REGULATION OF GENE EXPRESSION BY NO/CGMP

描述(研究人员摘要)：一氧化氮 (NO)cGMP/cGMP依赖的蛋白激酶(G-K)信号转导 通路对于许多生理和生理功能的调节是重要的。 病理生理过程，例如，在心血管系统中 平滑肌细胞、内皮细胞和血小板的主要决定因素 功能，并与高血压的发生和发展有关 动脉硬化。然而，与cAMP依赖的蛋白激酶相比， 已知G-激酶激活的下游效应。这个 研究人员发现，G-激酶调节基因表达：作为对 NO或cGMP，激酶转位到细胞核，诱导磷酸化 表达CREB相关蛋白并激活FOS、JunB和TNF-α 推动者。初步数据表明，G-激酶反式激活这些 转录识别的类似顺式作用元件的启动子 AP-1(Fos-jun)、Cre-和CCAAT-增强子结合蛋白的影响因素 家族及其亚细胞定位和生物活性 激酶受特定锚定蛋白的调节。的特殊目标 这一建议是：(1)确定NO/cGMP反应元件的DNA序列 (NGREs)：(Ii)确定靶向转录因子 NO/cGMP/G-激酶；和(Iii)鉴定关键底物和细胞质或 与G-激酶结合的核锚定蛋白。NGRE将由以下内容定义 基因中可能的增强子元件的缺失和定点突变 FOS和肿瘤坏死因子-α启动子。NO/cGMP靶向的转录因子将 在使用Ga14融合产物的反式激活研究中被鉴定 候选转录因子的显性负突变体以及 DNA结合研究和DNA亲和层析 编码NGRE的寡聚脱氧核苷酸。蛋白质相互作用克隆将是 用于鉴定与G-激酶相互作用的蛋白质及其作用 这些蛋白对G-激酶的亚细胞定位和功能的影响 测试过。一氧化氮作为一种信号分子的广泛重要性一直是 最近获得认可；这项拨款申请中建议的研究应 为NO和cGMP的作用机制提供新的见解 哺乳动物细胞。由于NO/cGMP信号的药理学操作 转导途径为多种肿瘤提供了治疗潜力 人类疾病，更好地了解其长期的下游影响 迫切需要NO和cGMP。

DESCRIPTION (Investigator's Abstract): The nitric oxide (NO)cGMP/cGMP-dependent protein kinase (G-kinase) signal transduction pathway is important for the regulation of many physiological and pathophysiological processes, e.g., in the cardiovascular system it is a major determinant of smooth muscle cell, endothelial cell and platelet functions and is implicated in the development of hypertension and atherosclerosis. However, compared to cAMP-dependent protein kinase, little is known about the downstream effects of G-kinase activation. The investigators found that G-kinase regulates gene expression: in response to NO or cGMP, the kinase translocates to the nucleus, induces phosphorylation of CREB-related proteins and activates the fos, junB and TNF-alpha promoters. Preliminary data suggest that G-kinase transactivates these promoters through similar cis-acting elements recognized by transcription factors of the AP-1 (Fos-Jun), CRE- and CCAAT-enhancer binding protein families and that subcellular localization and biological activity of the kinase is regulated by specific anchoring proteins. The speciic aims of this proposal are: (1) to define DNA sequences of NO/cGMP-response elements (NGREs): (II) to identify transcription factors targeted by NO/cGMP/G-kinase; and (III) to identify key substrates and cytoplasmic or nuclear anchoring proteins which bind to G-kinase. NGREs will be defined by deletion and site-directed mutagenesis of putative enhancer elements in the fos and TNF-alpha promoters. Transcription factors targeted by NO/cGMP will be identified in transactivation studies using Ga14-fusion products and dominant negative mutants of candidate transcription factors as well as in DNA binding studies and DNA affinity chromatography using oligodeoxynucleotides encoding NGREs. Protein interaction cloning will be employed to identify proteins interacting with G-kinase and the effect of these proteins on subcellular localization and function of G-kinase will be tested. The widespread importance of NO as a signaling molecule has been recognized recently; the studies proposed in this grant application should provide new insights into the mechanism of action of NO and cGMP in mammalian cells. Since pharmacological manipulation of the NO/cGMP signal transduction pathway offers therapeutic potential for a wide variety of human diseases, a better understanding of the long-term downstream effects of NO and cGMP is urgently needed.