Modulation of PLCgamma2-Mediated Signaling Via its C2-Domain in B Lymphocytes

描述（由申请人提供）：最佳的免疫反应需要充足的离子供应和仔细调节的离子稳态。低Mg2+条件对免疫的不利影响已被充分记录，但缺乏对这种Mg2+敏感性的机制见解。最近发现的蛋白TRPM7是一种独特的融合了活跃的丝氨酸/苏氨酸激酶和离子通道的蛋白，是Mg2+稳态的主要调节剂。TRPM7已被证明与几种磷脂酶C （PLC）同工酶相互作用。PLC蛋白是几乎所有免疫细胞类型（包括b淋巴细胞）发育和激活所需的关键信号通路的核心，b淋巴细胞是体液免疫反应的细胞建筑师。PLCg2是B细胞受体（BCR）信号传导的核心，介导B细胞的成熟和激活。我们提出trpm7激酶通过PLCg2的丝氨酸/苏氨酸磷酸化，根据Mg2+的可用性调节BCR-信号。已经充分表征了酪氨酸磷酸化对PLCg2的调节，但由于绝大多数细胞磷酸化事件涉及丝氨酸/苏氨酸残基，因此丝氨酸/苏氨酸磷酸化对PLCg2的调节只是假设，尽管可能性很大。我们在细胞系中收集了初步的实验证据，支持我们的主要假设，即PLCg2的c2结构域是trpm7激酶的底物，导致Mg2+敏感调节bcr诱导的Ca2+反应。本研究旨在进一步探索这一新的磷酸化事件对PLCg2定位、tyr1磷酸化和酶活性的影响，并在现有的PLCg2缺乏小鼠模型中利用互补方法研究其在体内的生理相关性。

DESCRIPTION (provided by applicant): Optimal immune responses require adequate ionic supply and carefully regulated ion-homeostasis. The adverse effects of low-Mg2+ conditions on immunity are well documented, but mechanistic insights into this Mg2+-sensitivity are lacking. The recently discovered protein TRPM7 is the unique fusion of an active Ser/Thr kinase with an ion channel, and a master regulator of Mg2+-homeostasis. TRPM7 has been shown to interact with several phospholipase C (PLC) isozymes. PLC proteins are at the heart of crucial signaling pathways required for the development and activation of virtually every immune cell type, including B-lymphocytes, which are the cellular architects of humoral immune responses. PLCg2 is central to B-cell receptor (BCR) signaling, and mediates B- cell maturation as well as activation. We propose that TRPM7-kinase modulates BCR- signaling in accordance to the availability of Mg2+ through Ser/Thr phosphorylation of PLCg2. The regulation of PLCg2 by Tyr-phosphorylation has been amply characterized, but its modulation by Ser/Thr phosphorylation is only postulated, although highly probable, since the vast majority of cellular phosphorylation events involve Ser/Thr residues. We have gathered preliminary experimental evidence in cell lines supporting our main hypothesis that the C2-domain of PLCg2 is a substrate of TRPM7-kinase, resulting in the Mg2+-sensitive modulation of BCR-elicited Ca2+-responses. This proposal aims at further exploring the effect of this novel phosphorylation event on PLCg2's localization, Tyr-phosphorylation and enzymatic activity, as well as to investigate its physiological relevance in vivo using a complementation approach in an existing mouse model of PLCg2 deficiency.