Novel Mechanisms of Calcium Signaling in B lymphocytes

描述(申请人提供)：钙(钙)是一种多功能的第二信使，调节淋巴细胞分化和功能。淋巴细胞中抗原受体结合后启动钙信号转导的途径被很好地描绘出来。例如，B细胞上的抗原受体结合，激活酪氨酸激酶Lyn/Syk，继而激活PLC32，导致IP3的产生，从而激活内质网(ER)膜上的通道(IP3受体)。IP3敏感的内质网钙离子储存库的耗尽触发了内质网钙离子感受器STIM1的重新定位，使其进入靠近质膜的连接内质网的点状结构。ORAI1是最近发现的位于质膜上的CRAC通道孔，激活后也聚集成点状，随后与STIM1相互作用，导致“商店操作”的CRAC通道激活。虽然长期以来人们一直认为IP3介导的存储耗尽是最大限度激活CRAC的必要和充分条件，但最近的数据，包括我们自己的数据，显然挑战了这一概念。在内质网钙库耗尽后的Lyn-/-SYK-/-B细胞中，观察到了IP3介导的库释放下游CRAC激活的调节。为了支持这一观点，我们的研究表明，CRAC的激活可以被Lyn/Syk抑制剂或中和抗Lyn和-Syk抗体引入单个B细胞胞浆而被取消。我们随后观察到Orai1和Syk在物理上相互作用，这让我们假设Syk参与了CRAC激活所需的Orai1重分布。因此，这项建议侧重于LYN和SYK在调节门店后耦合(Aim1)中的作用。CRAC活性可以独立调节的另一个迹象是我们的初步数据提供了PLC32与Orai1直接相互作用的证据，这表明。我们的数据表明，PLC32和Orai1的物理结合对CRAC的激活至关重要，这种相互作用可以被酪氨酸磷酸化的TFII-I抑制，TFII-I是一个已知的BTK靶标。根据这些数据和TFII-I在调节PLC31依赖的TRPC3钙通道激活中的类似作用，我们假设PLC3-2以一种脂肪酶非依赖的方式调节CRAC通道介导的钙内流，该方式受BTK依赖的TFII-I磷酸化的负调节(在Aim 2中测试)。最后，我们最近与Dan Billadeau的合作表明，适配器蛋白WAVE2调节T细胞激活后钙离子的进入，但不能调节细胞外钙的释放。我们的初步结果表明，WAVE2在B细胞中起着类似的作用。此外，在B细胞中，WAVE2与STIM1结合，并在激活后解离，这表明WAVE2在定位STIM1与Orai1的相互作用中发挥了作用。我们将在目标3中进一步探讨WAVE2在BCR诱导CRAC激活中的机制作用。钙信号的严格调控对淋巴细胞效应功能至关重要，其失调导致免疫缺陷和炎症性疾病。我们提议的研究将确定在疾病状态下钙信号可能出错的其他机制。这些研究的结果还将为改善免疫缺陷疾病和/或自身免疫和炎症状况的方法提供洞察力。 与公共健康相关：钙是一种多功能的第二信使，几乎调节淋巴细胞分化和功能的方方面面，从而调节免疫反应。这些研究的主要目的是了解钙离子进入淋巴细胞的新机制。这些研究的结果将确定积极和消极地操纵免疫反应的目标和策略，以改善免疫缺陷疾病和/或抑制自身免疫性和炎症性疾病的发展。

DESCRIPTION (provided by applicant): Calcium (Ca2+) is a multifunctional second messenger that regulates lymphocyte differentiation and function. The pathways that initiate Ca2+signaling following antigen receptor engagement in lymphocytes are well delineated. For example, antigen receptor engagement on B cells, activates tyrosine kinases Lyn/Syk and the consequent activation of PLC32 results in the generation of IP3, which activates channels (IP3 receptors) on the endoplasmic reticulum (ER) membrane. Depletion of IP3-sensitive ER Ca2+ stores triggers relocalization of STIM1, the ER Ca2+ sensor, into punctate structures in junctional ER adjacent to the plasma membrane. Orai1, the recently identified CRAC channel pore located in the plasma membrane, also aggregates into puncta following activation, and its subsequent interaction with STIM1 results in "store-operated" CRAC channel activation. While it has long been thought that IP3-mediated store depletion is both necessary and sufficient for maximal CRAC activation, recent data, including our own data, clearly challenge this notion. Regulation of CRAC activation downstream of IP3-mediated store release was observed in lyn-/-syk-/- B cells following ER Ca2+stores depletion. In support of this idea, our studies demonstrate that CRAC activation is abrogated by either Lyn/Syk inhibitors or neutralizing anti-Lyn and -Syk antibodies introduced into the cytoplasm of single B cells. Our subsequent observation that Orai1 and Syk physically interact led us to hypothesize that Syk is involved in Orai1 redistribution required for CRAC activation. Accordingly, this proposal focuses on the role of Lyn and Syk in the regulation of post-store coupling (Aim1). Another indication that CRAC activity could be independently regulated is provided by our preliminary data that PLC32 directly interacts with Orai1, suggesting. Our data suggest that physical association of PLC32 and Orai1 is critical for CRAC activation and that such interactions can be inhibited by tyrosine phosphorylated TFII-I, a known Btk target. Based on these data and an analogous role for TFII-I in regulating PLC31-dependent activation of TRPC3 Ca2+ channels, we hypothesize that PLC3-2 regulates CRAC channel mediated Ca2+ entry in a lipase-independent manner that is negatively regulated by Btk dependent phosphorylation of TFII-I (tested in Aim 2). Finally, our recent work with Dan Billadeau demonstrates that the adaptor protein WAVE2 regulates Ca2+ entry, but not Ca2+ release from stores following T cell activation. Our preliminary results demonstrate that WAVE2 plays an analogous role in B cells. Furthermore, WAVE2 associates with STIM1 in B cells and dissociates following activation, suggesting a role in orienting STIM1 for interactions with Orai1. We will further explore the mechanistic role of WAVE2 in BCR-induced CRAC activation in Aim 3. Tight regulation of Ca2+signaling is critical for lymphocyte effector function and its dysregulation contributes to immunodeficiency as well as inflammatory diseases. Our proposed studies will identify additional mechanisms by which Ca2+ signaling could go awry in disease states. Results from these studies will also provide insight into approaches to ameliorate immunodeficiency diseases and/or autoimmune and inflammatory conditions. PUBLIC HEALTH RELEVANCE: Calcium is a multifunctional second messenger that regulates nearly every aspect of lymphocyte differentiation and function and thereby the immune response. The primary objective of these studies is to understand novel mechanisms by which calcium entry into lymphocytes is regulated. Results from these studies will identify targets and strategies both positive and negative manipulation of the immune response to ameliorate immunodeficiency diseases and/or to suppress development of autoimmune and inflammatory disease.