Phospholipase D proteins in immunoreceptor-mediated signaling

描述（由申请人提供）：PLD（磷脂酶D）家族蛋白是催化磷脂酰胆碱水解，生成磷脂酸（PA）和胆碱的酶。近年来，已经取得了相当大的进展，证明PLD（磷脂酶D）家族蛋白PLD1和PLD2对白细胞的信号传导、激活和功能很重要；然而，以前的许多研究结果依赖于使用抑制剂或过表达系统的数据。因此，这些研究产生了不同的，有时是相互矛盾的数据。此外，由于缺乏小鼠模型，PLD1和PLD2在体内的生理作用尚未得到详细探讨。我们使用细胞系和PLD1和PLD2缺陷小鼠的初步数据支持这些酶在白细胞功能中起关键作用的断言。我们假设PLD1和PLD2在免疫受体介导的信号传导和细胞活化中都很重要。由于它们在亚细胞定位、酶活性和调控方面的差异，这两种蛋白很可能在信号传导中也有不同的作用。我们设计了三个具体目标来验证这一假设。在目标#1中，我们将研究PLD1、PLD2或两者的缺失对tcr介导的PA产生的影响。我们还将使用实时成像来检查抗原特异性T细胞中PA的亚细胞定位，并确定其定位是否受到PLD缺乏的影响。在Aim #2中，我们将使用PLD缺陷小鼠研究PLD在胸腺细胞发育和tcr介导的信号传导中的功能。在Aim #3中，我们将在体内和体外研究PLD1和PLD2在fc5ri介导的信号传导和肥大细胞功能中的作用。完成这些特定目标将增强我们对这两种进化上保守的酶在免疫系统中的功能的理解。此外，由于有报道称PLD活性或蛋白表达在人类癌症中大大增加，我们的研究也可以深入了解PLD在肿瘤发生中的潜在作用。

DESCRIPTION (provided by applicant): 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.