REGULATION OF GLUCOCORTICOID RECEPTOR BY PHOSPHORYLATION

通过磷酸化调节糖皮质激素受体

• 批准号: 2736871
• 负责人: Michael J. Garabedian
• 金额: $ 29.91万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-03-01 至 2003-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2736871
• 关键词: biological signal transduction; cyclin dependent kinase; gene expression; genetic regulation; genetic transcription; glucocorticoids; laboratory rabbit; phosphorylation; protein kinase; protein sequence; steroid hormone receptor; transcription factor

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.

该提案的总体目标是描述 磷酸化调节的分子机制 糖皮质激素受体（GR）活性。 GR是激素依赖性的 转录因子几乎在所有组织中表达，但它 显示出调节细胞类型基因的非凡能力- 具体方式。 虽然糖皮质激素是主要药物 激活 GR 转录调节功能的信号， GR 介导的转录活性还受 磷酸化。 GR 的氨基末端含有 转录激活结构域在四个位点被磷酸化 培养的哺乳动物细胞中的主要位点。 几种激酶具有 已被鉴定在体外磷酸化 GR 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) 和 糖原合酶激酶 3 (GSK3) 磷酸化苏氨酸 171 （T171）。 这些位点的磷酸化对于 GR 很重要 功能：S224和S232的丝氨酸到丙氨酸的突变减少 GR 转录激活，而 T171 的丙氨酸突变 S246 增加 GR 转录激活。 因此，GR- 介导的转录活性受到正向调节 并通过磷酸化产生负作用。 我们建议 多种蛋白激酶的磷酸化使 GR 能够做出反应 不同的细胞外信号。 这种整合能力 磷酸化形式的多个信号允许 GR 作用的灵活性，与类固醇结合 配体，可能对于协调细胞类型特异性至关重要 GR的行动。 我们进一步假设磷酸化 调节 GR 与相关蛋白质的相互作用 转录调控。 该假设将通过 表达 Cdk、JNK 和 GSK3 的激活剂或抑制剂 瞬时转染实验中培养的哺乳动物细胞 旨在监测 GR 依赖性转录调控。 在 此外，我们将鉴定和表征蛋白质 与 GR N 端转录激活结构域相互作用 利用蛋白质相互作用以磷酸化依赖性方式 筛选酵母。 获得对机械的理解 多个信号通路之间的通信，如实现的那样 通过 GR 及其调节激酶，是 了解 GR 调节基因表达的机制 可能会揭示可能的干预点 糖皮质激素耐药新疗法的开发 恶性肿瘤，例如乳腺癌和急性淋巴母细胞癌 白血病。