EGR-mediated STIM1-PMCA expression and function in T cell subsets

钙池操纵的钙内流（SOCE）是内质网（ER）中钙离子丢失导致细胞内钙离子浓度升高的过程。 Ca 2+穿过质膜（PM）进入细胞。STIM 1和STIM 2是关键的ER Ca 2+传感器， 控制Orai 1的激活，即钙库操纵的钙通道。STIM 1/2对T细胞受体（TCR）至关重要- 介导的信号传导及其在人类中的缺乏导致淋巴增生性疾病和自身免疫性疾病， 在T细胞发育和活化方面存在缺陷。除了ER Ca 2+释放和SOCE，Ca 2+信号 涉及Ca 2+挤出机制。在淋巴细胞中，质膜Ca 2 +/ATP酶4（PMCA 4）是主要的 Ca 2+分泌的介质，是正常功能所必需的。最近，我们将PMCA 4确定为进一步的靶点， STIM 1（而不是STIM 2）在淋巴细胞活化过程中的作用。与SOCE不同，STIM 1诱导的PMCA抑制是初始的。 通过早期生长反应1（EGR 1）和EGR 4介导的STIM 1上调来表达。EGR无处不在， 快速上调锌指转录因子。目前的建议是基于以下假设： TCR参与刺激EGFR介导的启动Ca 2+清除的幼稚T细胞中的STIM 1上调 抑制，对于维持NFAT/NF-κB活化和随后的T细胞活化至关重要。这项工作是 分为三个目标。目的1是表征EGFR 1/4介导的T细胞多样性和功能的控制。 虽然EGR 1 KO小鼠在早期T细胞发育中表现出先前描述的缺陷，但初步研究结果表明， 移植在这里是第一次揭示了T细胞表型在EGR 4KO小鼠。我们观察到 通过双阴性阶段，T细胞活化过程中Ca 2+清除的失调和缺陷性T细胞 扩张.这些研究将扩大，包括EGR 1/4dKO小鼠和包括分析 发育、常规和非常规亚型。目的2是评估STIM 1 - 597敲入AF- 影响T细胞活化。正如我们最近的研究所建立的，STIM 1 597位氨基酸截短（STIM 1 597） 消除PMCA关联而不影响Orai 1激活13。对从一个人身上获得的T细胞进行初步分析。 最近产生的STIM 1 β 597基因敲入小鼠揭示了Ca ~（2+）的有趣的T细胞亚群特异性变化。 2个以上 海绵。对T细胞活化的影响将通过测量转录因子活化、细胞因子 表达模式和T细胞多样性。目标3是定义POST协调的机制 功能性STIM 1-PMCA 4相互作用。我们发现，EGFR诱导的STIM 1上调导致减少 PMCA在T细胞活化期间在免疫突触内起作用。然而，第三种蛋白质称为POST 与这两种蛋白质相互作用，并在钙池耗竭期间调节Ca 2+清除。我们的初步调查结果提供了 对这些相互作用如何在T细胞活化过程中协调并定义其功能含义的新见解， 阳离子。这些相互作用的动力学将使用突变分析、FRET和Ca 2 + en. 尝试/清除测定。总的来说，这些研究揭示了一个新的概念，钙信号如何有助于 发生在秒至分钟时间段之外的事件，包括单个Ca 2+瞬变。

Store-operated Ca2+ entry (SOCE) is the process whereby loss of Ca2+ in the endoplasmic reticulum (ER) leads to Ca2+ entry into cells across the plasma membrane (PM). STIM1 and STIM2 are critical ER Ca2+ sensors that control the activation of Orai1, the store-operated Ca2+ channel. STIM1/2 are critical for T cell receptor (TCR)- mediated signaling and their deficiency in humans results in lymphoproliferative disorder and autoimmunity sug- gesting defects in T cell development and activation. In addition to ER Ca2+ release and SOCE, Ca2+ signals involve Ca2+ extrusion mechanisms. In lymphocytes, Plasma Membrane Ca2+/ATPase 4 (PMCA4) is the primary mediator of Ca2+ extrusion and is essential for normal function. Recently, we identified PMCA4 as a further target of STIM1 (but not STIM2) during lymphocyte activation. Unlike SOCE, STIM1-induced PMCA inhibition is initi- ated by Early Growth Response 1 (EGR1) and EGR4-mediated STIM1 upregulation. EGRs are ubiquitous and rapidly upregulated zinc finger transcription factors. The current proposal is based on the following hypothesis: TCR engagement stimulates EGR-mediated STIM1 upregulation in naïve T cells that initiates Ca2+ clearance inhibition, critical for the maintenance of NFAT/NF-κB activation and subsequent T cell activation. This work is organized into 3 aims. Aim 1 is to characterize EGR1/4-mediated control of T cell diversity and function. While EGR1KO mice exhibit previously described defects in early T cell development, preliminary findings re- ported here are the first to reveal a T cell phenotype in EGR4KO mice. We have observed altered progression through the double-negative stages, dysregulation of Ca2+ clearance during T cell activation and defective T cell expansion. These investigations will be expanded, both to include EGR1/4dKO mice and to include analysis of developmental, conventional and non-conventional subtypes. Aim 2 is to assess how STIM1∆597 knockin af- fects T cell activation. As established in our recent study, STIM1 truncation at amino acid 597 (STIM1∆597) eliminates PMCA association without affecting Orai1 activation13. Preliminary analysis of T cells obtained from a recently generated STIM1∆597 knock-in mouse reveals intriguing T cell subset-specific changes in Ca re- 2+ sponses. Implications to T cell activation will be determined by measuring transcription factor activation, cytokine expression patterns and T cell diversity. Aim 3 is to define the mechanisms whereby POST coordinates functional STIM1-PMCA4 interactions. We revealed that EGR-induced STIM1 upregulation causes decreased PMCA function within the immunological synapse during T cell activation. However, a third protein termed POST interacts with both proteins and regulates Ca2+ clearance during store depletion. Our preliminary findings provide new insight into how these interactions are coordinated during T cell activation and define their functional impli- cations. The dynamics of these interactions will be determined using mutational analysis, FRET and Ca2+ en- try/clearance assays. Collectively, these studies reveal a novel new concept for how Ca2+ signals contribute to events that occur beyond the seconds to minutes time periods encompassing a single Ca2+ transient.