REGULATION AND FUNCTION OF AP1 IN MATURE B CELLS

描述(改编自《调查者摘要》)：JunB合作 几种不同类别的转录因子对B细胞的调节 激活和发育及其表达控制在水平上 抄写。这个AP1成分似乎是主要的这样的成分 通过bcr调节刺激后的早期细胞周期事件。这个 目前拟议工作的重点是监管JunB 转录被bcr信号激活。第一个目标将是 了解JunB启动子中的Ets/Stat复合元件如何介导 Bcr诱导的JunB转录。定点突变将被用于 确定单个Ets和Stat基序的功能贡献 对BCR诱导性的影响。EMSA和DNA亲和层析将用于 确定连接该区域的反式作用因子。一个相邻的 CAMP反应元件(CRE)对于JunB BCR的诱导性和 结合CRE结合蛋白1(CREB1)。调查员发现了一本小说 Ser133磷酸化激活CREB1的途径。这 Bcr通过冈田酸敏感增加了磷酸化。 蛋白磷酸酶(PPase)途径。调查员假设 这是通过抑制PP1或PP-2A活性的BCR信号来实现的。 因此，提案的第二个目标将是测试以下几个方面 这一假设使用了抗PP1和抗PP-2A的抗体和层析 B细胞提取液分级鉴定和定量测定CREB1 PPase 活动。药物抑制剂和编码基因的表达载体 BCR信号通路的主要负干扰成分将是 用于识别抑制PPase所需的关键中间蛋白。 这些研究的数据预计将挑战现有的研究范式 哺乳动物细胞中CREB1的调控并应阐明一种新的BCR Ets和/或Stat结合的信号通路及其潜在的新作用 基因调控中的蛋白质。

DESCRIPTION (Adapted from the Investigator's Abstract): JunB cooperates with several different classes of transcription factors to regulate B cell activation and development and its expression is controlled at the level of transcription. This AP1 component appears to be the major such component regulating early cell cycle events after stimulation via the BCR. The current focus of the proposed work is on the regulation of junB transcription activation by BCR signals. The first goal will be to understand how a composite ets/Stat element in the junB promoter mediates BCR inducible junB transcription. Site directed mutagenesis will be used to determine the functional contribution of the individual ets and Stat motifs to BCR inducibility. EMSAs and DNA affinity chromotography will be used to identify the trans-acting factors that bind this region. An adjacent cAMP-response element (CRE) is also necessary for junB BCR-inducibility and binds CRE-binding protein 1 (CREB1). The investigator has uncovered a novel pathway for CREB1 activation by Ser133 phosphorylation. This phosphorylation is increased by the BCR via an okadaic acid-sensitive protein phosphatase (PPase) pathway. The investigator hypothesizes that this is accomplished by BCR signals inhibiting the activity of PP1 or PP-2A. Thus, the second goal of the proposal will be to test several aspects of this hypothesis using anti-PP1 and anti-PP-2A antibodies and chromatographic fractionation of B cell extracts to identify and quantitate CREB1 PPase activity. Pharmacological inhibitors and expression plasmids encoding dominant negative interfering components of BCR signaling pathways will be used to identify key intermediate proteins necessary for PPase inhibition. The data from these studies are expected to challenge existing paradigms for CREB1 regulation in mammalian cells and should elucidate a novel BCR signaling pathway and potentially new roles for ets and/or Stat binding proteins in gene regulation.