Transcription Cofactors of the Thyroid Hormone Receptor

描述(申请人提供)：TRAP/Mediator复合体包含至少18个不同的亚基，并在核激素受体(NRs)和广泛的其他激活剂的转录激活中发挥重要作用。单亚基TRAP220作为细胞生长和发育的关键信号调节因子，靶向于NRs和GATA转录因子家族的TRAP/Mediator。TRAP220还被认为在全复合体组装和稳定性方面发挥着重要的结构作用，并可能进一步作为其他基本共调节因子的结合表面。因此，TRAP220在小鼠体内的消融是胚胎致死的。我们最近发现TRAP220在体内被诱导磷酸化，并怀疑其功能活性受此类事件的调节。我们进一步假设，到目前为止，TRAP220包含对其共激活功能至关重要的未知调控结构域。这项建议的长期目标是清楚地了解TRAP220是如何被磷酸化的，以及磷酸化如何在体内调节TRAP220的活性。此外，我们还试图从全复合体组装和转录激活的角度更准确地描绘TRAP220‘S的内在功能结构域。我们的具体目标是：1)确定TRAP220在体内的磷酸化机制(S)，并确定TRAP220磷酸化位点的准确位置。特定的TRAP220PO4-位点和相关的激酶将通过电迁移率位移分析、32-PO4掺入研究和质谱学来确定。具体的PO4-位点和相关的激酶将通过突变和体外激酶反应来确认。2)研究TRAP220磷酸化的功能后果。TRAP220PO4位点突变体(每个目标1)将被测试有缺陷的NR结合、转录共激活、亚细胞定位以及与其他辅助因子的共定位。我们还将进行细胞同步化研究，以检查TRAP220是否显示细胞周期依赖的磷酸化。3)确定TRAP220全复合体组装和辅激活子功能所必需的结构元件。TRAP220缺失和定点突变将通过在培养细胞中瞬时转染和使用染色质模板进行体外转录试验来测试共激活的丧失。稳定表达的TRAP220缺失突变体也将用于生化鉴定特定的TRAP220相关的TRAP/Mediator亚基。鉴于TRAP/Mediator的全球重要性，不仅对于NR信号转导，而且对于其他调节因子的反式激活，这里的研究应该对整个转录领域具有重要的意义。值得注意的是，这些研究还可能有助于确定和定义治疗药物治疗激素依赖型癌症的新靶点。

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.