MOLECULAR ENDOCRINOLOGY OF PEPCK GENE REGULATION BY CAMP

CAMP 对 PEPCK 基因调控的分子内分泌学

• 批准号: 6124892
• 负责人: PATRICK G QUINN
• 金额: $ 20.25万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-05-01 至 2001-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6124892
• 关键词: DNA directed RNA polymerase; cAMP response element binding protein; cyclic AMP; gene expression; genetic promoter element; genetic regulation; genetic regulatory element; genetic transcription; glucose metabolism; laboratory rat; phosphoenolpyruvate carboxylase; protein kinase A; protein structure; tissue /cell culture; transcription factor; transfection; yeast two hybrid system

The long term objective of this research program is to determine how cAMP regulates gene expression, with particular emphasis on the mechanism of action of the cAMP regulatory element (CRE) binding protein (CREB). The CRE plays a crucial role in both basal and cAMP-stimulated transcription of the gene encoding phospho-enolpyruvate carboxykinase (PEPCK), which catalyzes the rate-limiting step in gluconeogenesis to maintain appropriate blood glucose concentrations. Protein kinase A is rapidly activated by cAMP and phosphorylates CREB on Ser 133, enhancing its ability to activate transcription. Transcription initiation involves: 1) assembly of a closed complex of general transcription factors (GTFs) and RNA polymerase II at the TATA site; 2) isomerization of the closed complex to an open complex capable of catalyzing RNA synthesis, accompanied by melting of the start site; and 3) release of the actively transcribing polymerase or promoter clearance. During the previous award, we showed that CREB contains distinct domains, a constitutive activation domain (CAD) and a kinase-inducible domain (KID), that act independently to regulate basal and hormone-induced PEPCK gene transcription. We also showed that CREB interacts with the GTFs, TFIIB and TFIID, through its CAD, and mapped three CAD subdomains that may bind to different targets in the transcription complex to enhance its assembly and stability on the promoter. Others have shown that regulatory factors are bound to the CRE and TATA sequences of the PEPCK gene in vivo, even in the absence of treatment with cAMP. Together, these data suggest the hypothesis to be tested in the current proposal: that the CAD in CREB acts at an early step to help, assemble a polymerase complex, whereas phosphorylation of KID causes a rapid change in the rate of transcription initiation by effecting the recruitment of a late factor, the isomerization/promoter melting step, or disassembly of the complex and promoter clearance by RNA polymerase II. The Specific Aims are: 1) to further define the components of the CAD that interact with the TFIIB and TFIID proteins in the initiation complex, and; 2) to determine the steps in transcription initiation that are affected by the CAD and KID, including analysis of closed and open complex formation and promoter clearance of the PEPCK gene, both in nuclear extracts with in vitro transcription assays and in hepatoma cells in vivo. These studies will help to elucidate the mechanism by which the CAD and KID in CREB maintain the PEPCK promoter in a ready state and then rapidly transmit signals to the polymerase complex in response to hormonal stimuli to maintain glucose homeostasis.

该研究计划的长期目标是确定训练营 调节基因表达，特别强调 CAMP调节元件（CRE）结合蛋白（CREB）的作用。这 CRE在基础和cAMP刺激的转录中起着至关重要的作用 编码磷酸 - 烯醇丙酮酸羧酶（PEPCK）的基因 催化糖异生的速率限制步骤以维持 适当的血糖浓度。蛋白激酶A迅速 被CAMP激活并在Ser 133上磷酸化CREB，从而增强其 激活转录的能力。转录启动涉及：1） 封闭的一般转录因子（GTF）和 TATA位点的RNA聚合酶II； 2）封闭的异构化 复合到一个能够催化RNA合成的开放式复合物， 伴随着开始现场的融化； 3）主动释放 转录聚合酶或启动子清除率。在上一个奖项中， 我们表明CREB包含不同的域，一种本构激活 域（CAD）和激酶诱导型域（KID），独立起作用 调节基底和激素诱导的PEPCK基因转录。我们也是 表明Creb通过其与GTFS，TFIIB和TFIID相互作用 CAD，并映射了三个可能结合不同目标的CAD子域 在转录复合物中，以增强其组装和稳定性 发起人。其他人则表明，调节因素与CRE结合 pepck基因的tata序列在体内，即使在没有的情况下 营地治疗。这些数据一起表明该假设是 在当前的提案中测试：CREB中的CAD在早期起作用 帮助，组装聚合酶复合物，而磷酸化的磷酸化 KID会导致转录率的迅速变化 影响早招的招募，即异构化/启动子 熔化步骤，或通过RNA拆卸复合物和启动子清除率 聚合酶II。具体目的是：1）进一步定义组件 与TFIIB和TFIID蛋白相互作用的CAD 启动综合体，; 2）确定转录的步骤 受CAD和KID影响的启动，包括分析 PEPCK的封闭和开放的复合形成和启动子清除率 基因，既在具有体外转录测定的核提取物中，又 体内肝癌细胞。这些研究将有助于阐明 CREB中的CAD和KID维护PEPCK启动子的机制 以准备状态，然后快速传输信号到聚合酶 响应激素刺激以维持葡萄糖稳态。