SIGNAL TRANSDUCTION PATHWAYS IN HEMATOPOIESIS

造血中的信号转导途径

• 批准号: 6537564
• 负责人: BERNARD MATHEY-PREVOT
• 金额: $ 30.96万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-10 至 2004-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6537564
• 关键词: Drosophilidae; JAK kinase; biological signal transduction; blood cells; cell proliferation; cell transformation; erythropoietin; genetic transcription; genetically modified animals; growth factor receptors; guanosine triphosphate; hematopoiesis; hematopoietic growth factor; molecular cloning; mutant; neoplastic transformation; nuclear factor kappa beta; phenotype; phosphorylation; protein structure function; protein tyrosine kinase; receptor expression; suppressor mutations; transcription factor; transfection

We postulate that there is extensive crosstalk between hematopoietic signal transduction pathways. We propose that this crosstalk helps to regulate the activation and termination of signals emanating from the surface of activated hematopoietic cells. To begin to address this hypothesis, two constitutively active receptors (cEpoR and CD8-cEyk), known to activate the JAK/STAT pathway, were introduced into murine hematopoietic (Ba/F3) cells. Both caused factor independence and transformation, as a result of activation of the JAK/STAT and Ras/Raf pathways. However, signaling events initiated by cEpoR and CD8-cEyk were clearly different. To further evaluate these differences, we have developed an in vivo assay in Drosophila blood cells. We take advantage of the fact that signaling pathways are remarkably conserved among metazoans. cDNAs for both receptors have been placed under the control of Gal4 transcriptional regulatory elements, and injected into Drosophila embryos. Transgenic flies were isolated and crossed with Drosophila lines expressing Gal4 protein under the control of a heat shock promoter. Upon heat shock, expression of the vertebrate receptors is induced, resulting in leukemia-like proliferation of blood cells and formation of melanotic tumors. This offers a powerful, novel assay to probe signal transduction circuits in vivo. We will further refine this assay, and determine the participation of the JAK/STAT, Ras/Raf, NF-kappaB and JNK pathways in hemocyte proliferation and tumorigenesis in genetic experiments. We will test whether loss of function mutants in these pathways can suppress the melanotic tumor phenotype. In addition, we will use this system to characterize a hyperphosphorylated STAT5 mutant that we have recently generated. We have shown that this STAT5 mutant is resistant to dephosphorylation. We expect to find that it confers a gain of function phenotype. Finally, we will exploit a genetic screen in Drosophila to isolate novel genes that interact with known signal transduction pathways to suppress the melanotic tumor phenotype. Viable and fertile flies, which all show melanotic tumors, will be crossed with mutant stocks carrying chromosomal aberrations, P-element insertions, or chemically- induced point mutations. Suppression of the melanotic tumor phenotype in the F1 generation will serve as the criterion to identify accessory factors that modulate known hematopoietic signal transduction pathways. This series of experiments should produce new insights into hematopoietic signal transduction that could not be obtained from standard biochemical experiments, or from gene manipulations in mice.

我们假设造血信号转导途径之间存在广泛的串扰。 我们建议该串扰有助于调节从活化造血细胞表面产生的信号的激活和终止。 为了开始解决这一假设，将两个组成型活性受体（CEPOR和CD8-CEYK）（已知激活JAK/STAT途径）引入到鼠造血（BA/F3）细胞中。 由于JAK/STAT和RAS/RAF途径的激活，两者都引起了因子独立性和转化。 但是，CEPOR和CD8-CEYK发起的信号传导事件明显不同。 为了进一步评估这些差异，我们在果蝇血细胞中开发了一个体内测定。 我们利用了这一事实，即信号通路在后生动物中明显保守。 两种受体的cDNA都置于GAL4转录调节元件的控制之下，并注射到果蝇胚胎中。 分离转基因苍蝇，并与果蝇线交叉，在热激启动子的控制下表达GAL4蛋白。 热休克后，诱导脊椎动物受体的表达，导致血清细胞的白血病样增殖并形成黑色素肿瘤。这提供了一个强大的新颖测定法，以探测体内信号转导电路。 我们将进一步完善该测定法，并确定JAK/STAT，RAS/RAF，NF-KAPPAB和JNK途径在血细胞增殖中的参与以及遗传实验中的肿瘤发生。 我们将测试这些途径中功能突变体的丧失是否可以抑制黑素肿瘤表型。 此外，我们将使用该系统来表征我们最近生成的高磷酸化STAT5突变体。 我们已经表明，该STAT5突变体对去磷酸化具有抗性。 我们希望发现它赋予了功能表型的增益。 最后，我们将利用果蝇中的遗传筛选来分离与已知信号转导途径相互作用的新型基因，以抑制黑色素的肿瘤表型。所有均表现出黑色素性肿瘤的可行和肥沃的果蝇将与携带染色体畸变，p元素插入或化学诱导的点突变的突变型杂交交叉。在F1生成中抑制黑色素肿瘤表型将作为确定调节已知造血信号转导途径的辅助因子的标准。 这一系列的实验应产生对造血信号转导的新见解，这些见解无法从标准的生化实验或小鼠的基因操纵中获得。