CSF-1R Regulation of Type I PIP5K

磷脂酰肌醇(PtdIns)是一种调节多种细胞过程的膜脂，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之所以能够调节这些不同的功能，是因为在一组特定的PtdIns激酶的催化下，在肌醇部分的特定位置增加了磷酸基团。因此，了解调控PtdIns信号转导的机制取决于这些PtdIns激酶的特性。该项目将剖析其中一种激酶的调控途径，即小鼠I型磷脂酰肌醇4-磷酸5-激酶(PIP5K-Iβ)的β亚型。初步研究表明，截短的、酶失活的PIP5K-Iβ可以恢复生长因子受体缺陷形式的细胞功能，即集落刺激因子-1受体。工作假说是，这种截断的激酶抑制了激活的受体从质膜上的移除，并干扰了内源性PIP5K-Iβ阻止受体降解的功能。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，不依赖于PIP5K功能，足以恢复有缺陷的集落刺激因子-1受体的活性，并且截断的激酶通过结合和隔离生物非活性复合体中的内源性PIP5K-Iβ而起作用。含有突变形式或天然形式的激酶的蛋白质复合体将通过蛋白质组成、亚细胞分布和酶活性来表征。目的2是确定PIP5K-Iβ在不同磷酸化的PtdIn产生中的作用，并确定调节PIP5K-I活性和亚细胞靶向的生理因素。PtdIns在细胞中的定点磷酸化将在克隆模拟因子-1受体激活之后以及在突变的激酶形式过表达之后进行表征。此外，PIP5K-Iβ的调节域将通过丙氨酸筛选激酶的突变并测试改变的蛋白质对细胞依赖于PtdIns的活动的影响来确定。

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.