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GENE CONTROL BY INSULIN-REGULATED PHOSPHORYLATION

通过胰岛素调节的磷酸化进行基因控制

基本信息

批准号：
2713399
负责人：
PATRICK G QUINN
金额：
$ 19.25万
依托单位：
PENNSYLVANIA STATE UNIV HERSHEY MED CTR
依托单位国家：
美国
项目类别：
财政年份：
1996
资助国家：
美国
起止时间：
1996-06-01 至 2000-05-31
项目状态：
已结题

项目摘要

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）：这是一个修订后的应用一种新的建议来研究胰岛素抑制H4肝癌细胞中cAMP对PEPCK基因的激活。的首席研究员建议研究如何交易因素，CREB，CBP 可能还有其他未知的蛋白质被磷酸化并激活通过PKA的cAMP，去磷酸化和抑制目前未知的胰岛素介质。在具体目标1中，主要研究者将确定CREB/CBP/其他蛋白质中的哪些氨基酸被磷酸化，或去磷酸化反应cAMP和胰岛素，这些研究将涉及磷酸肽作图和磷酸化位点突变体的使用。在具体目标2中，主要研究者将检验以下假设： CREB和PEPCK最小启动子之间形成的独特桥复合物在胰岛素作用期间被破坏，这将涉及顺式 - 最小启动子中的元素和生化分析的影响， cAMP和胰岛素对因子组成和磷酸化状态的影响与大桥建筑群有关。在具体目标3中，研究者将确定是否改变的磷酸化状态， CREB/CBP/其他蛋白是由于相关核激酶的变化，磷酸酶活性，这些研究将涉及使用抑制剂和纯化的磷酸酶和激酶，以改变反式激活因子及其体外转录活性测定。