Transcription Cofactors of the Thyroid Hormone Receptor

描述（由申请人提供）：TRAP/Mediator复合物包含至少18个不同的亚基，在核激素受体（nr）和其他多种激活剂的转录激活中起重要作用。单个亚基TRAP220作为细胞生长和发育的关键信号介质，它将TRAP/ mediator靶向于NRs和GATA转录因子家族。TRAP220也被认为在整体复合物的组装和稳定性方面起着重要的结构作用，并可能进一步作为其他必要的协同调节因子的结合表面。因此，小鼠的TRAP220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到这些事件的调节。我们进一步假设TRAP220包含对其辅激活子功能至关重要的尚未表征的调控结构域。本提案的长期目标是清楚地了解TRAP220如何被磷酸化以及磷酸化如何调节TRAP220在体内的活性。此外，我们试图更精确地描述TRAP220在全复合体组装和转录激活方面的内在功能域。我们的具体目标是：1)确定TRAP220在体内磷酸化的机制，确定TRAP220磷酸化位点的精确位置。具体的TRAP220 po4位点和相关激酶将通过电迁移位移测定、32-po4结合研究和质谱测定来确定。具体的po4位点和相关的激酶将通过诱变和体外激酶反应来确定。2)研究TRAP220磷酸化的功能后果。将测试TRAP220 po4位点突变体（详见目标1）是否有缺陷的nr结合、转录共激活、亚细胞定位以及与其他辅因子的共定位。我们还将进行细胞同步研究，以检查TRAP220是否显示细胞周期依赖性磷酸化。3)定义完整复合物组装和辅激活子功能所需的TRAP220结构元素。TRAP220缺失和位点定向突变体将通过在培养细胞中瞬时转染和用染色质模板进行体外转录测定来检测共激活的丧失。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TRAP/Mediator亚基。鉴于TRAP/Mediator在全球范围内的重要性，不仅对NR信号传导，而且对其他调控因子的反激活也具有重要意义，因此本文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确定激素依赖性癌症治疗药物的新靶点。

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.