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 局部与激酶活性的细胞核和肌动蛋白细胞骨架 在对遗传毒性损伤的压力反应中受到调节。 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，并激活ABL以成为白血病 通过几种机制，包括SH3缺失和染色体易位 在人类白血病中。 ABL SH3结合蛋白的酵母两杂交筛选 鉴定出一个称为PAG的基因，其蛋白质产物（PAG）具有许多 C-ABL的生理抑制剂期望的特性。 什么时候 与C-ABL共表达，PAG与ABL In Vivo相关联 抑制C-ABL诱导的酪氨酸磷酸化，取决于 ABL SH3和激酶域。 PAG还抑制C-ABL的激酶活性 在免疫复合激酶测定中。 但是，纯化的PAG是一个差 纯化ABL的抑制剂，表明其他蛋白质可能是 必需的。 有趣的是，PAG先前已被确定为 由氧化应激诱导的新型蛋白质家族 抗氧化剂和细胞周期调节特性。 长期目标 该应用程序是为了进一步了解法规和 C-ABL的生理功能，并更好地了解 ABL的白血病形式的激活。在第一个目标中，生化 将确定通过PAG抑制C-ABL的机制。 位置 PAG中的ABL SH3和激酶域相互作用位点将被映射。 这 在体内观察到的ABL的抑制作用将在体外使用PAG在体外重组 作为与细胞提取物分离C-ABL的亲和力试剂。 的身份 体外抑制所需的其他细胞蛋白将是 在生化上追踪，另外一个针对PAG的两种杂交屏幕将 被执行。 将测试PAG是否具有抗氧化活性和 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的亚细胞定位以及内源性PAG和ABL是否 将确定共同点，以及该关联是否受到调节 通过DNA损伤或氧化应激。 内源性PAG的功能将是 被反义和主导的阴性策略抑制， 确定ABL激活和表型反应的后果。 它 如果PAG的过表达可以通过 含SH3的致癌ABL。这些实验将产生重要的新 有关C-ABL的功能和调节的信息以及 激活ABL的白血病形式，并确定新的途径 抗白血病疗法。