SIGNAL TRANSDUCTION MECHANISMS OF ERYTHROPOIETIN

促红细胞生成素的信号转导机制

• 批准号: 6348752
• 负责人: BARBARA A. MILLER
• 金额: $ 17.18万
• 依托单位: GEISINGER CLINIC
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-03-01 至 2002-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6348752
• 关键词: CHO cells; DNA binding protein; JAK kinase; biological signal transduction; calcium channel; calcium flux; calmodulin; cell differentiation; cell proliferation; digital imaging; enzyme activity; erythroid stem cell; erythropoiesis; erythropoietin; fluorescence microscopy; genetic transcription; growth factor receptors; human tissue; microinjections; nuclear factor kappa beta; phosphatidylinositol 3 kinase; phosphorylation; protein structure; transfection /expression vector; western blottings

The long term goal of this project is to understand the signal transduction mechanisms through which growth factors control hematopoietic proliferation and differentiation. This knowledge is essential to understand disorders of hematopoietic regulation including aplastic anemia and leukemia. The major goal of this grant is to understand the mechanisms through which erythropoietin (Epo) regulates ion channels during erythroid differentiation and to determine the functional role of calcium influx in erythropoiesis. This system is a model to delineate the immediate signaling events which follow interaction of Epo with its receptor on normal cells. The following specific aims will be addressed: Specific Aim 1: Identification of the signaling mechanisms through which Epo regulates calcium channels. We have characterized the Epo-modulated calcium channel with patch-clamp methodology and have determined that tyrosine phosphorylation and the G protein subunit Gialpha2 are required. We have also shown that Jak2 is involved. (A) Here, we will determine the Epo signaling pathways which link Jak2 to calcium channel activation. Involvement of STAT, Ras, or the IRS-2/PI 3-kinase pathways will be examined using microinjection of single BFU-E derived erythroblasts and quantitative fluorescence microscopy coupled digital video imaging. If Ras is required, the role of other transducers in the Ras pathway will be examined. (B) We will determine the domains of erythropoietin receptor required for Epo modulation of calcium channel opening. Specific Aim 2: Determination of the role of [Cai] in regulation of transcription factor activation in erythropoiesis. The functions of the NF-kappaB and bHLH transcription factors and the c-Jun N-terminal kinases (JNK) are modulated by calcium. We will determine if the amplitude or duration of the Ca++ response in erythroid cells affects NF-kappaB transcription factor activation, or if calcium/calmodulin levels influence DNA binding of bHLH proteins, particularly SCL. We will also determine the effect of [Cai] on JNK activation and the role of PI 3-kinase in this pathway.

该项目的长期目标是了解信号 生长因子控制的转导机制 造血细胞增殖和分化。 这种知识是 对于理解造血调节障碍至关重要， 再生障碍性贫血和白血病。 该补助金的主要目标是 了解促红细胞生成素（Epo）调节 离子通道在红细胞分化过程中，并确定 钙内流在红细胞生成中的功能作用。 该系统是一 模型来描述紧接着的信号事件， 正常细胞上Epo与其受体的相互作用。 以下 具体目标如下： 具体目标1：确定信号传导机制， 促红细胞生成素调节钙通道。 我们已经表征了Epo调制的 用膜片钳方法研究钙通道，并确定 酪氨酸磷酸化和G蛋白亚基Gialpha 2是 必需的. 我们还证明了Jak 2也参与其中。(A)在这里，我们将 确定连接Jak 2和钙通道的Epo信号通路 activation. STAT、Ras或IRS-2/PI 3-激酶通路的参与 将使用显微注射单个BFU-E衍生的 成红细胞和定量荧光显微镜耦合数字 视频成像 如果需要Ras， 将检查Ras途径。 (B)我们将确定 促红细胞生成素调节钙通道所需的促红细胞生成素受体 开放后 具体目标2：确定[Cai]在调节 转录因子在红细胞生成中的活化。 的职能 NF-κ B和bHLH转录因子和c-Jun N-末端 激酶（JNK）受钙调节。 我们将确定 红系细胞中Ca++反应的幅度或持续时间影响 NF-κ B转录因子激活，或如果钙/钙调蛋白 水平影响bHLH蛋白的DNA结合，特别是SCL。 我们 还将确定[Cai]对JNK激活的影响以及 PI 3-激酶在这条通路中的作用。