A TRANSCRIPTION FACTOR WITHIN LIPID RAFTS MODULATES B CELL SIGNALING VIA THE BCR

描述(申请人提供)：B细胞抗原受体(BCR)复合体的不同信号强度如何转化为生化信号尚不清楚。越来越多的证据表明，脂筏起着通过BCR传递信号的平台的作用。Bright是一种B细胞限制性转录因子，通过与免疫球蛋白重链(IGH)内含子增强子两侧的核基质附着区结合，反式激活免疫球蛋白重链(IGH)基因座。Bright的DNA结合和转录活性是由它与Xla/Xid中涉及的分子Bruton‘s Tyroine Kinase(BTK)直接结合而刺激的。Bright在细胞核和细胞质之间经历依赖于细胞周期的穿梭，并与G1/S细胞周期进程有关。我们发现，一小部分(1-2%)的光亮物质组成地存在于脂筏中。在那里，它与BTK和BCR信号复合体联系在一起。在替代抗原刺激BCR后，Bright从木筏上以与信号强度成正比的速度排出。Bright与总甲基化E2和E3组分的可诱导结合导致Bright的Sumo-I修饰，并随后将其从筏子释放到质膜。BCR刺激诱导Bright和F-肌动蛋白连接蛋白Ezrin的瞬时、筏子特异性结合和随后的共同释放，这表明Bright在微丝解聚中可能发挥作用-BCR信号转导中的一个关键事件。这是第一次在脂筏中发现转录因子的功能。我们假设Bright具有减弱BCR信号的功能，即Bright耗尽的信号小体比含有Bright的信号小体更活跃。在这个R21的应用中，我们建议(1)定义Bright to Liped Right的规范要求；(2)构建和启动针对脂筏局部Bright的特异性改变的小鼠的分析；以及(3)确定脂筏参与的途径-局部Bright以调节BCR信号强度。这些研究提出了在膜和核之间运输货物的另一种途径。它们对免疫耐受、自身免疫和免疫缺陷具有长期意义。 转录因子是与细胞核内的DNA结合以启动基因表达的蛋白质。我们发现，已知具有这种特性的转录因子(Bright)也具有出人意料的特性，即定位于细胞膜(脂筏)的特定结构中。Bright以一种未知的方式调节B淋巴细胞对抗原的反应能力。了解脂筏定位的Bright如何调节免疫反应将影响我们对免疫耐受的理解，免疫耐受是防止免疫系统对自身反应的免疫机制，即自身免疫。

DESCRIPTION (provided by applicant): How variant signaling strength of the B cell antigen receptor (BCR) complex is transformed into biochemical signals is not well understood. A growing body of evidence indicates that lipid rafts function as platforms for signalling through the BCR. Bright is a B cell-restricted transcription factor that transactivates the immunoglobulin heavy chain (IgH) locus by binding to nuclear matrix attachment regions flanking the IgH intronic enhancer. Bright's DNA binding and transcriptional activities are stimulated by its direct association with Bruton's tyrosine kinase (Btk), the implicated molecule in XLA/xid. Bright undergoes a cell cycle- dependent shuttle between the nucleus and the cytoplasm and is implicated in G1/S cell cycle progression. We discovered that a small (1-2%) pool of Bright resides constitutively within lipid rafts. There it associates with Btk and the BCR signaling complex. Following BCR stimulation by surrogate antigen, Bright is discharged from rafts at a rate proportional to signal strength. An inducible association of Bright with sumoylation E2 and E3 components leads to Sumo-I-modification of Bright and its subsequent discharge from rafts into plasma membranes. BCR stimulation induces a transient, rafts-specific association and subsequent co-discharge of Bright and the F-actin linker protein Ezrin, suggesting a potential role for Bright in microfilament depolymerization - a key event in BCR signaling. This is the first functional demonstration of a transcription factor in lipid rafts. We hypothesize that Bright functions to attenuate BCR signaling; i.e., Bright-depleted signalosomes are more active than Bright-containing signalosomes. In this R21 application, we propose (1) to define the requirements for specification of Bright to lipid rafts; (2) to construct and initiate analysis of mice specifically altered for lipid-rafts-localized Bright; and (3) to identify pathways engaged by lipid rafts-localized Bright to regulate BCR signaling strength. These studies suggest another avenue for the transport of cargo between the membrane and the nucleus. They provide long-term significance for immunological tolerance, autoimmunity and immunodeficiency. Transcription factors are proteins that bind to DNA within the nucleus to initiate gene expression. We discovered that a transcription factor (Bright), which is known to possess this property, also has the unexpected property of localizing within specific structures of the cell membrane (lipid rafts). There Bright functions in an unknown manner to regulate the ability of the B lymphocyte to respond to antigen. Understanding how lipid rafts-localized Bright modulates immune responses will impact on our understanding of immunological tolerance, the immune mechanism that prevents the immune system from reacting against self; i.e., autoimmunity.