REGULATION OF GLUCOCORTICOID RECEPTOR BY PHOSPHORYLATION

通过磷酸化调节糖皮质激素受体

• 批准号: 6350717
• 负责人: Michael J. Garabedian
• 金额: $ 32.53万
• 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/6350717
• 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表征 磷酸化调节的分子机制 糖皮质激素受体（GR）活性。 GR是一种依赖激素 转录因子几乎在所有组织中表达 显示出显着调节细胞类型基因的能力 - 具体方式。 虽然糖皮质激素是主要的 激活GR的转录调节功能时的信号， GR介导的转录活性也受 磷酸化。 GR的氨基末端包含一个 转录激活结构域在四个处磷酸化 培养的哺乳动物细胞中的主要部位。 几个激酶有 已被鉴定在鉴定出的体外磷酸化GR 站点。 其中，细胞周期蛋白依赖性激酶（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 糖原合酶激酶3（GSK3）磷酸化苏氨酸171 （T171）。 这些位点的磷酸化对GR很重要 功能：S224和S232的甲氨酸突变的丝氨酸降低 GR转录激活，而T171的丙氨酸突变 S246增加了GR转录激活。 因此，gr- 介导的转录活性受到正面调节 并因磷酸化而负。 我们提出了这一点 多种蛋白激酶的磷酸化使GR能够反应 到各种细胞外信号。 这种整合的能力 磷酸化形式的多个信号允许A GR动作的灵活性，与类固醇结合 配体，对于配位特定于细胞类型可能至关重要 GR的动作。 我们进一步假设磷酸化 调节GR与涉及的蛋白质的相互作用 转录调节。 这个假设将由 表达CDK，JNK和GSK3的激活剂或抑制剂 瞬时转染测定中培养的哺乳动物细胞 旨在监视GR依赖性的转录调节。 在 此外，我们将确定并表征蛋白质 与GR N末端转录激活结构域相互作用 使用蛋白质相互作用以磷酸化依赖性方式 筛子在酵母中。 对机械理解 正如实现的那样，多个信号通路之间的通信 通过GR及其调节激酶，对 了解GR调节基因表达的机制和 可能揭示了在 开发抗糖皮质激素的新疗法 恶性肿瘤，例如乳腺癌和急性淋巴细胞 白血病。