REGULATION AND FUNCTION OF AP1 IN MATURE B CELLS

描述（改编自研究者摘要）：JunB合作 与几种不同类型的转录因子一起调节B细胞 激活和发育，其表达控制在 转录。 该AP 1组分似乎是主要的此类组分 通过BCR调节刺激后的早期细胞周期事件。 的 目前建议的工作重点是调节junB 通过BCR信号激活转录。 第一个目标是 了解junB启动子中的复合ets/Stat元件如何介导 BCR诱导的junB转录。 定点诱变将用于 确定单个ets和Stat基序的功能贡献 到BCR感应器。 EMSA和DNA亲和色谱法将用于 找出与该区域结合的反式作用因子。 相邻 cAMP反应元件（CRE）也是junB BCR诱导所必需的， 结合CRE结合蛋白1（CREB 1）。 调查员发现了一本小说 通过Ser 133磷酸化的CREB 1活化途径。 这 BCR通过冈田酸敏感的磷酸化酶增加磷酸化， 蛋白磷酸酶（PPase）途径。 研究者假设， 这是通过BCR信号抑制PP 1或PP-2A的活性来实现的。 因此，该提案的第二个目标将是测试以下几个方面： 该假设使用抗PP 1和抗PP-2A抗体和色谱法， 分离B细胞提取物以鉴定和定量CREB 1 PPase 活动 药理学抑制剂和编码 BCR信号通路的显性负干扰成分将是 用于鉴定PPase抑制所必需的关键中间蛋白。 这些研究的数据预计将挑战现有的范式， CREB 1在哺乳动物细胞中的调节，并应阐明一种新的BCR ets和/或Stat结合的信号通路和潜在的新作用 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.