat J L, Taneja R, Chambon P
Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP/College de France, Illkirch, CU de Strasbourg.
Mol Endocrinol. 2000 Sep;14(9):1398-410. doi: 10.1210/mend.14.9.0527.
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.
视黄酸（RA）可诱导单层培养的F9细胞分化为原始内胚层样细胞，而RA与cAMP联合使用则会导致壁内胚层分化。在视黄酸受体α基因缺失的F9细胞（RARα-/-细胞）中，RA仍能有效地触发RARγ介导的原始内胚层分化，但壁内胚层分化明显延迟。为了研究RARα1激活功能AF-1和AF-2及其磷酸化位点在RA和cAMP诱导的壁内胚层分化过程中的作用，我们在RARα-/-细胞中建立了重新表达野生型或突变型RARα1的细胞系。我们发现，RARα1的蛋白激酶A（PKA）磷酸化位点和AF-2AD核心（螺旋12）是壁内胚层高效分化所必需的，而AF-1脯氨酸定向激酶磷酸化位点则是可有可无的。有趣的是，缺失AF-1激活结构域（A/B区域）而非AF-2AD核心，会产生一个显性负突变体，当在RARα-/-细胞中表达时，该突变体会消除原始内胚层分化。我们还表明，RARα的AF-1和AF-2激活功能而非其磷酸化位点，以启动子背景依赖性方式参与RA反应基因的诱导。
