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。 公共卫生相关性：离子稳态的改变对人类健康具有严重影响，损害免疫反应的有效性和适当性，并引起或加剧严重疾病，如癌症或糖尿病。计划中的研究将有助于扩大我们对分子机制的了解，允许调整免疫反应以获得必需的和最丰富的细胞内二价阳离子Mg 2+。了解这些调节过程代表了开发用于治疗干预的新型免疫调节策略的潜在机会。鉴于其重点是B淋巴细胞，这一建议是相关的条件，如自身免疫性疾病，或各种形式的免疫缺陷。

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.