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

描述（由申请人提供）：B细胞抗原受体（BCR）复合物的变异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是通过BCR信号传导的平台。Bright是一种B细胞限制性转录因子，通过结合IgH内含子增强子两侧的核基质附着区来激活免疫球蛋白重链（IgH）位点。Bright与布鲁顿酪氨酸激酶（Btk） （XLA/xid中涉及的分子）的直接关联刺激了其DNA结合和转录活性。Bright在细胞核和细胞质之间进行细胞周期依赖的穿梭，并与G1/S细胞周期进程有关。我们发现一小部分（1-2%）的Bright存在于脂筏中。在那里它与Btk和BCR信号复合物相关联。通过替代抗原刺激BCR后，Bright以与信号强度成比例的速率从筏中排出。Bright与sumoylation E2和E3组分的诱导关联导致Bright的sumo - i修饰及其随后从筏中排放到质膜中。BCR刺激诱导了短暂的筏特异性结合和随后的Bright和f -肌动蛋白连接蛋白Ezrin的共放电，这表明Bright在微丝解聚中可能发挥作用，这是BCR信号传导的关键事件。这是转录因子在脂筏中的第一个功能演示。我们假设Bright具有减弱BCR信号的功能；即，耗尽bright的信号体比含有bright的信号体更活跃。在本R21申请中，我们建议(1)定义Bright对脂筏的规格要求；(2)构建并启动脂筏定位型Bright特异性改变小鼠的分析；(3)确定脂质筏定位的Bright参与调节BCR信号强度的途径。这些研究提示了在膜和细胞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。

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.