Transcription Cofactors of the Thyroid Hormone Receptor

描述（由申请人提供）：TRAP/介体复合物含有至少18种不同的亚基，并在核激素受体（NR）以及广泛的其他激活剂的转录激活中发挥重要作用。单个亚基TRAP 220作为细胞生长和发育的关键信号传导介体，因为它将TRAP/介体靶向NR和转录因子的加塔家族。TRAP 220也被认为在全复合物组装和稳定性方面发挥重要的结构作用，并且可以进一步充当其他必需共调节因子的结合表面。因此，小鼠中的TRAP 220消融是胚胎致死的。我们最近发现TRAP 220在体内可诱导磷酸化，并怀疑其功能活性受这些事件的调节。我们进一步假设，TRAP 220包含尚未表征的调控结构域，其辅激活因子功能的关键。该提案的长期目标是清楚地了解TRAP 220如何磷酸化以及磷酸化如何调节体内TRAP 220活性。此外，我们试图更精确地描绘TRAP 220的内在功能域的整体组装和转录激活。我们的具体目标是：1）鉴定TRAP 220在体内磷酸化的机制并确定TRAP 220磷酸化位点的精确位置。将使用电迁移率变动试验、32-po 4-掺入研究和质谱法测定特异性TRAP 220 PO 4-位点和相关激酶。特异性PO 4位点和相关激酶将通过诱变和体外激酶反应进行确认。2)研究TRAP 220磷酸化的功能后果。将检测TRAP 220 PO 4位点突变体（根据目的1）的NR结合缺陷、转录共激活、亚细胞定位和与其他辅因子的共定位。我们还将进行细胞同步化研究，以检查TRAP 220是否显示细胞周期依赖性磷酸化。3)确定TRAP 220的结构元件，这些元件是全复合物组装和辅激活因子功能所必需的。将通过在培养细胞中瞬时转染和通过使用染色质模板的体外转录测定来检测TRAP 220缺失和定点突变体的共激活丧失。稳定表达的TRAP 220缺失突变体也将用于生物化学鉴定特异性TRAP 220相关TRAP/介体亚基。鉴于TRAP/Mediator的全球重要性，不仅对于NR信号传导，而且对于其他调节因子的反式激活，这里的研究应该对转录领域具有重要意义。值得注意的是，这些研究也可能有助于确定和定义治疗药物治疗依赖性癌症的新靶点。

DESCRIPTION (provided by applicant): The TRAP/Mediator complex contains at least 18 different subunits and plays an essential role in transcriptional activation by nuclear hormone receptors (NRs) as well as a broad range of other activators. A single subunit, TRAP220, acts as a pivotal signaling mediator of cellular growth and development in that it targets TRAP/Mediator to NRs and to the GATA family of transcription factors. TRAP220 is also thought to play an important structural role in terms of holocomplex assembly and stability, and may further act as a binding surface for other essential coregulatory factors. Accordingly, TRAP220 ablation in mice is embryonic lethal. We recently found that TRAP220 is inducibly phosphorylated in vivo and suspect that its functional activity is regulated by such events. We further hypothesize that TRAP220 contains as of yet uncharacterized regulatory domains critical for its coactivator function. The long-term objectives of this proposal are to clearly understand how TRAP220 becomes phosphorylated and how phosphorylation regulates TRAP220 activity in vivo. Furthermore, we seek to more precisely delineate TRAP220's intrinsic functional domains in terms of holocomplex assembly and transcriptional activation. Our specific goals are: 1) To identify the mechanism(s) of TRAP220 phosphorylation in vivo and to determine the precise location of TRAP220 phosphorylation sites. Specific TRAP220 PO4-sites and relevant kinases will be determined using electromobility shift assays, 32-po4-incorporation studies, and mass spectrometry. The specific PO4-sites and relevant kinases will be confirmed via mutagenesis and in vitro kinase reactions. 2) To investigate the functional consequences of TRAP220 phosphorylation. TRAP220 PO4-site mutants (per aim 1) will be tested for defective NR-binding, transcriptional coactivation, subcellular localization and colocalization with other cofactors. We will also carry out cell synchronization studies to examine whether TRAP220 displays cell cycle-dependent phosphorylation. 3) To define the structural elements of TRAP220 necessary for holocomplex assembly and coactivator function. TRAP220 deletion and site-directed mutants will be tested for loss of coactivation by transient transfection in cultured cells and by in vitro transcription assays with chromatin templates. Stably expressed TRAP220 deletion mutants will also be used to biochemically identify specific TRAP220-associated TRAP/Mediator subunits. Given the global importance of TRAP/Mediator, not only for NR signaling but also for transactivation by other regulatory factors, the studies here should have important implications for the transcription field in general. Significantly, these studies may also help to identify and define new targets for therapeutic agents in the treatment of hormone-dependent cancers.