REGULATION OF GLUCOCORTICOID RECEPTOR BY PHOSPHORYLATION

这项提案的总体目标是描述 磷酸化调控的分子机制 糖皮质激素受体活性。GR是一种激素依赖型激素 转录因子几乎在所有组织中都有表达，但它 显示出非凡的能力来调节一种细胞类型的基因- 具体的方式。虽然糖皮质激素是主要的 激活GR转录调节功能的信号， GR介导的转录活性也受 磷酸化。GR的氨基末端含有一个 转录激活结构域在第4位被磷酸化 培养的哺乳动物细胞中的主要部位。有几种激酶具有 已在体外鉴定出磷酸化GR 网站。其中，细胞周期蛋白依赖性蛋白激酶(CDK) 磷酸化丝氨酸232(S232)和丝氨酸(S224)，而c-jun N- 末端激酶(JNK)磷酸化丝氨酸246(S246)和 糖原合成酶激酶-3(GSK3)使苏氨酸171磷酸化 (T171)。这些位点的磷酸化对GR很重要 功能：S224和S232丝氨酸到丙氨酸突变减少 GR转录激活，而T171的丙氨酸突变 和S246增强GR转录活性。因此，GR- 介导的转录活性既受正向调节 以及通过磷酸化产生的负效应。我们建议 多个蛋白激酶的磷酸化使GR能够 不同的细胞外信号。这种整合的能力 磷酸化形式的多个信号允许 GR作用的灵活性，与类固醇联合使用 配体，可能在协调细胞类型特异性中起关键作用 GR的行动。我们进一步假设，磷酸化 调节GR与相关蛋白的相互作用 转录调控。这一假设将通过以下方式得到解决 CDK、JNK和GSK3激活剂或抑制物在大肠杆菌中的表达 瞬时转染法中培养的哺乳动物细胞 旨在监控依赖GR的转录调控。在……里面 此外，我们还将鉴定和鉴定那些 与GR N末端转录激活域相互作用 使用蛋白质相互作用以依赖于磷酸化的方式 在酵母中筛选。从机械论的角度理解 如所实现的，多个信令路径之间的通信 通过GR及其调节激酶，是 了解糖皮质激素受体调节基因表达的机制 可能会揭示可能被利用的干预点 糖皮质激素耐药新疗法的研究进展 恶性肿瘤，如乳腺癌和急性淋巴母细胞瘤 白血病。

The overall objectives of this proposal are to characterize the molecular mechanisms by which phosphorylation regulates glucocorticoid receptor (GR) activity. GR is a hormone-dependent transcription factor expressed in virtually all tissues, yet it displays a remarkable capacity to regulate genes in a cell type- specific manner. Although glucocorticoids act as the primary signal in activating GR's transcriptional regulatory functions, GR-mediated transcriptional activity is also regulated by phosphorylation. The amino terminus of GR contains a transcriptional activation domain that is phosphorylated at four major sites in cultured mammalian cells. Several kinases have been identified that phosphorylate GR in vitro at the identified sites. Of these, the cyclin-dependent kinases (Cdks) phosphorylate serine 232 (S232) and serine (S224), while c-Jun N- terminal kinase (JNK) phosphorylates serine 246 (S246) and glycogen synthase kinase-3 (GSK3) phosphorylates threonine 171 (T171). Phosphorylation of these sites is important for GR function: serine to alanine mutations of S224 and S232 decrease GR transcriptional activation, whereas alanine mutations of T171 and S246 increase GR transcriptional activation. Thus, GR- mediated transcriptional activity is regulated both positively and negatively by phosphorylation. We propose that phosphorylation by multiple protein kinases enables GR to respond to diverse extracellular signals. This ability to integrate multiple signals in the form of phosphorylation permits a flexibility in GR action that, in conjunction with the steroid ligands, may be crucial in coordinating the cell type specific actions of GR. We further hypothesize that phosphorylation regulates GR's interaction with proteins involved in transcriptional regulation. This hypothesis will be addressed by expressing activators or inhibitors of Cdk, JNK and GSK3 in cultured mammalian cells in transient transfection assays designed to monitor GR-dependent transcriptional regulation. In addition, we will identify and characterize proteins that interact with the GR N-terminal transcriptional activation domain in a phosphorylation-dependent manner using a protein interaction screen in yeast. Gaining a mechanistic understanding of the communication between multiple signaling pathways, as realized through GR and its regulatory kinases, is fundamental to understanding the mechanism of GR-regulated gene expression and may reveal likely points of intervention to be exploited in the development of new therapies for glucocorticoid-resistant malignancies, such as breast cancer and acute lymphoblastic leukemia.