MOLECULAR ENDOCRINOLOGY OF PEPCK GENE REGULATION BY CAMP

这项研究计划的长期目标是确定cAMP 调节基因表达，特别强调 cAMP调节元件（CRE）结合蛋白（CREB）的作用。的 CRE在基础转录和cAMP刺激的转录中都起着至关重要的作用 编码磷酸烯醇丙酮酸羧激酶（PEPCK）的基因， 催化异生过程中的限速步骤， 适当的血糖浓度。蛋白激酶A迅速 通过cAMP激活并磷酸化Ser 133上的CREB，增强其 激活转录的能力。转录起始涉及：1） 组装通用转录因子（GTF）的封闭复合物， RNA聚合酶II在TATA位点; 2）封闭的 复合物转化为能够催化RNA合成的开放复合物， 伴随着起始位点的熔化;以及3）活性的 转录聚合酶或启动子清除。在上一次颁奖时， 我们发现CREB包含不同的结构域， 结构域（CAD）和激酶诱导结构域（KID），其独立地起作用 调节基础和酶诱导的PEPCK基因转录。我们也 显示CREB通过其与GTF，TFIIB和TFIID相互作用， CAD，并映射了可能绑定到不同目标的三个CAD子域 在转录复合物中，以增强其在 启动子其他研究表明，监管因素与CRE有关 和TATA序列的PEPCK基因在体内，即使在缺乏 用cAMP治疗。总之，这些数据表明， 在目前的建议中测试：CREB中的CAD在早期 步骤来帮助组装聚合酶复合物，而 KID引起转录起始速率的快速变化， 影响后期因子的募集，异构化/促进剂 解链步骤，或复合物的分解和启动子被RNA清除 聚合酶II。具体目标是：1）进一步定义组件 与TFIIB和TFIID蛋白相互作用的CAD的 起始复合物，和; 2）确定转录步骤 受CAD和KID影响的初始化，包括分析 闭合和开放复合物的形成以及PEPCK的启动子清除 基因，无论是在核提取物与体外转录测定和 体内肝癌细胞。这些研究将有助于阐明 CREB中的CAD和KID维持PEPCK启动子的机制 然后迅速将信号传输到聚合酶 葡萄糖复合物是对激素刺激的反应，以维持葡萄糖稳态。

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.