The PLD (phospholipase D) family proteins are enzymes that catalyze the hydrolysis of phosphatidylcholine, generating phosphatidic acid (PA) and choline. In recent years, considerable progress has been made demonstrating that the PLD (phospholipase D) family proteins, PLD1 and PLD2, are important for the signaling, activation, and function of leukocytes;however, many of the results from previous studies rely upon data using inhibitors or overexpression systems. As a result, these studies have produced varying and sometimes conflicting data. In addition, the physiological roles of PLD1 and PLD2 in vivo have not been explored in detail due to a lack of mouse models. Our preliminary data using cell lines and PLD1- and PLD2- deficient mice, which we have generated, support the assertion that these enzymes are critical in leukocyte function. We hypothesize that both PLD1 and PLD2 are important in immunoreceptor-mediated signaling and cellular activation. Due to their differences in subcellular localization, enzymatic activity, and regulation, these two proteins most likely also have distinct roles in signaling. We have designed three specific aims to test this hypothesis. In Aim #1, we will examine the effect of the deletion of PLD1, PLD2, or both on TCR-mediated PA production. We will also use live imaging to examine the subcellular localization of PA in antigen-specific T cells and to determine if its localization is affected by PLD deficiency. In Aim #2, we will investigate PLD function in thymocyte development and TCR-mediated signaling using PLD-deficient mice. In Aim #3, we will investigate the role of PLD1 and PLD2 in Fc5RI-mediated signaling and mast cell function in vivo and in vitro. Completion of these specific aims will enhance our understanding on the function of these two evolutionally conserved enzymes in the immune system. In addition, since it has been reported that PLD activity or protein expression is greatly increased in human cancers, our study can also provide insight into the potential role of PLDs in tumorigenesis. PUBLIC HEALTH RELEVANCE: The proposed work will have a positive impact on our understanding on the function of these two evolutionally conserved enzymes in the signaling and activation of leukocytes. This study will also facilitate the identification of intracellular targets for drug design to modulate leukocyte function during infection, transplantation, and treatment of cancer.