REGULATION OF MEMBRANE BIOSYNTHESIS BY CSF 1

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The long term goal of the research plan is to understand the interrelationship between the biogenesis of membrane phospholipids and growth factor regulation of hematopoietic cell physiology. Phosphatidylcholine (PtdCho) is recognized as a major structural building block of biological membranes and the precursor to diacylglycerol and eicosanoid second messengers following stimulation of cell surface receptors. The research will focus on CTP:phosphocholine cytidylyltransferase (CT), the enzyme that governs the rate of PtdCho biosynthesis, and the relationship between PtdCho metabolism, growth factor stimulation and cell cycle progression in a colony-stimulating factor 1 (CSF-1)-dependent murine macrophage cell line. Membrane phospholipid synthesis is a cell cycle-regulated process governed by the interaction between PtdCho synthesis and degradation. PtdCho degradation is regulated by growth factor stimulation in GI phase and PtdCho synthesis is controlled by periodic CT phosphorylation. PtdCho, in turn, influences cell cycle regulation. The inhibition of S phase PtdCho synthesis with an antineoplastic lysophosphatidylcholine analog leads to the arrest of BAC1.2F5 macrophage cells in G2 phase followed by apoptosis. This research demonstrates that PtdCho synthesis is an essential process inhibited by this unique class of anticancer drugs, thus defining their mechanism of action and identifying CT as a novel anticancer drug target. These data also reveal an interaction between PtdCho synthesis and the cell cycle machine that ensures the coordination of membrane formation with cell growth. The goals of the proposed project are to clarify the mechanistic details that underlie this regulatory loop. The experimental plan is organized around three specific aims that will address: 1) the role of CT phosphorylation at Ser-315, a major phosphorylation site in vivo, by cyclin-dependent protein kinases in the regulation of PtdCho metabolism, 2) the mechanism for the inactivation of CT and PtdCho synthesis in CSF-1- deprived cells, and 3) the regulation of the cell cycle by membrane PtdCho content and the role of CT in CSF-1-stimulated PtdCho degradation. These experiments are designed to generate new information that will contribute to the understanding of membrane phospholipid formation during entry, transit and exit from the cell cycle. Since there is only a single CT gene and protein, the proposed research on this ubiquitous regulator of phospholipid synthesis is directly relevant to other mammalian cell systems. Defining the role of CT and PtdCho in cell growth and function will provide important information that will be useful in the development of new approaches to exploiting CT and other enzymes in the CDP-choline pathway as therapeutic targets.