IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION OF P210 BCR/ABL SUBSTRATE

这个项目的广泛的长期目标是了解，在分子上， 水平，p210 bcr/abl蛋白酪氨酸激酶活性如何最终 导致慢性期CML的骨髓扩张。 为了实现这一 （a）确定和描述 相关p210 bcr/abl底物及其相互作用，在原发性 免疫沉淀法检测原始慢性期CML母细胞， 用已知蛋白质的可用抗体或通过蛋白质 纯化，测序和克隆，产生抗体， （B）确定潜在的新蛋白的生物学功能， 相关基质。 确定组成型酪氨酸 在原发性原始慢性期CML中底物的磷酸化 通过检查原始胚和成熟胚， 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 在原发性原始慢性期CML原始细胞中受影响的途径：（1a） 其他已知相关底物的表征;（1c）进一步 相关底物的鉴别;（id）对-酪氨酸的鉴别 原代原始正常母细胞中kit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. p155是一种酪氨酸蛋白， 在原发性CML母细胞中磷酸化升高（3a） p155在慢性期CML原始细胞和p210中的特征 bcr/abl表达细胞系;（3b）p155的纯化;（3c）克隆和 测序p155（艾德）功能分析。

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.