INHIBITOR OF THE C ABL TYROSINE KINASE

C ABL 酪氨酸激酶抑制剂

• 批准号: 2601273
• 负责人: RICHARD A. VAN ETTEN
• 金额: $ 25.96万
• 依托单位: IMMUNE DISEASE INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-04 至 2003-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2601273
• 关键词: 3T3 cells; DNA damage; SDS polyacrylamide gel electrophoresis; active sites; antioxidants; binding proteins; cell growth regulation; chimeric proteins; enzyme activity; enzyme inhibitors; enzyme structure; epitope mapping; flow cytometry; gene expression; immunoprecipitation; ionizing radiation; mutant; oncogenes; oxidative stress; phosphorylation; protein purification; protein sequence; protein tyrosine kinase; western blottings; yeast two hybrid system

DESCRIPTION: c-Abl is a ubiquitously expressed non-receptor tyrosine kinase localized to the nucleus and actin cytoskeleton whose kinase activity is regulated in the stress response to genotoxic insults. The kinase activity of c-Abl appears to be negatively regulated in vivo by a cellular inhibitor binding via the Abl SH3 domain, and Abl is activated to become leukemogenic by several mechanisms, including SH3 deletion and chromosomal translocations in human leukemias. A yeast two-hybrid screen for Abl SH3-binding proteins identified a gene called PAG, whose protein product (Pag) has many properties expected of a physiological inhibitor of c-Abl. When co-expressed with c-Abl, Pag associates with Abl in vivo and potently suppresses the tyrosine phosphorylation induced by c-Abl, dependent on the Abl SH3 and kinase domains. Pag also inhibits the kinase activity of c-Abl in an immune complex kinase assay. However, purified Pag is a poor inhibitor of purified Abl, suggesting that additional proteins might be required. Interestingly, Pag has been previously identified as a member of a novel family of proteins induced by oxidative stress which have antioxidant and cell cycle regulatory properties. The long-term goals of this application are to further our understanding of the regulation and physiological function of c-Abl, and to better understand the mechanism of activation of leukemogenic forms of Abl. In the first aim, the biochemical mechanism of inhibition of c-Abl by Pag will be determined. The location of the Abl SH3 and kinase domain interaction sites in Pag will be mapped. The inhibition of Abl observed in vivo will be reconstituted in vitro using Pag as an affinity reagent to isolate c-Abl from cell extracts. The identity of an additional cellular protein required for in vitro inhibition will be pursued biochemically, and an additional two-hybrid screen against Pag will be carried out. It will be tested whether Pag has antioxidant activity and whether oxidative stress activates c-Abl. In the second aim, it will be determined whether Pag is a physiological inhibitor of c-Abl in vivo. The subcellular localization of Pag and whether endogenous Pag and Abl co-associate will be determined, and whether this association is regulated by DNA damage or oxidative stress. The function of endogenous Pag will be inhibited by antisense and dominant-negative strategies, and the consequences for Abl activation and phenotypic responses determined. It will be tested if overexpression of Pag can inhibit transformation by SH3-containing oncogenic Abl. These experiments will yield important new information about the function and regulation of c-Abl and the mode of activation of leukemogenic forms of Abl, and identify new avenues for anti-leukemic therapies.

描述：c-Abl 是一种普遍表达的非受体酪氨酸激酶 定位于细胞核和肌动蛋白细胞骨架，其激酶活性为 对基因毒性损伤的应激反应进行调节。 激酶活性 c-Abl 的体内似乎受到细胞抑制剂的负调节 通过 Abl SH3 结构域结合，Abl 被激活成为白血病细胞 通过多种机制，包括 SH3 缺失和染色体易位 在人类白血病中。 Abl SH3 结合蛋白的酵母双杂交筛选 鉴定出一个名为PAG的基因，其蛋白质产物（Pag）有许多 c-Abl 生理抑制剂的预期特性。 什么时候 Pag 与 c-Abl 共表达，在体内与 Abl 有效结合 抑制 c-Abl 诱导的酪氨酸磷酸化，依赖于 Abl SH3 和激酶结构域。 Pag 还抑制 c-Abl 的激酶活性 在免疫复合物激酶测定中。 然而，纯化的 Pag 是一种较差的 纯化 Abl 的抑制剂，表明可能存在其他蛋白质 必需的。 有趣的是，帕格此前已被确认为 由氧化应激诱导的一个新的蛋白质家族，具有 抗氧化和细胞周期调节特性。 的长期目标 该应用程序旨在加深我们对法规的理解和 c-Abl的生理功能，并更好地了解其机制 Abl 的白血病形成形式的激活。第一个目标是生化 将确定Pag抑制c-Abl的机制。 的位置 Pag 中的 Abl SH3 和激酶结构域相互作用位点将被绘制。 这 体内观察到的 Abl 抑制将使用 Pag 在体外重建 作为亲和试剂从细胞提取物中分离 c-Abl。 身份 体外抑制所需的额外细胞蛋白将是 进行生化研究，另外针对 Pag 的两种杂交筛选将 进行。 将测试Pag是否具有抗氧化活性以及 氧化应激是否激活c-Abl。 在第二个目标中，它将是 确定 Pag 是否是体内 c-Abl 的生理抑制剂。 这 Pag 的亚细胞定位以及是否为内源性 Pag 和 Abl 将确定共同关联，以及该关联是否受到监管 DNA 损伤或氧化应激。 内源性 Pag 的功能为 被反义和显性失活策略抑制，并且 确定 Abl 激活和表型反应的后果。 它 将测试 Pag 的过度表达是否可以通过以下方式抑制转化： 含有 SH3 的致癌 Abl。这些实验将产生重要的新成果 有关c-Abl的功能和调节以及作用方式的信息 激活白血病形式的 Abl，并确定新的途径 抗白血病治疗。