PIP PHOSPHATASE AND INOSITOL PHOSPHOLIPID HOMEOSTATSIS

PIP 磷酸酶和肌醇磷脂稳态

• 批准号: 6240981
• 负责人: Linda Joy Pike
• 金额: $ 19.27万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-12-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6240981
• 关键词: 3T3 cells; Escherichia coli; biological signal transduction; cell cycle proteins; cell differentiation; cell growth regulation; complementary DNA; enzyme activity; enzyme mechanism; gene expression; genetic library; growth /development; homeostasis; immunoprecipitation; insulinlike growth factor; laboratory rabbit; lipid metabolism; messenger RNA; neoplastic cell culture for noncancer research; nucleic acid sequence; oligonucleotides; phosphatidylinositols; polymerase chain reaction; protein sequence; protein structure function

Many growth factors and hormones elicit their biological response by stimulating the hydrolysis of phosphatidylinositol 4,5-P2 to produce diacylglycerol and inositol 1,4,5-P3. Adequate levels of the polyphosphoinositides used in such signaling appear to be crucial to the maintenance of proper cell function. Decreases in the levels of inositol-containing phospholipids have been associated with a variety of pathological conditions including diabetic neuropathy and Respiratory Distress Syndrome. Phosphoinositides are present not only in the plasma membrane but also occur as part of a distinct nuclear PI cycle. Nuclear PI turnover has been shown to be regulated by IGF-1 and the levels of nuclear inositol phospholipids appear to change in cells undergoing differentiation. The levels of polyphosphoinositides are determined by their relative rates of synthesis and degradation. While much research has focused on the synthesis of polyphosphoinositides, comparatively little is known of the phosphoinositide phosphatases that degrade phosphoinositides. Recent work has suggested that alterations in these enzymes may be the basis for changes in nuclear PI homeostasis associated with differentiation. Furthermore, preliminary data have indicated that activity of several enzymes involved in PI turnover and cell signaling are modulated in a cell cycle-dependent fashion suggesting the possibility that inositol phospholipid homeostasis may be modulated during the cell cycle. Based on the above considerations, the specific aims of this proposal are: 1. To clone and sequence a PI4-P 4-phosphatase. 2. To define the role of the PI 4-P phosphatase in inositol phospholipid homeostasis and cell signaling. 3. To examine the expression of PI 4-P 4-phosphatase mRNA and protein throughout the cell cycle. 4. To elucidate the role of the PI 4-P phosphatase in the nuclear PI cycle and characterize the effects of IGF's on this cycle. A PIP phosphatase from rat brain has been purified 76,000-fold in the laboratory and partially characterized. In the proposed experiments, protein sequence will be obtained from the purified PIP phosphatase and used to design probes for the cloning and sequencing of a cDNA corresponding to this enzyme. The role of the PIP phosphatase in maintaining inositol phospholipid homeostasis will then be investigated by overexpressing the PIP phosphatase in 3T3 cells and evaluating its effect on inositol phosphate and phospholipid levels. The involvement of the PIP phosphatase in the nuclear PI cycle and the effects of IGF's on this novel signaling pathway will also be investigated. Insight into the structure, function and regulation of a PIP phosphatase will aid in understanding the mechanisms involved in regulating the levels of phosphoinositides within cells.

许多生长因子和激素通过以下方式引起生物反应： 刺激磷脂酰肌醇4,5-P2的水解产生 二酰基甘油和肌醇1,4,5-P3。 足够的水平 用于此类信号传导的多磷酸肌醇似乎对于 维持正常的细胞功能。 水平下降 含肌醇的磷脂与多种 病理状况，包括糖尿病神经病变和呼吸系统疾病 苦恼综合症。 磷酸肌醇不仅存在于质膜中，还存在于质膜中。 作为独特的核 PI 循环的一部分发生。 核PI营业额已 已被证明受 IGF-1 和核肌醇水平的调节 磷脂在细胞分化过程中似乎发生变化。 这 多磷酸肌醇的水平由其相对比率决定 的合成和降解。 虽然很多研究都集中在 多磷酸肌醇的合成，目前人们所知甚少 降解磷酸肌醇的磷酸肌醇磷酸酶。 最近的 研究表明这些酶的改变可能是 与分化相关的核 PI 稳态的变化。 此外，初步数据表明，一些活动 参与 PI 周转和细胞信号转导的酶在 细胞周期依赖性时尚表明肌醇的可能性 磷脂稳态可能在细胞周期期间受到调节。 基于 综合上述考虑，本提案的具体目标是： 1. 克隆并测序 PI4-P 4-磷酸酶。 2. 明确PI 4-P磷酸酶在肌醇磷脂中的作用 稳态和细胞信号传导。 3.检测PI 4-P 4-磷酸酶mRNA和蛋白的表达情况 整个细胞周期。 4. 阐明PI 4-P磷酸酶在核PI中的作用 周期并描述 IGF 对该周期的影响。 来自大鼠大脑的 PIP 磷酸酶已在纯化中纯化了 76,000 倍 实验室和部分表征。 在提议的实验中， 将从纯化的 PIP 磷酸酶中获得蛋白质序列，并 用于设计用于 cDNA 克隆和测序的探针 对应于该酶。 PIP磷酸酶的作用 然后将研究维持肌醇磷脂稳态 通过在 3T3 细胞中过表达 PIP 磷酸酶并评估其 对磷酸肌醇和磷脂水平的影响。 参与程度 PIP 磷酸酶在核 PI 循环中的作用以及 IGF 的作用 还将对这种新的信号通路进行研究。 洞察 PIP 磷酸酶的结构、功能和调节将有助于 了解调节水平的机制 细胞内的磷酸肌醇。