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

描述（由申请方提供）：最佳免疫应答需要充足的离子供应和仔细调节的离子稳态。低Mg 2+条件对免疫力的不利影响有充分的记录，但缺乏对这种Mg 2+敏感性的机制见解。最近发现的蛋白质TRPM 7是活性Ser/Thr激酶与离子通道的独特融合，并且是Mg 2 +-稳态的主要调节剂。TRPM 7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）的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架构师。PLC g 2是B细胞受体（BCR）信号传导的中心，并且介导B细胞成熟以及活化。我们认为TRPM 7激酶通过PLCg 2的Ser/Thr磷酸化调节Mg 2+的可用性，从而调节BCR信号传导。通过Tyr-磷酸化对PLC g2的调节已被充分表征，但其通过Ser/Thr磷酸化的调节仅被假定，尽管可能性很高，因为绝大多数细胞磷酸化事件涉及Ser/Thr残基。我们已经收集了初步的细胞系实验证据，支持我们的主要假设，即PLCg 2的C2结构域是TRPM 7激酶的底物，导致BCR引起的Ca 2+反应的Mg 2+敏感性调节。该提案旨在进一步探索这种新的磷酸化事件对PLCg 2的定位，Tyr-磷酸化和酶活性的影响，以及在现有的PLCg 2缺陷小鼠模型中使用互补方法研究其体内生理相关性。

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.