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 转导机制，生长因子控制 造血增殖和分化。 这些知识是 了解造血调节的疾病至关重要 性贫血和白血病。 这笔赠款的主要目标是 了解红细胞生成素（EPO）调节的机制 红细胞分化过程中的离子通道并确定 钙涌入在红细胞生成中的功能作用。 这个系统是 描绘随后的即时信号事件的模型 EPO与其受体在正常细胞上的相互作用。 下列 具体目标将被解决： 特定目标1：识别信号传导机制 EPO调节钙通道。 我们已经表征了Epo修饰 钙通道采用贴片钳方法，并确定 酪氨酸磷酸化和G蛋白亚基Gialpha2是 必需的。 我们还表明JAK2参与其中。 （a）在这里，我们将 确定将JAK2连接到钙通道的EPO信号通路 激活。 STAT，RAS或IRS-2/PI 3-激酶途径的参与 将使用单个BFU-E得出的显微注射来检查 红细胞和定量荧光显微镜耦合数字 视频成像。 如果需要RAS，则其他传感器在 将检查RAS路径。 （b）我们将确定 钙通道的EPO调节所需的红细胞生成素受体 开场。 特定目的2：确定[CAI]在调节中的作用 红细胞生成的转录因子激活。 功能 NF-KAPPAB和BHLH转录因子以及C-Jun N末端 激酶（JNK）通过钙调节。 我们将确定是否 红细胞细胞中Ca ++反应的幅度或持续时间影响 NF-kappab转录因子激活，或者如果钙/钙调蛋白 水平影响BHLH蛋白，特别是SCL的DNA结合。 我们 还将确定[CAI]对JNK激活和角色的影响 在此途径中的Pi 3-激酶。