NUCLEAR TARGETING OF SIGNAL TRANSDUCTION ENZYMES

信号转导酶的核靶向

• 批准号: 2018002
• 负责人: VINCENT M COGHLAN
• 金额: $ 19.48万
• 依托单位: GOOD SAMARITAN HOSP & MED CTR(PRTLND,OR)
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-22 至 1998-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2018002
• 关键词: DNA binding protein; biological signal transduction; gene expression; hormone regulation /control mechanism; intermolecular interaction; intracellular transport; nuclear matrix; pituitary hormones; protein kinase A; protein structure function; protein transport; tissue /cell culture

Many hormones exert their effect through receptor-coupled pathways that result in production of the intracelllular second messenger cAMP. The diverse physiological responses induced by elevated cAMP levels are mediated by the cAMP-dependent protein kinase )PKA) through phosphorylation of metabolic enzymes, channel proteins, and transcriptional regulators. Selectivity of PKA action is achieved in part through compartmentalization of the kinase with preferred substrates. A family of anchoring proteins, called AKAPs, have been characterized that maintain PKA at particular subcellular sites and target the kinase for specific functional roles. In this application we propose to investigate nuclear targeting of PKA and its role in mediating the hormone-induced expression of genes. We have discovered a novel anchoring protein, called AKAP 95, that binds PKA and is exclusively localized to the nuclear matrix. The properties of this AKAP will be characterized as part of a long term goal to define the relationship between structural organization in the cell nucleus and the regulation of nuclear signaling pathways. Molecular and genetic approaches are proposed that will define the nuclear complexes forced by aKAP 95 and identify new proteins that may be structural components of the nuclear matrix. As AKAPs are known to be multifunction, experiments are proposed that will determine whether association of other kinases and phosphatases with the nuclear matrix occurs through AKAP 95. We have also discovered that AKAP 95 is a DNA binding protein and we will establish whether the protein preferentially binds a specific DNA sequence, and whether binding affects transcriptional activity. Targeting of PKA and other signaling enzymes to the nucleus may be important for normally-regulated gene expression. We will use inhibitor peptide and DNA constrictors to determine the effect of disrupting PKA anchoring on cAMP-induced expression of the prolactin gene in cultured rat pituitary tumor GH cells. Antisense oligonucleotides and constructs will be used to specifically reduce PKA binding states at the nuclear matrix to establish the role of AKAP 95-mediated targeting in regulating ranscriptional events. These studies have relevance to cancer, as malignant progression is often accompanied by disruption of both nuclear organization and signal transduction mechanisms.

许多激素通过受体耦合途径发挥作用 这导致了细胞内第二信使训练营的生产。 营地水平升高引起的多种生理反应 由cAMP依赖性蛋白激酶）通过 代谢酶，通道蛋白和 转录调节器。 PKA动作的选择性已在 通过首选激酶的隔室化。 基材。 一个称为Akap的锚定蛋白的家族一直是 表征在特定亚细胞部位保持PKA的特征 靶向激酶来发挥特定的功能作用。 在此应用中，我们 建议研究PKA的核靶向及其在 介导激素诱导的基因表达。 我们发现了一种新颖的锚定蛋白，称为AKAP 95， 绑定PKA并仅定位于核基质。 这 该AKAP的属性将被描述为长期的一部分 定义结构组织之间关系的目标 细胞核和核信号通路的调节。 提出了分子和遗传方法，以定义 AKAP 95强迫的核复合物，并鉴定出新的蛋白质 可以是核基质的结构成分。 作为Akap 已知是多功能的，提出了将 确定其他激酶和磷酸酶的关联是否与 核基质通过AKAP 95发生。我们也有 发现AKAP 95是DNA结合蛋白，我们将 确定蛋白质是否优先结合特定的DNA 序列以及结合是否影响转录活性。 将PKA和其他信号酶靶向核可能是 对于正常调节的基因表达很重要。 我们将使用 抑制剂肽和DNA收缩器以确定 破坏PKA锚定在营地引起的表达 培养大鼠垂体肿瘤GH细胞中的催乳素基因。 反义 寡核苷酸和构建体将用于特别减少 核基质处的PKA结合状态以确立AKAP的作用 95介导的靶向调节重述事件。 这些 研究与癌症有关，因为恶性进展通常是 伴随着核组织和信号的破坏 转导机制。