IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION OF P210 BCR/ABL SUBSTRATE

P210 BCR/ABL 底物的鉴定和功能表征

• 批准号: 6316960
• 负责人: BAYARD D CLARKSON
• 金额: $ 24.43万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-06-01 至 2002-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6316960
• 关键词: biological signal transduction; cell growth regulation; cell line; cell transformation; cellular oncology; chronic myelogenous leukemia; cyclins; enzyme activity; enzyme substrate; growth factor; immunoprecipitation; molecular cloning; molecular oncology; nucleic acid sequence; oncoproteins; phosphorylation; protein purification; protein sequence; protein structure function; protein tyrosine kinase; protooncogene; western blottings

The broad long term goal of this project is to understand, on a molecular level, how the p210 bcr/abl protein tyrosine kinase activity ultimately causes a myeloid expansion in chronic phase CML. In order to achieve this objective, it becomes essential to: (a) Identify and characterize the relevant p210bcr/abl substrates, and their interactions, in primary primitive chronic phase CML blasts by immunoprecipitation and immunoblotting with available antibodies to known proteins or by protein purification, sequencing and cloning of, an generation of antibodies to, potential novel proteins; (b) Ascertain the biological functions of the relevant substrates. Ascertain whether the constitutive tyrosine phosphorylation of a substrate in primary primitive chronic phase CML blasts is aberrant or untimely by examining primary primitive and maturing normal subpopulations; (d) Determine the growth factor signal transduction pathways in which the relevant substrates may be involved. The specific aims of the project are: Aim 1. To further identify and characterize the relevant p210 bcr/abl substrates and signal transduction pathways affected in primary primitive chronic phase CML blasts: (1a) Characterization of other known relevant substrates; (1c) Further identification of relevant substrates; (id) Identification of P-tyr proteins in kit ligand pathway in primary primitive normal blasts. Aim 2. To isolate, sequence and characterize p56; (2a) cDNA cloning and sequencing; (2b) Antibody production; (2c) Functional analyses. Aim 3. To analyze the structure and function of p155, a protein whose tyrosine phosphorylation is elevated in primary CML blasts (3a) Further characterization of p155 in primary chronic phase CML blasts and p210 bcr/abl expressing cell lines; (3b) Purification of p155; (3c) Cloning and sequencing p p155 (ed) Functional analyses.

该项目的广泛长期目标是了解分子 p210 bcr/abl 蛋白酪氨酸激酶活性最终如何 导致慢性期 CML 的骨髓扩张。 为了实现这一目标 为实现这一目标，必须： (a) 确定并描述 初级中相关的 p210bcr/abl 底物及其相互作用 通过免疫沉淀法原始慢性期 CML 母细胞 使用已知蛋白质或蛋白质的可用抗体进行免疫印迹 纯化、测序和克隆，一代抗体， 潜在的新型蛋白质； (b) 确定生物功能 相关基材。 确定是否是组成型酪氨酸 原发性原始慢性期 CML 中底物的磷酸化 通过检查初级原始和成熟细胞是否异常或不合时宜 正常亚群； (d) 确定生长因子信号 可能涉及相关底物的转导途径。 该项目的具体目标是： 目标 1. 进一步确定和 表征相关 p210 bcr/abl 底物和信号转导 原发性慢性期 CML 原始细胞受影响的途径：(1a) 其他已知相关底物的表征； (1c)进一步 相关基材的识别； (id) P-tyr 的鉴定 原代正常原始细胞中试剂盒配体途径中的蛋白质。 目标2。 分离、测序和表征 p56； (2a) cDNA克隆和 测序； (2b)抗体产生； (2c) 功能分析。 目标3。 分析 p155（一种酪氨酸蛋白）的结构和功能 原代 CML 母细胞中磷酸化水平升高 (3a) 进一步 原发性慢性期 CML 母细胞中 p155 和 p210 的特征 bcr/abl 表达细胞系； (3b)p155的纯化； (3c) 克隆和 测序 p p155 (ed) 功能分析。