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反应元件结合蛋白的作用机制 (CREBP)及其调控因子之间的相互作用 磷酸烯醇式丙酮酸羧激酶(PEPCK)基因。CRE扮演着至关重要的角色 在基础转录和cAMP刺激的PEPCK基因转录中的作用。 PEPCK的调控表达，催化限速步骤 糖异生，对维持适当的血糖至关重要 浓度。基因转录受相互作用的调节 相互作用的蛋白质因子和RNA聚合酶，这是 通过这些因子与顺式作用的DNA元件的结合而促进。循环 AMP激活蛋白激酶A，导致几个 蛋白质，包括Cre结合蛋白。CREBP最近一直在 纯化、克隆并被证明直接参与基因调控 表情。 为了了解CREBP如何与其他因素相互作用来调节 基因表达，有必要确定CREBP的结构域是 参与变态激活。CREBP以二聚体的形式与Cre结合，并有两个 不同的14个氨基酸的异构体，具有不同的转录 由于CREBP的选择性剪接，细胞中存在能力 MRNA.目的1是确定CREBP二聚化的意义 用于反式激活的异构体，并确定 异构体的分布是受调控的。这将通过检查 每种多肽单独的贡献与其相互作用的比较 以及二聚体中不同的异构体，并通过确定CREBP 合成或剪接是由激素调节的。目标2是确定 CREBP参与基础和cAMP激活的功能结构域 刺激转录。这将通过创造特定的突变来实现 在CREBP cDNA中，通过将潜在的激活结构域与GAL4融合 DNA结合域。突变的CREBP的反式激活能力 蛋白质和CREBP/GAL4融合因子将在转染过程中进行评估 体外转录检测。目标3是识别和描述 Cre与异源启动子元件的相互作用 那些参与监管PEPCK的人。这将确定潜在的 CREBP相互作用的靶点。所需的CREBP域 不同转录因子之间的相互作用可以通过 检测不同CREBP变异体和融合因子的能力 通过这些其他因素来增强活性。

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.