MOLECULAR ENDOCRINOLOGY OF PEPCK GENE REGULATION BY CAMP

这里描述的研究计划的长期目标是 了解cAMP如何调节基因表达，特别强调 cAMP反应元件（CRE）结合蛋白的作用机制 （CREBP）和调节CREBP表达的因子的相互作用， 磷酸烯醇丙酮酸羧激酶（PEPCK）基因。 综合招聘考试是一项重要的 在PEPCK基因的基础和cAMP刺激的转录中起作用。 调节PEPCK的表达，其催化细胞凋亡中的限速步骤。 葡萄糖代谢是维持适当血糖的关键 浓度的 基因转录是由以下物质的相互作用调节的： 相互作用的蛋白质因子和RNA聚合酶， 通过这些因子与顺式作用DNA元件的结合而促进。 环状 AMP激活蛋白激酶A，导致几个磷酸化的蛋白激酶A的磷酸化。 蛋白质，包括CRE结合蛋白。 CREBP最近在 纯化、克隆并显示直接参与基因调控 表情 为了了解CREBP如何与其他因素相互作用， 基因表达，有必要确定CREBP的结构域， 参与转录激活。 CREBP作为二聚体与CRE结合， 同种型相差14个氨基酸，具有不同的转录 由于CREBP的选择性剪接， mRNA。 目的1是确定CREBP二聚化的意义 异构体的反式激活，并确定是否量或 异构体的分布受到调节。 这将通过检查 每种肽单独的贡献与相同肽的相互作用相比， 和不同的亚型，并通过确定CREBP是否 合成或剪接受激素调节。 目标2是确定 CREBP的功能结构域参与基础和cAMP的激活， 受激转录 这将通过创造特定的突变来实现 在CREBP cDNA和融合潜在的激活结构域的GAL 4 DNA结合域。 突变的CREBP的反式激活能力 蛋白和CREBP/GAL 4融合因子的表达将在转染中进行评估 和体外转录测定。 目标3是识别和表征 CRE与异源启动子元件的相互作用，特别是 参与PEPCK调控的人员。 这将确定潜在的 CREBP相互作用的目标。 需要的CREBP域 不同转录因子之间的相互作用可以通过 检查不同CREBP变体和融合因子的能力， 通过这些其他因素增强激活。

The long-term objective of the research program described here is to understand how cAMP regulates gene expression with particular emphasis on the mechanism of action of the cAMP response element (CRE) binding protein (CREBP) and the interplay of factors regulating expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene. The CRE plays an essential role in both basal and cAMP-stimulated transcription of the PEPCK gene. Regulated expression of PEPCK, which catalyzes the rate-limiting step in gluconeogenesis, is essential for maintaining appropriate blood glucose concentrations. Gene transcription is regulated by the interaction of trans-acting protein factors with each other and RNA polymerase, which is facilitated by binding of these factors to cis-acting DNA elements. Cyclic AMP activates protein kinase A, resulting in phosphorylation of several proteins, including the CRE binding protein. CREBP has recently been purified, cloned and shown to be directly involved in regulation of gene expression. In order to understand how CREBP interacts with other factors to regulate gene expression, it is necessary to identify the domains of CREBP that are involved in transactivation. CREBP binds to the CRE as a dimer, and two isoforms that differ by 14 amino acids and possess distinct transcriptional capacities are present in cells due to alternative splicing of the CREBP mRNA. Aim 1 is to determine the significance of the dimerization of CREBP isoforms for transactivation and to establish whether the amount or distribution of isoforms is regulated. This will be done by examining the contribution of each peptide alone compared to the interactions of the same and different isoforms in a dimer and by determining whether CREBP synthesis or splicing is regulated by hormones. Aim 2 is to identify the functional domains of CREBP involved in activation of basal and cAMP- stimulated transcription. This will be done by creating specific mutations in the CREBP cDNA and by fusing potential activation domains to the GAL4 DNA-binding domain. The transactivational capacity of mutated CREBP proteins and of CREBP/GAL4 fusion factors will be assessed in transfection and in vitro transcription assays. Aim 3 is to identify and characterize the interactions of the CRE with heterologous promoter elements, especially those involved in regulation of PEPCK. This will identify potential targets of CREBP interactions. Domains of CREBP that are required for the interplay of different transcription factors can then be identified by examining the ability of different CREBP variants and fusion factors to potentiate activation by these other factors.