REGULATION OF GLUCOCORTICOID RECEPTOR BY PHOSPHORYLATION

本提案的总体目标是， 磷酸化调控的分子机制 糖皮质激素受体（GR）活性。 GR是一种依赖于 转录因子几乎在所有组织中表达，但它 显示出调节细胞基因的非凡能力 具体方式。 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 转录激活结构域是磷酸化的四个 在培养的哺乳动物细胞中的主要位点。 有几种激酶 已经确定，在体外磷酸化糖皮质激素受体在确定的 网站. 其中，细胞周期蛋白依赖性激酶（Cdks） 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）， 糖原合成酶激酶-3（GSK 3）磷酸化苏氨酸171 （T171）。 这些位点的磷酸化对于GR 功能：S224和S232的丝氨酸至丙氨酸突变减少 GR转录激活，而T171的丙氨酸突变 和S246增加GR转录激活。 因此，GR- 介导的转录活性受到正向调节， 而磷酸化则呈阴性。 我们建议 多种蛋白激酶的磷酸化使GR能够响应 多种细胞外信号。 这种整合能力 磷酸化形式的多种信号允许 GR作用的灵活性，与类固醇结合， 配体，可能是至关重要的协调细胞类型特异性 我们进一步假设，磷酸化 调节GR与蛋白质的相互作用， 转录调控 这一假设将通过以下方式加以解决： 表达Cdk、JNK和GSK 3的激活剂或抑制剂， 在瞬时转染测定中培养的哺乳动物细胞 用于监测GR依赖的转录调节。 在 此外，我们将鉴定和表征蛋白质， 与GR N-末端转录激活结构域相互作用 以磷酸化依赖的方式使用蛋白质相互作用 在酵母中筛选。 获得机械的理解 多个信号通路之间的通信，如所实现的 通过GR及其调节激酶，是至关重要的， 了解GR调节基因表达的机制， 可能会揭示可能的干预点， 糖皮质激素抵抗的新疗法的开发 恶性肿瘤，如乳腺癌和急性淋巴细胞癌 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.