CSF-1R Regulation of Type I PIP5K

I 型 PIP5K 的 CSF-1R 调节

• 批准号: 9982410
• 负责人: Nathan Davis
• 金额: $ 27.21万
• 依托单位: LSU Health Sciences Center -Shreveport
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-05-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9982410&HistoricalAwards=false
• 关键词: CSF; 1R; Regulation; Type; PIP5K

Phosphatidylinositols (PtdIns) are membrane lipids that regulate diverse cellular processes including cell survival and proliferation, receptor internalization, intracellular membrane trafficking, secretion, and remodeling of the actin cytoskeleton. The ability of PtdIns to regulate such diverse functions is due to the addition of phosphate groups at specific sites on the inositol moiety as catalyzed by a set of specific PtdIns kinases. Understanding the mechanisms that regulate PtdIns signaling therefore hinges upon the characterization of these PtdIns kinases. This project will dissect a regulatory pathway for one of these kinases, the beta isoform of the murine type-I phosphatidylinositol 4-phosphate 5-kinase (PIP5K-I beta). As shown in preliminary studies, a truncated and enzymatically inactive form of PIP5K-I beta can restore the function in cells of a defective form of growth factor receptors, namely colony-stimulating factor-1 receptor. The working hypothesis is that this truncated kinase inhibits the removal of the activated receptor from the plasma membrane and interferes with the function of endogenous PIP5K-I beta to block receptor degradation. The objective of these studies is to test this hypothesis by the following specific aims. Aim 1 is to demonstrate that a block of receptor internalization and degradation, independent of PIP5K function, is sufficient to restore the activity of the defective colony-stimulating factor-1 receptor, and that the truncated kinase acts by binding and sequestering endogenous PIP5K-I beta in biologically inactive complexes. Protein complexes containing either mutant or native forms of the kinase will be characterized by protein composition, subcellular distribution and enzyme activity. Aim 2 is to define the contribution of PIP5K-I beta in the production of different phosphorylated PtdIns and to define the physiological factors that regulate PIP5K-I beta activity and subcellular targeting. Site-specific phosphorylation of PtdIns in cells will be characterized after activation of the colony simulating factor-1 receptor, as well as after overexpression of mutant kinase forms. Additionally, the regulatory domains of PIP5K-I beta will be determined by alanine-screening mutagenesis of the kinase and testing the effects of the altered proteins on the PtdIns-dependent activities of the cell.

磷脂酰肌醇（PtdIns）是调节多种细胞过程的膜脂质，包括细胞存活和增殖、受体内化、细胞内膜运输、分泌和肌动蛋白细胞骨架的重塑。 PtdIns调节这些不同功能的能力是由于在肌醇部分的特定位点上添加磷酸基团，如由一组特定的PtdIns激酶催化的。 因此，了解调节PtdIns信号传导的机制取决于这些PtdIns激酶的特性。这个项目将剖析这些激酶之一的调节途径，小鼠I型磷脂酰肌醇4-磷酸5-激酶（PIP 5 K-I β）的β亚型。 如初步研究所示，截短和酶失活形式的PIP 5 K-I β可以恢复细胞中生长因子受体缺陷形式的功能，即集落刺激因子-1受体。 工作假设是，这种截短的激酶抑制从质膜上去除活化的受体，并干扰内源性PIP 5 K-I β的功能以阻断受体降解。这些研究的目的是通过以下具体目标来检验这一假设。 目的1是证明受体内化和降解的阻断，独立于PIP 5 K功能，足以恢复有缺陷的集落刺激因子-1受体的活性，并且截短的激酶通过结合和隔离内源性PIP 5 K-I β在生物学上无活性的复合物中起作用。 含有突变或天然形式的激酶的蛋白质复合物将通过蛋白质组成、亚细胞分布和酶活性来表征。 目的2是确定PIP 5 K-I β在不同磷酸化PtdIn产生中的贡献，并确定调节PIP 5 K-I β活性和亚细胞靶向的生理因子。 细胞中PtdIns的位点特异性磷酸化将在殖民地-1受体活化后以及突变激酶形式过表达后表征。 此外，通过激酶的丙氨酸筛选诱变并测试改变的蛋白质对细胞的PtdIn依赖性活性的影响，确定PIP 5 K-I β的调节结构域。