Novel Mechanisms of Calcium Signaling in B lymphocytes

描述（由申请人提供）：钙（Ca2+）是调节淋巴细胞分化和功能的多功能第二信使。在淋巴细胞抗原受体参与后启动Ca2+信号的途径被很好地描述。例如，抗原受体与B细胞结合，激活酪氨酸激酶Lyn/Syk，随后PLC32的激活导致IP3的产生，IP3激活内质网（ER）膜上的通道（IP3受体）。ip3敏感的ER Ca2+储存的耗尽触发STIM1 （ER Ca2+传感器）重新定位到靠近质膜的连接性ER的点状结构中。最近发现的位于质膜上的CRAC通道孔Orai1在激活后也聚集成点状，其随后与STIM1的相互作用导致“储存操作”的CRAC通道激活。虽然长期以来人们一直认为ip3介导的存储耗竭是最大CRAC激活的必要和充分条件，但最近的数据，包括我们自己的数据，清楚地挑战了这一概念。在ER Ca2+储存耗尽后，在lyn-/-syk-/- B细胞中观察到ip3介导的储存释放下游CRAC活化的调节。为了支持这一观点，我们的研究表明，将Lyn/Syk抑制剂或中和的抗Lyn和-Syk抗体引入单个B细胞的细胞质中，可以消除CRAC的激活。我们随后观察到Orai1和Syk的物理相互作用，这使我们假设Syk参与了CRAC激活所需的Orai1再分配。因此，本文将重点研究Lyn和Syk在储存后耦合（Aim1）调控中的作用。我们的初步数据表明，PLC32直接与Orai1相互作用，这表明CRAC活性可以独立调节。我们的数据表明PLC32和Orai1的物理关联对于CRAC激活至关重要，并且这种相互作用可以被酪氨酸磷酸化的TFII-I（已知的Btk靶点）抑制。基于这些数据和TFII-I在调节plc31依赖的TRPC3 Ca2+通道激活中的类似作用，我们假设PLC3-2以脂酶不依赖的方式调节CRAC通道介导的Ca2+进入，这是由Btk依赖的TFII-I磷酸化负调控的（在Aim 2中进行了测试）。最后，我们最近与Dan Billadeau的工作表明，衔接蛋白WAVE2调节Ca2+的进入，但不调节T细胞活化后Ca2+从储存中的释放。我们的初步结果表明，WAVE2在B细胞中起着类似的作用。此外，WAVE2在B细胞中与STIM1结合，并在激活后解离，这表明它在定向STIM1与Orai1的相互作用中起作用。我们将在Aim 3中进一步探讨WAVE2在bcr诱导的CRAC激活中的机制作用。Ca2+信号的严格调节对淋巴细胞效应功能至关重要，其失调有助于免疫缺陷和炎症性疾病。我们提出的研究将确定Ca2+信号在疾病状态下可能出错的其他机制。这些研究的结果也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见解。

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.