MOLECULAR ENDOCRINOLOGY OF PEPCK GENE REGULATION BY CAMP

这项研究计划的长期目标是确定坎普如何 调节基因表达，特别强调 CAMP调节元件(CRE)结合蛋白(CREB)的作用。这个 Cre在基础转录和cAMP刺激的转录中都起着关键作用。 编码磷酸烯醇式丙酮酸羧激酶(PEPCK)的基因 催化糖异生中的限速步骤以维持 适当的血糖浓度。蛋白激酶A迅速 CAMP激活并磷酸化Ser133上的CREB，增强其 激活转录的能力。转录启动包括：1) 组装通用转录因子(GTF)和封闭的复合体 TATA位点的RNA聚合酶II；2)闭合的异构化 能够催化RNA合成的开放的络合物， 伴随着起始点的熔融；3)活性的释放 转录聚合酶或启动子清除。在上一次颁奖期间， 我们证明了CREB包含不同的结构域，这是一种结构性激活 结构域(CAD)和激酶诱导结构域(KID)，它们独立发挥作用 调节基础和激素诱导的PEPCK基因转录。我们也 显示CREB与GTF、TFIIB和TFIID通过其 CAD，并映射了可能绑定到不同目标的三个CAD子域 在转录复合体中增强其在 推动者。其他人则表明，监管因素与CRE有关 和体内PEPCK基因的TATA序列，即使在缺乏 用夏令营治疗。总而言之，这些数据表明，假设是 在目前的方案中得到了检验：CREB中的CAD在早期作用 步骤帮助，组装一个聚合酶复合体，而磷酸化 KID通过以下方式引起转录启动速度的快速变化 影响后期因子--异构化/启动子的招募 熔融步骤，或通过RNA拆解复合体和启动子清除 聚合酶II.具体目标是：1)进一步定义成分 与TFIIB和TFIID蛋白相互作用的CAD的 起始复合体，以及；2)确定转录的步骤 受CAD和KID影响的启动，包括分析 PEPCK闭合和开放复合体的形成及启动子清除 基因，无论是在核提取的体外转录实验中，还是在 体内的肝癌细胞。这些研究将有助于阐明 CREB中CAD和KID维持PEPCK启动子的机制 处于就绪状态，然后将信号快速传输到聚合酶 对荷尔蒙刺激作出反应的复合体，以维持葡萄糖动态平衡。

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.