IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION OF P210 BCR/ABL SUBSTRATE

这个项目广泛的长期目标是理解，在一个分子上 水平，p210BCR/ABL蛋白酪氨酸激酶活性最终如何 导致慢性期CML的髓样扩张。为了实现这一目标 为了实现这一目标，必须：(A)确定和描述 相关的p210bcr/abl底物及其相互作用，在初级 免疫沉淀诱导原始慢性期CML细胞分化 用已知蛋白质或蛋白质的有效抗体进行免疫印迹 一代抗体的纯化、测序和克隆， 潜在的新蛋白质；(B)确定该蛋白的生物学功能 相关底物。确定构成酪氨酸是否 慢性粒细胞白血病原发原始慢性期一种底物的磷酸化 通过检查原始的原始的和成熟的是异常的或不合时宜的 正常亚群；(D)确定生长因子信号 相关底物可能参与的转导途径。 该项目的具体目标是：目标1.进一步确定和 鉴定相关的p210 BCR/ABL底物和信号转导 原发原始慢性期CML细胞影响的通路：(1A) 其他已知相关底物的特性；(1c)进一步 相关底物的鉴定；(Id)P-Tyr的鉴定 原代原始正常母细胞中KIT配体途径中的蛋白质。目标2. P56基因的分离、测序和鉴定；(2a)cdna克隆和 测序；(2b)抗体生产；(2c)功能分析。目标3. 分析其酪氨酸蛋白p155的结构和功能 原代CML原始细胞(3a)的磷酸化水平进一步升高 原发慢性期CML细胞中p155和p210的表达特征 表达bcr/abl的细胞系；(3b)p155的纯化；(3c)克隆和 对p155(Ed)功能分析进行排序。

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.