GENE CONTROL BY INSULIN-REGULATED PHOSPHORYLATION

通过胰岛素调节的磷酸化进行基因控制

• 批准号: 2430233
• 负责人: PATRICK G QUINN
• 金额: $ 18.51万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-06-01 至 1999-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2430233
• 关键词: biological signal transduction; cAMP response element binding protein; cyclic AMP receptors; enzyme induction /repression; genetic promoter element; genetic transcription; hormone regulation /control mechanism; insulin; liver cells; messenger RNA; mutant; nucleic acid sequence; phosphoenolpyruvate carboxylase; phosphoprotein phosphatase; phosphorylation; protein kinase; protein kinase A; site directed mutagenesis; transcription factor; western blottings

DESCRIPTION (Adapted from the applicant's abstract): This is a revised application of a new proposal to study the mechanism whereby insulin inhibits activation of the PEPCK gene by cAMP in H4 hepatoma cells. The principal investigator proposes to study how transacting factors, CREB, CBP and possibly other unidentified proteins, are phosphorylated and activated by cAMP via PKA, and dephosphorylated and inhibited by presently unknown insulin mediators. In Specific Aim 1, the principal investigator will determine which amino acids in CREB/CBP/other proteins are phosphorylated or dephosphorylated in response to cAMP and insulin, and these studies will involve phosphopeptide mapping and the use of phosphorylation-site mutants. In Specific Aim 2, the principal investigator will test the hypothesis that a unique bridge complex formed between CREB and the PEPCK minimal promoter is disrupted during insulin action, and this will involve analysis of cis -elements in the minimal promoter and biochemical analysis of the effects of cAMP and insulin on the composition and phosphorylation state of factors associated with the bridge complex. In Specific Aim 3, the principal investigator will determine whether an altered phosphorylation state in CREB/CBP/other proteins is due to changes in relevant nuclear kinase and phosphatase activities, and these studies will involve the use of inhibitors and purified phosphatases and kinases to alter the phosphorylation state of trans-activating factors and their activity in in vitro transcription assays.

描述(改编自申请人的摘要)：这是一个修改后的 一种新的方案在研究胰岛素作用机制中的应用 抑制H4肝癌细胞中cAMP对PEPCK基因的激活。这个 首席研究员建议研究交易因素、CREB、CBP 可能还有其他未知的蛋白质被磷酸化并被激活 由cAMP通过PKA，并被目前未知的去磷酸化和抑制 胰岛素调节剂。在具体目标1中，首席调查员将 确定CREB/CBP/其他蛋白质中哪些氨基酸是磷酸化的或 去磷酸化对cAMP和胰岛素的反应，这些研究将 涉及磷酸化多肽图谱和磷酸化位点突变体的使用。 在具体目标2中，首席调查员将检验假设 CREB与PEPCK最小启动子之间形成的独特桥联复合体 在胰岛素作用期间被破坏，这将涉及到对顺式的分析 -微量启动子中的元件及生化分析的影响 CAMP和胰岛素对因子组成和磷酸化状态的影响 与桥梁建筑群有关。在具体目标3中，校长 研究人员将确定一种改变的磷酸化状态是否 CREB/CBP/其他蛋白是由于相关核蛋白和蛋白的变化 磷酸酶活性，这些研究将涉及到抑制剂的使用 和纯化的磷酸酶和激酶来改变细胞的磷酸化状态 反式激活因子及其体外转录活性 化验。