PIP PHOSPHATASE AND INOSITOL PHOSPHOLIPID HOMEOSTATSIS

许多生长因子和激素引起他们的生物反应， 刺激磷脂酰肌醇4，5-P2的水解以产生 甘油二酯和肌醇1，4，5-P3。 适当水平的 在这种信号传导中使用的多磷酸肌醇似乎是至关重要的， 维持正常的细胞功能。 减少的水平 含有肌醇的磷脂已经与多种 病理状况，包括糖尿病性神经病变和呼吸系统疾病 痛苦综合症。 磷酸肌醇不仅存在于质膜中， 作为一个独特的核PI周期的一部分发生。 核PI营业额 已被证明受IGF-1和核肌醇水平的调节 磷脂似乎在经历分化的细胞中发生变化。 的 多磷酸肌醇的水平由它们的相对速率决定 合成和降解的过程。 虽然许多研究都集中在 合成的聚磷酸肌醇，相对知之甚少， 降解磷酸肌醇的磷酸肌醇磷酸酶。 最近 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， 参与PI周转和细胞信号传导的酶被调节成 细胞周期依赖性的方式表明肌醇 在细胞周期中可以调节磷脂体内平衡。 基于 基于上述考虑，这项建议的具体目标是： 1.目的：克隆一个PI 4-P4-磷酸酶基因，并进行序列测定. 2.确定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-P4-磷酸酶mRNA和蛋白的表达 在整个细胞周期中。 4.阐明PI 4-P磷酸酶在核PI中的作用， 周期和特点的影响IGF的对这个周期。 一种来自大鼠脑的PIP磷酸酶已经纯化了76，000倍， 实验室和部分特征。 在拟议的实验中， 蛋白质序列将从纯化的PIP磷酸酶获得， 用于设计用于cDNA克隆和测序的探针 对应于这种酶。 PIP磷酸酶在 维持肌醇磷脂稳态将被研究 通过在3 T3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 PIP磷酸酶在核PI周期中的作用和IGF的影响 对这种新的信号通路也将进行研究。 洞察 PIP磷酸酶结构、功能和调节将有助于 了解参与调节水平的机制， 细胞内的磷酸肌醇。

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.