Spatial control of Ca2+ signals in lymphocytes

描述（由申请人提供）：淋巴细胞激活由多个细胞内信号传导过程组成。这种复杂性使淋巴细胞能够以各种方式调节其功能，从而正确地进行分化和激活。这种复杂系统的损伤可能导致免疫系统的功能失调，如自身免疫、过敏和免疫缺陷。活化t细胞核因子（Nuclear factor of activated t cells, NF-AT）是控制淋巴细胞活化的重要转录因子之一。NF-AT被Ca2+/钙调素依赖性磷酸酶钙调磷酸酶（CN）激活。NF-AT与CN的相互作用被FK506和环孢素A所抑制，它们被广泛用于抑制免疫应答。最近，一些细胞和病毒蛋白也被确定为CN/NF-AT相互作用的抑制剂。在初步研究中，我们发现了一种结合淋巴细胞特异性Src家族激酶Lck的蛋白Reps1，并在淋巴细胞中NF-AT激活中发挥关键作用。Reps1在胸腺细胞和其他淋巴器官中高度表达。在鸡b细胞系DT-40中敲除Reps1基因，由于缺乏NF-AT去磷酸化，导致NF-AT激活被消除。即使通过绕过近端信号事件的药物刺激细胞，NF-AT的激活也没有恢复。此外，在T细胞中表达突变型Reps1的转基因小鼠显示成熟T细胞产生IL-2的量显著降低。综上所述，这些数据表明Reps1在淋巴细胞活化中起着重要作用。在本研究中，我们将详细分析Reps1的体外功能机制及其在体内的生物学意义。

DESCRIPTION (provided by applicant): Lymphocyte activation consists of multiple intracellular signaling processes. This complexity allows lymphocytes to regulate their function in various ways so that their differentiation and activation are properly carried out. Impairment in this complex system could lead to malfunctioning of the immune system, such as autoimmunity, allergy, and immunodeficiency. Nuclear factor of activated T-cells (NF-AT) is regarded as one of the most important transcription factors that controls lymphocyte activation. NF-AT is activated by Ca2+/calmodulin-dependent phosphatase calcineurin (CN). The interaction between NF-AT and CN is inhibited by FK506 and Cyclosporin A, which are broadly utilized for suppression of the immune responses. Recently, several cellular and viral proteins were also determined as the inhibitors of CN/NF-AT interaction. In the preliminary study, we identify a protein Reps1 that binds the lymphoid specific Src family kinase Lck and plays a critical role in NF-AT activation in lymphocytes. Reps1 is highly expressed in thymocytes and other lymphoid organs. Gene knockout of Reps1 in a chicken B-cell line DT-40 resulted in abolishment of NF-AT activation due to lack of NF-AT dephosphorylation. NF-AT activation was not restored even when cells were stimulated by pharmacological agents that bypass proximal signaling events. Further, transgenic mice expressing a mutant form of Reps1 in T cells showed significantly reduced IL-2 production by mature T cells. Together, the data indicates that Reps1 plays an essential role in lymphocyte activation. In this study, we will analyze the detailed mechanism of Reps1 function in vitro and its biological significance in vivo.