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足够的离子供应和仔细调节的离子稳态。低镁条件对免疫的不利影响已有很好的文献记载，但缺乏对这种镁敏感性的机械性见解。最近发现的TRPM7蛋白是一种活性丝氨酸/苏氨酸激酶与离子通道的独特融合，是镁离子稳态的主要调节者。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发育和激活所需的关键信号通路的核心，包括B淋巴细胞，它们是体液免疫反应的细胞构建者。PLCG2是B细胞受体(BCR)信号转导通路的核心，调节B细胞的成熟和激活。我们认为，TRPM7-激酶通过PLCG2的丝氨酸/苏氨酸磷酸化，根据镁离子的可利用性来调节BCR-信号。酪氨酸磷酸化对PLCG2的调节已经得到了充分的表征，但Ser/Thr磷酸化对PLCG2的调节只是假设的，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BCR诱导的钙反应对镁敏感的调制。本研究旨在进一步探讨这一新的磷酸化事件对PLCG2的S定位、酪氨酸磷酸化和酶活性的影响，并在已有的PLCG2缺乏症小鼠模型中采用互补的方法研究其在体内的生理意义。 公共卫生相关性：离子动态平衡的改变严重影响人类健康，损害免疫反应的有效性和适当性，并导致或加剧癌症或糖尿病等严重疾病。计划中的研究将有助于扩大我们对分子机制的了解，允许调节免疫反应，以获得必要的和最丰富的细胞内二价阳离子镁离子。了解这些调节过程为治疗干预开发新的免疫调节策略提供了潜在的机会。由于它的重点是B淋巴细胞，这一建议与自身免疫性疾病或各种形式的免疫缺陷等疾病有关。

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. PUBLIC HEALTH RELEVANCE: Alterations in ion homeostasis have severe effects on human health, impairing the effectiveness and appropriateness of immune responses, and causing or exacerbating grave diseases such as cancer or diabetes. The planned studies will contribute to expanding our knowledge about molecular mechanisms allowing for the adjustment of immune responses to the availability of the essential and most abundant intracellular divalent cation Mg2+. Understanding these regulatory processes represent potential opportunities to develop novel immunomodulatory strategies for therapeutic intervention. Given its focus on B-lymphocytes, this proposal is relevant to conditions such as auto-immune diseases, or various forms of immunodeficiencies.