CSF-1R Regulation of Type I PIP5K

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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.