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

存储操作的钙内流(SOCE)是内质网(ER)内的钙离子丢失导致的过程 通过质膜(PM)进入细胞。STIM1和STIM2是关键的内质网钙离子传感器 控制Orai1的激活，Orai1是存储操作的钙通道。STIM1/2对T细胞受体(TCR)至关重要。 人类介导的信号及其缺陷导致淋巴增殖性疾病和自身免疫提示。 T细胞发育和激活过程中存在缺陷。除内质网钙释放和SOCE外，钙信号 涉及钙离子排出机制。在淋巴细胞中，质膜钙/ATPase 4(PMCA4)是主要的 Ca~(2+)排出的中介体，是正常功能所必需的。最近，我们将PMCA4确定为进一步的目标 在淋巴细胞激活过程中STIM1(但不是STIM2)的表达。与SOCE不同的是，STIM1诱导的PMCA抑制是无创性的。 早期生长反应1(Egr1)和EGR4介导的STIM1上调。排污权无处不在，而且 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EGR介导的STIM1在启动钙清除的幼稚T细胞中的上调 抑制，对于维持NFAT/NF-κB的激活和随后的T细胞激活至关重要。这项工作是 组织成3个目标。目的1是研究Egr1/4对T细胞多样性和功能的调控。 虽然EGR1KO小鼠在早期T细胞发育中表现出先前描述的缺陷，但初步发现 在这里，我们首次发现了EGR4KO小鼠的T细胞表型。我们观察到了变化的进程 通过双阴性阶段，T细胞活化和缺陷T细胞中钙离子清除的失调 扩张。这些研究将扩大到包括Egr1/4dKO小鼠和分析 发展型、常规型和非常规型。目标2是评估STIM1∆597如何敲打- 影响T细胞的激活。根据我们最近的研究，STIM1在氨基酸第597位截断(STIM1∆597) 消除PMCA关联而不影响Orai1激活13。人外周血中T细胞的初步分析 最近产生的STIM1STIM597敲入小鼠显示了耐人寻味的T细胞亚群特异性Ca Re-∆变化 2+ 海绵。T细胞激活的意义将通过测量转录因子激活、细胞因子来确定 表达模式和T细胞多样性。目标3是定义员额坐标的机制 功能STIM1-PMCA4相互作用。我们发现，EGR诱导的STIM1上调原因减少 PMCA在T细胞活化的免疫突触中发挥作用。然而，第三种名为POST的蛋白质 与这两种蛋白质相互作用，并在储存耗尽过程中调节钙的清除。我们的初步发现提供了 对T细胞激活过程中这些相互作用是如何协调的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通过突变分析、FRET和Ca~(2+)EN- 尝试/清除化验。总而言之，这些研究揭示了一个新的概念，即钙信号如何参与 超过几秒到几分钟的时间段发生的事件包括单个钙离子瞬变。

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.